Differential responses of weaned piglets to supplemental porcine or chicken plasma in diets without inclusion of antibiotics and zinc oxide.

This study was conducted to investigate the effects of spray-dried porcine plasma protein (SDPP) or spray-dried chicken plasma protein (SDCP) supplementation in diets without the inclusion of antibiotics and zinc oxide (ZnO) on growth performance, fecal score, and fecal microbiota in early-weaned piglets. A total of 192 healthy weaning piglets (Duroc × Landrace × Yorkshire, 21 d old) were blocked by BW (6.53 ± 0.60 kg) and randomly assigned to 4 dietary treatments: negative control (NC, basal diet), positive control (PC), basal diet + ZnO at 2 g/kg and antibiotics at 0.8 g/kg), SDPP (containing 5% SDPP), and SDCP (containing 5% SDCP). The experiment lasted 14 d. The SDPP group had higher (P < 0.05) final BW, average daily gain and average daily feed intake than the NC and SDCP groups. The percentage of piglets with fecal scores at 2 or ≥2 was higher (P < 0.05) in the NC and SDCP groups than in the PC group. A decreased (P < 0.05) bacterial alpha diversity and Bacteroidetes abundance, but increased (P < 0.05) Firmicutes abundance were observed in the PC and SDPP groups when compared to the NC group. The relative abundance of Lactobacillus was higher (P < 0.05) in the SDPP than in the SDCP group, and that of Streptococcus was higher (P < 0.01) in the PC and SDPP groups than in the NC group. The PC group also had higher (P < 0.01) Faecalibacterium abundance than the NC and SDCP groups. Additionally, the SDCP group had higher (P < 0.05) serum urea nitrogen than those fed other diets, and lower (P < 0.10) short-chain fatty acids to branched-chain fatty acids ratio than the PC and SDPP groups. Overall, SDPP was a promising animal protein for piglets in increasing feed intake, modifying gut microbiota profile, reducing gut protein fermentation and alleviating diarrhea frequency, thus promoting growth performance, under the conditions with limited in-feed utilization of antibiotics and ZnO.

Description and determinants of the faecal resistome and microbiome of farmers and slaughterhouse workers: A metagenome-wide cross-sectional study.

BACKGROUND: By studying the entire human faecal resistome and associated microbiome, the diversity and abundance of faecal antimicrobial resistance genes (ARGs) can be comprehensively characterized. Prior culture-based studies have shown associations between occupational exposure to livestock and carriage of specific antimicrobial resistant bacteria. Using shotgun metagenomics, the present study investigated 194 faecal resistomes and bacteriomes from humans occupationally exposed to ARGs in livestock (i.e. pig and poultry farmers, employees and family members and pig slaughterhouse workers) and a control population (Lifelines cohort) in the Netherlands. In addition, we sought to identify determinants for the human resistome and bacteriome composition by applying a combination of multivariate (NMDS, PERMANOVA, SIMPER and DESeq2 analysis) and multivariable regression analysis techniques. RESULTS: Pig slaughterhouse workers and pig farmers carried higher total ARG abundances in their stools compared to broiler farmers and control subjects. Tetracycline, ß-lactam and macrolide resistance gene clusters dominated the resistome of all studied groups. No significant resistome alpha diversity differences were found among the four populations. However, the resistome beta diversity showed a separation of the mean resistome composition of pig and pork exposed workers from broiler farmers and controls, independent of their antimicrobial use. We demonstrated differences in resistome composition between slaughter line positions, pig versus poultry exposed workers, as well as differences between farmers and employees versus family members. In addition, we found a significant correlation between the bacteriome and resistome, and significant differences in the bacteriome composition between and within the studied subpopulations. Finally, an in-depth analysis of pig and poultry farms - of which also farm livestock resistomes were analysed - showed positive associations between the number of on-farm working hours and human faecal AMR loads. CONCLUSION: We found that the total normalized faecal ARG carriage was larger in persons working in the Dutch pork production chain compared to poultry farmers and controls. Additionally, we showed significant differences in resistome and bacteriome composition of pig and pork exposed workers compared to a control group, as well as within-population (farms, slaughterhouse) compositional differences. The number of on-farm working hours and the farm type (pig or broiler) that persons live or work on are determinants for the human faecal resistome. Overall, our results may suggest direct or indirect livestock contact as a determinant for human ARG carriage. Future studies should further focus on the connection between the human and livestock resistome (i.e. transmission routes) to substantiate the evidence for livestock-associated resistome acquisition.

Occupational Exposure and Carriage of Antimicrobial Resistance Genes (tetW, ermB) in Pig Slaughterhouse Workers.

OBJECTIVES: Slaughterhouse staff is occupationally exposed to antimicrobial resistant bacteria. Studies reported high antimicrobial resistance gene (ARG) abundances in slaughter pigs. This cross-sectional study investigated occupational exposure to tetracycline (tetW) and macrolide (ermB) resistance genes and assessed determinants for faecal tetW and ermB carriage among pig slaughterhouse workers. METHODS: During 2015-2016, 483 faecal samples and personal questionnaires were collected from workers in a Dutch pig abattoir, together with 60 pig faecal samples. Human dermal and respiratory exposure was assessed by examining 198 carcass, 326 gloves, and 33 air samples along the line, next to 198 packed pork chops to indicate potential consumer exposure. Samples were analyzed by qPCR (tetW, ermB). A job exposure matrix was created by calculating the percentage of tetW and ermB positive carcasses or gloves for each job position. Multiple linear regression models were used to link exposure to tetW and ermB carriage. RESULTS: Workers are exposed to tetracycline and macrolide resistance genes along the slaughter line. Tetw and ermB gradients were found for carcasses, gloves, and air filters. One packed pork chop contained tetW, ermB was non-detectable. Human faecal tetW and ermB concentrations were lower than in pig faeces. Associations were found between occupational tetW exposure and human faecal tetW carriage, yet, not after model adjustments. Sampling round, nationality, and smoking were determinants for ARG carriage. CONCLUSION: We demonstrated clear environmental tetracycline and macrolide resistance gene exposure gradients along the slaughter line. No robust link was found between ARG exposure and human faecal ARG carriage.

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.

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.

Interaction of Skatole and Androstenone in the Olfactory Perception of Boar Taint.

This study analyzed odor-odor interactions of two malodorous volatile substances, androstenone and skatole, that may accumulate in fat and meat of uncastrated male (boar) pigs. Therefore, fat samples were collected from 1000+ entire male pig carcasses for sensory evaluation and quantification of boar taint compounds using gas chromatography-mass spectrometry (GC-MS). Each sample was sniffed by 10 trained assessors, resulting in 11 000+ individual ratings, which were subjected to statistical analysis. Pearson correlations of chemical traits and sensory traits (panel average) were higher for skatole [r(1029) = 0.59; p < 0.001] than for androstenone [r(1029) = 0.44; p < 0.001]. Linear terms of androstenone and skatole as well as their interaction significantly (p < 0.05) contributed to perception of deviant smell (R(2) = 0.43). Standardized regression coefficients illustrate the higher importance of skatole (ß = 0.68) than androstenone (ß = 0.39). Interindividual differences in the responses of assessors to androstenone and skatole are confirmed. A new curved approach is suggested because it better accounts for the interaction of androstenone and skatole than the "safe box" approach. On the basis of these data, sorting strategies using instrumental measurements are discussed. An automated detection based on only skatole measurements is recommended because its performance is only slightly inferior to a sorting based on both androstenone and skatole. Sorting thresholds need to be calibrated against consumer acceptance though.

Serological examination of fattening pigs reveals associations between Ascaris suum, lung pathogens and technical performance parameters.

Diagnosing the presence of the highly prevalent and economically important pig parasite Ascaris suum on fattening farms has so far been challenging. Currently, only the number of livers affected at slaughter is routinely used to measure parasite exposure. However, recently, a new serological test was developed based on the detection of antibodies to the A. suum haemoglobin molecule. The test showed to be highly sensitive for the detection of exposure to A. suum in fattening pigs. In this study we first compared the performance of A. suum serology versus the percentage of affected livers at slaughter, subsequently we investigated potential associations between A. suum infection levels and exposure to important lung pathogens and finally we identified correlations between serological data and technical performance parameters (TPIs) from 20 Belgian and 20 German pig fattening farms. In both Belgian and German farms, a significant relationship was detected between elevated average Ascaris serology and percentages of affected livers (ρ=0.63 and ρ=0.75, respectively). On the Belgian farms, both Ascaris serology and the percentage of affected livers were negatively correlated with average daily gain (ADG) (ρ=-0.69 and ρ=-0.56, respectively). Using the German dataset, only a borderline negative association was detected between the percentage of affected livers and the ADG (ρ=-0.44, P=0.053). In contrast, only in the German farms, correlations between the percentage of affected lungs at slaughter and elevated presence of A. suum and several other airway pathogens were detected. To conclude, this study indicates that serological screening for A. suum on fattening farms is an attractive new diagnostic tool that can be used to indicate the presence of roundworm infection by measuring infection intensity. Furthermore the results of this study also add weight to the evidence that both roundworm infections as well as herd exposure to airway pathogens have a significant impact on farm productivity and hence, that all these factors should to be taken into account when assessing pig health and farm productivity.

Prevalence of Mycobacterium avium in slaughter pigs based on serological monitoring results and bacteriological validation.

Mycobacterium avium (MA) is a potential food safety hazard in pigs. Blood samples of slaughtered pigs in the Netherlands and Germany were tested for the presence of MA antibodies to estimate the serological prevalence in the tested population. In the Dutch and German population 1.0% and 1.7% samples were positive, and 0.5% and 17.4% of the herds were at risk for having a MA infection respectively. The validity of the applied MA-ELISA was evaluated under field conditions. The specificity of the MA-ELISA was high (>98.4%). The average herd sensitivity was 18%. In the affected herds on average 50% of the animals were tested bacteriological positive for MA. It can be concluded that serological screening for the presence of MA antibodies is capable of identifying pig populations that are at risk for a MA infection.

Livestock-associated MRSA ST398 carriage in pig slaughterhouse workers related to quantitative environmental exposure.

OBJECTIVES: To assess livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) carriage among workers in pig slaughterhouses and assess associated risk factors, including occupational exposure to LA-MRSA. METHODS: A cross-sectional study in three Dutch pig slaughterhouses was undertaken. Nasal swabs of participants were taken. Nasal swabs and surface wipes, air and glove samples were screened for presence of methicillin-resistant Staphylococcus aureus (MRSA). MRSA was quantitatively determined on gloves and in air samples by culturing and real-time PCR. RESULTS: 11 of 341 (3.2%) participants were identified as nasal MRSA carriers. MRSA-positive workers were predominantly found at the start of the slaughter process. Major risk factors for carriage were working in the lairage and working in the scalding and dehairing area. Most nasal isolates (73%) belonged to the LA-MRSA clone ST398. MRSA ST398-positive environmental samples were found throughout the slaughter process. A clear decrease was seen along the slaughterline in the number of MRSA-positive samples and in the MRSA amount per sample. CONCLUSIONS: This study showed that working in the lairage area or scalding and dehairing area were the major risk factors for MRSA carriage in pig slaughterhouse workers, while the overall prevalence of MRSA carriage is low. Occupational exposure to MRSA decreased along the slaughterline, and the risk of carriage showed a parallel decrease.

A quantitative approach towards a better understanding of the dynamics of Salmonella spp. in a pork slaughter-line.

Pork contributes significantly to the public health disease burden caused by Salmonella infections. During the slaughter process pig carcasses can become contaminated with Salmonella. Contamination at the slaughter-line is initiated by pigs carrying Salmonella on their skin or in their faeces. Another contamination route could be resident flora present on the slaughter equipment. To unravel the contribution of these two potential sources of Salmonella a quantitative study was conducted. Process equipment (belly openers and carcass splitters), faeces and carcasses (skin and cutting surfaces) along the slaughter-line were sampled at 11 sampling days spanning a period of 4 months. Most samples taken directly after killing were positive for Salmonella. On 96.6% of the skin samples Salmonella was identified, whereas a lower number of animals tested positive in their rectum (62.5%). The prevalence of Salmonella clearly declined on the carcasses at the re-work station, either on the cut section or on the skin of the carcass or both (35.9%). Throughout the sampling period of the slaughter-line the total number of Salmonella per animal was almost 2 log lower at the re-work station in comparison to directly after slaughter. Seven different serovars were identified during the study with S. Derby (41%) and S. Typhimurium (29%) as the most prominent types. A recurring S. Rissen contamination of one of the carcass splitters indicated the presence of an endemic 'house flora' in the slaughterhouse studied. On many instances several serotypes per individual sample were found. The enumeration of Salmonella and the genotyping data gave unique insight in the dynamics of transmission of this pathogen in a slaughter-line. The data of the presented study support the hypothesis that resident flora on slaughter equipment was a relevant source for contamination of pork.

The BSE risk of processing meat and bone meal in nonruminant feed: a quantitative assessment for the Netherlands.

The total ban on use of meat and bone meal (MBM) in livestock feed has been very successful in reducing bovine spongiform encephalopathy (BSE) spread, but also implies a waste of high-quality proteins resulting in economic and ecological loss. Now that the BSE epidemic is fading out, a partial lifting of the MBM ban might be considered. The objective of this study was to assess the BSE risk for the Netherlands if MBM derived from animals fit for human consumption, i.e., category 3 MBM, would be used in nonruminant feed. A stochastic simulation model was constructed that calculates (1) the probability that infectivity of undetected BSE-infected cows ends up with calves and (2) the quantity of infectivity (Q(inf)) consumed by calves in case of such an incident. Three pathways were considered via which infectivity can reach cattle: (1) cross-contamination in the feed mill, (2) cross-contamination on the primary farm, and (3) pasture contamination. Model calculations indicate that the overall probability that infectivity ends up with calves is 3.2%. In most such incidents the Q(inf) is extremely small (median = 6.5 x 10(-12) ID(50); mean = 1.8 x 10(-4) ID(50)), corresponding to an average probability of 1.3 x 10(-4) that an incident results in >or=1 new BSE infections. Cross-contamination in the feed mill is the most risky pathway. Combining model results with Dutch BSE prevalence estimates for the coming years, it can be concluded that the BSE risk of using category 3 MBM derived from Dutch cattle in nonruminant feed is very low.

Spatial analysis of BSE cases in the Netherlands.

BACKGROUND: In many of the European countries affected by Bovine Spongiform Encephalopathy (BSE), case clustering patterns have been observed. Most of these patterns have been interpreted in terms of heterogeneities in exposure of cattle to the BSE agent. Here we investigate whether spatial clustering is present in the Dutch BSE case data. RESULTS: We have found three spatial case clusters in the Dutch BSE epidemic. The clusters are geographically distinct and each cluster appears in a different birth cohort. When testing all birth cohorts together, only one significant cluster was detected. The fact that we found stronger spatial clustering when using a cohort-based analysis, is consistent with the evidence that most BSE infections occur in animals less than 12 or 18 months old. CONCLUSION: Significant spatial case clustering is present in the Dutch BSE epidemic. The spatial clusters of BSE cases are most likely due to time-dependent heterogeneities in exposure related to feed production.

Identification of the characteristics and risk factors of the BSE epidemic in the Netherlands.

It is generally accepted that the bovine spongiform encephalopathy (BSE) epidemic is transmitted by feed contamination with meat and bone meal (MBM). Whether in some cases substances other than MBM have caused the spread of this disease cannot be excluded at present. Detailed knowledge about country-specific transmission routes and relevant risk factors is important to perform accurate risk analyses and to control BSE. In the present study all possible information of Dutch BSE cases was collected. The general epidemiological data were first used for a comprehensive descriptive analysis of the Dutch BSE epidemic. A case-control study was performed to identify the risk factors associated with the occurrence of BSE cases in the Netherlands. Sixty-eight of the 76 BSE cases enrolled in the case-control study. With an oral questionnaire information was collected about cow and farm management. The same questionnaire was used in interviews at 128 randomly selected control farms. Detailed analysis of the case anamneses showed that all affected cows could have been exposed to MBM from pig or poultry feed as a result of cross-contamination. In the case-control study, only feed producer appeared to be a relevant risk factor. Differences in log odds between feed producers were related to the moment of separation of production lines and to the origin of the MBM. The results suggest that there were meaningful differences in the level of infectivity in MBM from different origins at the time that cross-contamination was still possible. No other risk factors, either farm or cow related, were significantly associated with the occurrence of BSE.

[Campylobacter and Salmonella control in chickens and the role of fermented food]. / Campylobacter- en Salmonella- beheersing in vleeskuikens en de rol van brijvoer.

Salmonella and Campylobacter are undesirable pathogens on poultry. Therefore the effect of fermented feed on the colonization in the gastro-intestinal tract of the chicken, the introduction of both bacteria in a chicken flocks, and the transmission between chickens was studied. Broilers that were fed with fermented feed were significantly less susceptible for Salmonella and Campylobacter than chickens on a standard chicken feed. The spread of Salmonella between broiler chickens was reduced. However, the results also showed, like for other known control measures, that this feed can not absolutely guarantee the absence of Salmonella and Campylobacter. Therefore fermented feed must be seen as one of the hurdles in a so called multiple hurdle strategy. The combination of different hurdles should prevent the introduction and transmission. The effect of fermented feed on Campylobacter and Salmonella is partially caused by the presence of high concentrations of organic acids. In chickens fed with liquid feed the acidic barrier in the first part of the GI-tract was clearly improved. Besides organic acids there are other changes in the GI-tract. Changes in colonization levels of indicator organisms, changes in levels of organic acids and an increased pH in ileum and ceacum. These changes indicate a stabilised GI-flora in fermented feed fed poultry. The research confirmed that by changes in the composition of the feed (carbohydrates, acids, or micro-organisms) the GI-health can be promoted and therewith can contribute to the control of food pathogens in farmed animals.

Effect of fermented feed on the susceptibility for Campylobacter jejuni colonisation in broiler chickens with and without concurrent inoculation of Salmonella enteritidis.

Fermented liquid feed (FLF) protects broiler chickens against colonisation with Salmonella. While Campylobacter causes more disease cases in humans than Salmonella, the effect of FLF on Campylobacter was assessed. The fermented liquid feed is a moistened feed with a high number of lactobacilli, a high concentration of lactic acid, and a pH of 4. In three experiments Campylobacter was orally applied to individually housed 9-day-old broiler chickens. A significant reduction of susceptibility, as determined by cloacal swabs, was observed. At any moment where an animal has not started to shed Campylobacter yet, the probability to start shedding Campylobacter in a subsequent small time interval was nine times as high for the control chickens than for the animals that were fed FLF. FLF did not consistently change the Campylobacter colonisation level in the caeca. It was concluded that FLF could reduce the probability of introduction of Campylobacter in broiler flocks. In an experiment where some chickens were simultaneously inoculated with Salmonella enteritidis and Campylobacter, no interaction on susceptibility or caecal colonisation level was observed.

Passage of Salmonella through the crop and gizzard of broiler chickens fed with fermented liquid feed.

In vivo experiments were conducted in order to investigate the passage and bacterial reduction of Salmonella in the crop and gizzard of chickens when fed two different feeds. The chickens were fed dry conventional feed and fermented liquid feed. The fermented feed contains a relatively high concentration of lactic and acetic acid and lactobacilli. One and three week old broiler chickens were necropsied at short intervals after inoculation with Salmonella Enteritidis. Counts of Salmonella from the crop, gizzard, duodenum, caecum and colon/rectum were obtained. This revealed a sharper decrease of Salmonella in the anterior parts of the gastro-intestinal tract in chickens fed with fermented feed than in chickens fed dry feed. It is therefore concluded that fermented feed improves the barrier formed by the crop and gizzard. The reduction of Salmonella is fully realised in the crop and gizzard. The lower intestinal compartment did not show a substantial effect on the reduction of Salmonella. The performed in vivo method appeared to be an appropriate way to study intervention strategies that aim to control Salmonella by improving the barrier function of the upper gastro-intestinal tract.