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.

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.

Impact of test sensitivity and specificity on pig producer incentives to control Mycobacterium avium infections in finishing pigs.

In this paper we analyze the impact of the sensitivity and specificity of a Mycobacterium avium (Ma) test on pig producer incentives to control Ma in finishing pigs. A possible Ma control system which includes a serodiagnostic test and a penalty on finishing pigs in herds detected with Ma infection was modelled. Using a dynamic optimization model and a grid search of deliveries of herds from pig producers to slaughterhouse, optimal control measures for pig producers and optimal penalty values for deliveries with increased Ma risk were identified for different sensitivity and specificity values. Results showed that higher sensitivity and lower specificity induced use of more intense control measures and resulted in higher pig producer costs and lower Ma seroprevalence. The minimal penalty value needed to comply with a threshold for Ma seroprevalence in finishing pigs at slaughter was lower at higher sensitivity and lower specificity. With imperfect specificity a larger sample size decreased pig producer incentives to control Ma seroprevalence, because the higher number of false positives resulted in an increased probability of rejecting a batch of finishing pigs irrespective of whether the pig producer applied control measures. We conclude that test sensitivity and specificity must be considered in incentive system design to induce pig producers to control Ma in finishing pigs with minimum negative effects.

Usefulness of food chain information provided by Dutch finishing pig producers to control antibiotic residues in pork.

The EU prescribes that food business operators must use food chain information to assist in food safety control. This study analyses usefulness of food chain information about antibiotic usage covering the 60-day period prior to delivery of pigs to slaughter in the control of antibiotic residues in pork. A dataset with 479 test results for antibiotic residues in tissue samples of finishing pigs delivered to a Dutch slaughter company was linked to information provided by pig producers about antibiotic usage in these finishing pigs. Results show that twice as many producers reported using antibiotics in the group of 82 producers with antibiotic residues (11.0%) compared to the group without antibiotic residues (5.5%) (p=0.0686). For 89% of consignments with a finishing pig with antibiotic residues, the producer reported 'did not use antibiotics'. Food chain information about antibiotic usage provided by Dutch pig producers was no guarantee for absence of antibiotic residues in delivered finishing pigs.

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.

Serodiagnosis of Mycobacterium avium infections in pigs.

The aim of this study is the development and evaluation of a serodiagnostic assay for Mycobacterium avium (MA). After screening MA lipid fractions in an ELISA format, a polar lipid fraction was selected as antigen because of its superior recognition by serum antibodies in experimentally infected pigs. The resulting MA-ELISA was evaluated as an alternative for detection of MA infection by traditional pathological examination of pig lymph nodes for granulomatous lesions by meat inspectors. By comparing with bacteriological examination, the MA-ELISA showed significantly better sensitivity (69%) as compared to pathological examination (31%) in experimentally infected pigs. The MA-ELISA also appeared significantly more specific in a set of serum samples from MA negative pigs: only 1 out of these 153 serum samples reacted positive, whereas 99 (65%) of these had displayed false positive results by detection of lymph nodes lesions that appeared not to be associated with MA (Komijn et al., 2007). The MA-ELISA was subsequently evaluated using serum samples from two farms with pigs known to be infected with MA. Bacteriological examination of the sub-maxillary and mesenteric lymph nodes showed that 56% (103/184) and 35% (41/117) of the pigs, respectively were positive for MA in these farms. In the first farm, 16% (29/184) of the pigs tested positive in MA-ELISA and 31% (57/184) by pathological examination. On the contrary, in the second farm, more pigs tested positive 17% (15/117) in MA-ELISA with 8% (9/117) positivity by pathological examination. Taking the results on both farms together, the sensitivity of the MA-ELISA was 14% and the specificity 83%, whereas the sensitivity of the pathological examination was 31% and the specificity 86%. For practical reasons use of a serological test as the MA-ELISA may be preferred over pathological or bacteriological examinations. Our studies in experimentally infected and negative "field" sera indicate that the MA-ELISA is significantly more specific and more sensitive than detection by classical pathological examination. However, the studies in two MA infected farms show a variable picture with pathological examination overall performing better. Study in a wider range of "positive" farms will be needed to provide a more comprehensive view of the quality of both tests for detection of MA in infected farms. At the same time further optimization of MA-ELISA with use of lipid antigens from a broader range of serotypes may improve its performance in the face of infections with different MA serotypes.

Granulomatous lesions in lymph nodes of slaughter pigs bacteriologically negative for Mycobacterium avium subsp. avium and positive for Rhodococcus equi.

The prevalence of granulomatous lesions in lymph nodes of pigs was studied. From January till August 2004 in two slaughterhouses in The Netherlands 2,116,536 pigs were examined for the presence of granulomatous lesions in the sub-maxillary lymph nodes. In 15,900 (0.75%) of these pigs, lesions could be detected. Nine farms with the highest incidence of lesions were selected for a more detailed pathological and bacteriological examination. On these farms, the prevalence of lesions in sub-maxillary lymph nodes ranged from 2.3 to 5.7% with a mean of 3.0%. From 1276 pigs that were sampled, 98 (7.7%) displayed granulomatous lesions in the sub-maxillary lymph nodes and one (0.1%) pig showed lesions in its mesenteric lymph node. Mycobacterium avium subsp. avium (MAA) could not be isolated from the lymph nodes of the 99 pigs with lesions and from a selection of lymph nodes (n=61) of pigs without lesions. Rhodococcus equi was isolated from 44 out of 98 (44.9%) of the sub-maxillary lymph nodes with granulomatous lesions and from two mesenteric lymph nodes without lesions. A comparison of former studies and the current results indicate that the prevalence of MAA infections in slaughter pigs has strongly decreased over the last decade, whereas R. equi is highly prevalent. The high incidence of granulomatous lesions associated with the bacteriological presence of R. equi could be considered as a serious cause of misdiagnosis of MAA infections in cases where meat inspection is carried out by inspection for granulomatous changes of lymph nodes only.

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.

Effect of fermented feed on shedding of Enterobacteriaceae by fattening pigs.

Epidemiological studies showed that the use of fermented feed could significantly reduce Salmonella prevalence in pigs compared to the use of normal feed. Experimental challenge experiments with Salmonella have however never been conducted to reveal the efficacy of fermented feed in reducing Salmonella shedding and/or reducing the number of Salmonella-positive pigs. A longitudinal study was conducted to measure the effect of fermented feed, in particular of its components lactic acid and Lactobacillus plantarum, on gastrointestinal bacterial ecology (Salmonella, Enterobacteriaceae, lactobacilli, volatile fatty acids (VFAs), pH). Seeder pigs were used as a mode for Salmonella transmission within a pig herd. Bacteriological measurements were performed in faeces of the pigs. The results showed that fermented feed affected/reduced the Enterobacteriaceae population in faeces of the pigs. No differences were found in the number of positive pigs infected or in the number of shedding with Salmonella serovar Typhimurium fed fermented feed and between the normal feed group. S. serovar Goldcoast could not establish an infection in the seeder pigs in the fermented feed group as well as in the normal feed group. The pH of the faeces in the fermented feed groups was significantly higher than the pH of the faeces of the normal feed groups. The role of the undissociated form of the faecal VFAs on the significantly lower Enterobacteriaceae number in faeces of the pigs of the fermented feed groups could not be demonstrated because of the significant higher pH in the faeces of the pigs fed fermented feed.