Longitudinal study of ESBL Escherichia coli carriage on an organic broiler farm.

Objectives: To determine the molecular characteristics of ESBL-producing Escherichia coli (ESBL-E) collected during a longitudinal study on an organic broiler farm in order to investigate clonal expansion and horizontal gene transfer. Methods: Isolates were obtained from a longitudinal study performed previously on an organic broiler fattening farm. Samples from individually followed-up broilers, the broiler house, the transport van and persons that took the samples, taken at several timepoints (days 1, 3, 4, 7, 10, 42 and 70) within a production round and during the consecutive one (days 1, 2, 3 and 70), had been investigated for the occurrence of ESBL-E. In the current study, ESBL genes and MLST STs of these ESBL-E were determined. Plasmids were characterized and subtyped. Results: On arrival in round_1, ESBL-E of ST88 predominated, while on days 3, 4, 7 and 10 ST10 was most often found and at slaughter age ST155 and ST1551 prevailed. A shift in STs was also observed in round_2. None of the 35 individually selected broilers followed up in round_1 was positive for the same ESBL-E ST at all sampling times. All isolates carried CTX-M-1 group genes, confirmed as blaCTX-M-1 in 158 isolates. Further analysis of 36 isolates of different STs showed blaCTX-M-1 on IncI1/ST3 plasmids. Conclusions: The rapid dissemination of ESBL-E on this broiler farm was not due to the spread of one specific E. coli clone, but most likely the result of horizontal transfer of an IncI1/ST3 plasmid carrying blaCTX-M-1 resulting in a shift in the predominant ESBL-E population in broilers.

Role of the Environment in the Transmission of Antimicrobial Resistance to Humans: A Review.

To establish a possible role for the natural environment in the transmission of clinically relevant AMR bacteria to humans, a literature review was conducted to systematically collect and categorize evidence for human exposure to extended-spectrum ß-lactamase-producing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus spp. in the environment. In total, 239 datasets adhered to inclusion criteria. AMR bacteria were detected at exposure-relevant sites (35/38), including recreational areas, drinking water, ambient air, and shellfish, and in fresh produce (8/16). More datasets were available for environmental compartments (139/157), including wildlife, water, soil, and air/dust. Quantitative data from exposure-relevant sites (6/35) and environmental compartments (11/139) were scarce. AMR bacteria were detected in the contamination sources (66/66) wastewater and manure, and molecular data supporting their transmission from wastewater to the environment (1/66) were found. The abundance of AMR bacteria at exposure-relevant sites suggests risk for human exposure. Of publications pertaining to both environmental and human isolates, however, only one compared isolates from samples that had a clear spatial and temporal relationship, and no direct evidence was found for transmission to humans through the environment. To what extent the environment, compared to the clinical and veterinary domains, contributes to human exposure needs to be quantified. AMR bacteria in the environment, including sites relevant for human exposure, originate from contamination sources. Intervention strategies targeted at these sources could therefore limit emission of AMR bacteria to the environment.

Survival of heterophyid metacercaria in common carp (Cyprinus carpio).

Heterophyidae are small intestinal trematodes that infect vertebrates worldwide. Common carp (Cyprinus carpio) is one of the most preferred freshwater fish species by consumers in Asia, the region where fish-borne trematodes like Heterophyidae are most prevalent. How long Heterophyidae survive in common carp is unknown. The objective of this study was to quantify survival of Heterophyidae in common carp after experimental exposure. Fish of 0.18 g were either used as controls or exposed to 250 heterophyid cercaria for 24 h. Control fish did not become infected. Percentage infection of exposed fish at 0-2 (n = 53), >2-10 (n = 15), >10-20 (n = 11), and >20-27 (n = 33) weeks post exposure was 98, 80, 100, and 100 % respectively. The number of metacercaria per fish did not significantly decrease (P = 0.19) during 27 weeks after exposure: exp [3.6200-0.0193 × weeks post exposure]. All developed metacercaria were identified as Haplorchis spp. It was concluded that Heterophyidae may persist in carp for a long time, implying that harvestable carp are a risk to human health.

Individual and combined effects of deoxynivalenol (DON) with other Fusarium mycotoxins on rainbow trout (Oncorhynchus mykiss) growth performance and health.

This study assessed whether the toxicological effects of deoxynivalenol (DON) produced by Fusarium graminearum in rainbow trout (Oncorhynchus mykiss) are altered by the co-exposure to a mixture of toxins produced by Fusarium verticillioides (FUmix). This FUmix contained fusaric acid and fumonisin B1, B2 and B3. Four diets were formulated according to a 2 × 2 factorial design: CON-CON; CON-FUmix; DON-CON; and DON-FUmix. Diets with and without DON contained on average 2700 and 0 µg/kg feed, respectively. The sum of the analysed FUmix toxins was 12,700 and 100 µg/kg feed in the diets with and without FUmix, respectively. The experiment consisted of a 6-week restrictive feeding period immediately followed by a 2-week ad libitum feeding period. Growth performance measurements were taken per feeding period. Histopathological measurements in the liver and gastrointestinal tract (pyloric caeca, midgut and hindgut) were assessed at the end of week 1 and week 6 of the restrictive feeding period and at week 8, the last day of the ad libitum feeding period. During both restrictive and ad libitum feeding, the effects of FUmix and DON on growth performance were additive (no interaction effect; p > 0.05). During the restrictive feeding period, exposure to DON (p ≤ 0.001) and FUmix (p ≤ 0.01) inhibited growth and increased feed conversion ratio (FCR). During this period, DON exposure decreased the protein (p ≤ 0.001) and energy retention (p ≤ 0.05) in the trout. During the ad libitum feeding period, FUmix affected HSI (p ≤ 0.01), while DON exposure reduced feed intake (p ≤ 0.001) and growth (p ≤ 0.001) and increased FCR (p ≤ 0.01). In general, for both liver and intestinal tissue measurements, no interaction effects between DON and FUmix were observed. In the liver, histopathological analysis revealed mild alterations, increased necrosis score by DON (p ≤ 0.01), increased glycogen vacuolization by FUmix (p ≤ 0.05) and decreased percentage of pleomorphic nuclei by FUmix (p ≤ 0.01). DON had a minor impact on the intestinal histological measurements. Over time, some of the liver (glycogen vacuolization score, pleomorphic nuclei; p ≤ 0.01) and intestinal measurements (mucosal fold and enterocyte width; p ≤ 0.01) were aggravated in fish fed the FUmix contaminated diets, with the most severe alterations being noted at week 8. Overall, the co-exposure to FUmix and DON gave rise to additive effects but showed no synergistic or antagonistic effects for the combination of DON with other Fusarium mycotoxins.

Longitudinal study on transmission of MRSA CC398 within pig herds.

UNLABELLED: ABSBACKGROUND: Since the detection of MRSA CC398 in pigs in 2004, it has emerged in livestock worldwide. MRSA CC398 has been found in people in contact with livestock and thus has become a public health issue. Data from a large-scale longitudinal study in two Danish and four Dutch pig herds were used to quantify MRSA CC398 transmission rates within pig herds and to identify factors affecting transmission between pigs. RESULTS: Sows and their offspring were sampled at varying intervals during a production cycle. Overall MRSA prevalence of sows increased from 33% before farrowing to 77% before weaning. Overall MRSA prevalence of piglets was>60% during the entire study period. The recurrent finding of MRSA in the majority of individuals indicates true colonization or might be the result of contamination. Transmission rates were estimated using a Susceptible-Infectious-Susceptible (SIS-)model, which resulted in values of the reproduction ratio (R0) varying from 0.24 to 8.08. Transmission rates were higher in pigs treated with tetracyclins and ß-lactams compared to untreated pigs implying a selective advantage of MRSA CC398 when these antimicrobials are used. Furthermore, transmission rates were higher in pre-weaning pigs compared to post-weaning pigs which might be explained by an age-related susceptibility or the presence of the sow as a primary source of MRSA CC398. Finally, transmission rates increased with the relative increase of the infection pressure within the pen compared to the total infection pressure, implying that within-pen transmission is a more important route compared to between-pen transmission and transmission through environmental exposure. CONCLUSION: Our results indicate that MRSA CC398 is able to spread and persist in pig herds, resulting in an endemic situation. Transmission rates are affected by the use of selective antimicrobials and by the age of pigs.

Higher attack rate of fish-borne trematodes (Heterophyidae) in common carp fingerlings (Cyprinus carpio) at lower fish weight.

Fish-borne zoonotic trematodes (FZTs) can cause pathology in humans. Fish weight was reported as important risk factor for transmission from snail to fish. However, in fingerlings, the relation between fish weight and infection is unknown. Aim was quantifying the effect of fish weight on infection probability, attack rate, and metacercariae burden of FZTs in common carps (Cyprinus carpio) between 1 and 20 g. Fish were either used as controls (n = 66) or exposed to 250 parapleurolophocercous cercariae (n = 254). Fish weight was analysed as continuous explanatory variable or classified in four categories with average weights of 0.7 g (n = 116), 4.0 g (n = 58), 8.2 g (n = 57) and 14.2 g (n = 23). The inverse relation between percentage of fish with metacercariae and fish weight is reflected in lower percentages of infected fish at higher weights [%infected = 100/(1 + e([-2.02+0.15 fish weight (g)])); p < 0.01], i.e. 89 %, 85 %, 63 % and 61 %, respectively, in the four groups. Control fish did not get infected. Attack rates were 0.0087, 0.0073, 0.0040 and 0.0033 fish infected per cercariae, respectively; the first two attack rates being significantly higher than the latter two. Mean number of metacercariae per weight group was 5, 5, 2 and 1, respectively, (p < 0.05), with an inverse relation using weight as continuous explanatory variable [p < 0.01; number metacercariae = e(1.76-0.13fish weight(g))]. Concluding, an inverse relation exists between fish weight and probability of infection, attack rate and parasite burden in common carp fingerlings. Reducing transmission to fingerlings might be an effective intervention method to improve food safety, reduce the absolute amount of FZTs in the environment and eventually reduce incidence in humans.

Time- and Dose-Dependent Effects of Dietary Deoxynivalenol (DON) in Rainbow Trout (Oncorhynchus mykiss) at Organism and Tissue Level.

This study with juvenile rainbow trout evaluated the effects of dietary exposure to deoxynivalenol (DON) at industrially relevant doses (up to 1.6 mg/kg) on growth performance, the liver, and the gastrointestinal tract. Fifteen groups of 30 fish each were given one of five dietary treatments in triplicate: (1) control diet (CON; DON < 100 µg/kg feed), (2) naturally DON-contaminated diet (ND1) with a DON content of 700 µg/kg in the feed, (3) ND2 with a DON content of 1200 µg/kg feed, (4) a pure DON-contaminated diet (PD1) with 800 µg/kg of DON in the feed, and (5) PD2 with DON at a concentration of 1600 µg/kg in the feed. The feeding trial lasted eight weeks: six weeks of restrictive feeding followed by two weeks of ad libitum feeding. Exposure to DON during restrictive feeding for six weeks did not affect the growth performance of trout but did lead to a reduction in retained protein in fish fed with higher doses of DON in the ND2 and PD2 groups. During the two following weeks of ad libitum feeding, feed intake was similar among all groups, but body weight gain was lower in the ND2 and PD2 groups and feed efficiency was higher in PD2 (week 8). Histopathological assessment revealed liver damage, including altered nuclear characteristics and haemorrhages, in groups fed higher doses of natural DON (ND2) after just one week of restrictive feeding. Liver damage (necrosis and haemorrhage presence in ND2) was alleviated over time (week 6) but was again aggravated after ad libitum exposure (week 8). In contrast, gastrointestinal tract damage was generally mild with only a few histopathological alterations, and the absence of an inflammatory cytokine response was demonstrated by PCR at week 8. In conclusion, ad libitum dietary exposure of rainbow trout to either natural or pure DON resulted in reduced growth (dose-dependent), while restrictive exposure revealed time-dependent effects of natural DON in terms of liver damage.

Seroprevalence and risk factors of Q fever in goats on commercial dairy goat farms in the Netherlands, 2009-2010.

BACKGROUND: The aim of this study was to estimate the seroprevalence of Coxiella burnetii in dairy goat farms in the Netherlands and to identify risk factors for farm and goat seropositivity before mandatory vaccination started. We approached 334 eligible farms with more than 100 goats for serum sampling and a farm questionnaire. Per farm, median 21 goats were sampled. A farm was considered positive when at least one goat tested ELISA positive. RESULTS: In total, 2,828 goat serum samples from 123 farms were available. Farm prevalence was 43.1% (95%CI: 34.3%-51.8%). Overall goat seroprevalence was 21.4% (95%CI: 19.9%-22.9%) and among the 53 positive farms 46.6% (95%CI: 43.8%-49.3%). Multivariable logistic regression analysis included 96 farms and showed that farm location within 8 kilometres proximity from a bulk milk PCR positive farm, location in a municipality with high cattle density (≥ 100 cattle per square kilometre), controlling nuisance animals through covering airspaces, presence of cats or dogs in the goat stable, straw imported from abroad or unknown origin and a herd size above 800 goats were independent risk factors associated with Q fever on farm level. At animal level almost identical risk factors were found, with use of windbreak curtain and artificial insemination as additional risk factors. CONCLUSION: In 2009-2010, the seroprevalence in dairy goats in the Netherlands increased on animal and farm level compared to a previous study in 2008. Risk factors suggest spread from relatively closely located bulk milk-infected small ruminant farms, next to introduction and spread from companion animals, imported straw and use of artificial insemination. In-depth studies investigating the role of artificial insemination and bedding material are needed, while simultaneously general biosecurity measures should be updated, such as avoiding companion animals and vermin entering the stables, next to advice on farm stable constructions on how to prevent introduction and minimize airborne transmission from affected dairy goat farms to prevent further spread to the near environment.

The Occurrence of Mycotoxins in Raw Materials and Fish Feeds in Europe and the Potential Effects of Deoxynivalenol (DON) on the Health and Growth of Farmed Fish Species-A Review.

The first part of this study evaluates the occurrence of mycotoxin patterns in feedstuffs and fish feeds. Results were extrapolated from a large data pool derived from wheat (n = 857), corn (n = 725), soybean meal (n = 139) and fish feed (n = 44) samples in European countries and based on sample analyses by liquid chromatography/tandem mass spectrometry (LC-MS/MS) in the period between 2012-2019. Deoxynivalenol (DON) was readily present in corn (in 47% of the samples) > wheat (41%) > soybean meal (11%), and in aquafeeds (48%). Co-occurrence of mycotoxins was frequently observed in feedstuffs and aquafeed samples. For example, in corn, multi-mycotoxin occurrence was investigated by Spearman's correlations and odd ratios, and both showed co-occurrence of DON with its acetylated forms (3-AcDON, 15-AcDON) as well as with zearalenone (ZEN). The second part of this study summarizes the existing knowledge on the effects of DON on farmed fish species and evaluates the risk of DON exposure in fish, based on data from in vivo studies. A meta-analytical approach aimed to estimate to which extent DON affects feed intake and growth performance in fish. Corn was identified as the ingredient with the highest risk of contamination with DON and its acetylated forms, which often cannot be detected by commonly used rapid detection methods in feed mills. Periodical state-of-the-art mycotoxin analyses are essential to detect the full spectrum of mycotoxins in fish feeds aimed to prevent detrimental effects on farmed fish and subsequent economic losses for fish farmers. Because levels below the stated regulatory limits can reduce feed intake and growth performance, our results show that the risk of DON contamination is underestimated in the aquaculture industry.

Transgenerational effects of innate immune activation in broiler breeders on growth performance and immune responsiveness.

The impact of transgenerational effects on growth performance and immunity has not yet been studied extensively within the poultry husbandry sector. An important factor is the impact of the hens on the physical well-being and fitness to the environment of the offspring. This study is the first to investigate the effect of stimulating the maternal innate immune system with lipopolysaccharides (LPS) or ß-glucan on growth performance and immune responses in the next generation. Transgenerational effects and consequences of these maternal treatments were further examined using a necrotic enteritis (NE) challenge model in the offspring. We show that offspring of LPS-treated broiler breeders have a higher feed efficiency from 14 to 21 days of age, that is, the period just after the NE challenge. Moreover, more broiler chickens with intestinal lesions after the NE challenge were found in the offspring of the LPS-treated broiler breeders. Both the LPS and ß-glucan maternal treatments resulted in transgenerational effects on blood-derived monocytes by showing a tendency of decreased IL1ß mRNA levels after ex vivo LPS stimulation. These data are a first indication that broiler breeder hens can affect immune responsiveness and feeding efficiency of their offspring in a transgenerational manner.

Transmission dynamics of extended-spectrum ß-lactamase and AmpC ß-lactamase-producing Escherichia coli in a broiler flock without antibiotic use.

Extended-spectrum and AmpC ß-lactamase-producing Escherichia coli (ESBL/AmpC-EC) are found throughout the broiler production pyramid. Transmission of resistance between broilers and humans could occur at any point, representing a potential public health issue. Insight in farm transmission dynamics could provide a basis for control, leading to fewer contaminated broilers. The aim was quantifying transmission rates and routes of ESBL/AmpC-EC, and specific phylogenetic groups, in an organic broiler flock without antibiotic use. In each of two consecutive production rounds, 80 randomly chosen broilers were followed individually. Cloacal swabs from these, 20 other randomly chosen broilers, and 11 environmental samples were taken at several moments from arrival till slaughter. ESBL/AmpC-EC were isolated by selective pre-enrichment, and ESBL/AmpC-genes and E. coli phylogenetic groups were determined. Transmission parameters (ß) were estimated using a Generalised Linear Model with a susceptible-infectious-susceptible model. Effect of direct broiler contact as compared to contact through the environment and previous carriage c.q. infectious status and their interaction were included as explanatory variables. Multiplying ß by the length of the infectious period gives the reproduction ratio (R). On day 1, prevalence was 28.8% (95%CI 19.2-40.0%) and 0.0% (95%CI 0.0-4.5%) among individually followed broilers, in round 1 and 2 respectively. In round 2, the environment was positive before arrival of day-old chicks. After 3 days, almost 100% of broilers and environmental samples were positive in both rounds. Most samples were positive for CTX-M-1 group genes, and A1 and B1 were predominant phylogenetic groups. From day 3 there was a shift towards more phylogenetic groups. R was 1.70 (95%CI 0.55-5.25) for total ESBL/AmpC-EC. Risk for broilers to become infectious was lower if previously infectious (ßpreviously infectious=0.02 vs. ßnot previously infectious=3.41; P<0.0001). For phylogenetic groups separately, R was 0.88 (95%CI 0.38-2.07), 0.51 (95%CI 0.27-0.98), 0.99 (95%CI 0.65-1.51) for A1, B1 and rest (i.e. A0, B2, D1, D2) groups, respectively. The interaction effect for A1 and B1 was reflected in the fact that when broilers were previous infectious, the environment was relatively more important for transmission of the A1 group, while this was direct contact between broilers for the B1 group. Positive day-old chicks and the environment both play a role in introduction and transmission of ESBL/AmpC-EC in flocks. These results suggest that, even without selective pressure from antibiotics, total ESBL/AmpC-EC persistence, and resulting endemic situation, seem to be caused by shifts in carriage of different phylogenetic groups. It implies that contaminated broilers enter the slaughterhouse.

Methicillin-resistant Staphylococcus aureus and extended-spectrum and AmpC ß-lactamase-producing Escherichia coli in broilers and in people living and/or working on organic broiler farms.

The aim of this study was to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum and AmpC ß-lactamase (ESBL/AmpC)-producing Escherichia coli among broilers, and humans living and/or working on organic broiler farms; further characterise isolates; and compare these results with those from conventional farms. In the Netherlands, only 9 certified organic broiler farms were present. On 8 of these farms, 60 throat swabs and 20 cloacal swabs were taken per farm for MRSA and ESBL/AmpC-E. coli detection, respectively, at an average age of both 34 (T1) and 68 (T2) days. Faecal swabs and questionnaires were returned by 27 out of 36 humans. For selected ESBL/AmpC-producing E. coli isolates, phylogenetic groups, ß-lactamase genes, plasmid families, and sequence types were determined. MRSA was not detected in broiler and human samples. ESBL/AmpC-producing E. coli were isolated from broilers on 7/8 farms at T1 and on all farms at T2. Furthermore, 3 farmers at T1, and 2 farmers and 1 family member at T2 were positive. Genes found in broilers and humans were almost exclusively blaCTX-M-1 and blaCMY-2. Given the high overall human ESBL/AmpC-prevalence (18.5%), which is similar to conventional farms, contact with live broilers is assumed a risk factor for carriage. Farm and sample-level prevalence at T1 are consistent with those from conventional farms. At T2, just before slaughter, sample-level prevalence of ESBL/AmpC-E. coli appears to have decreased (94.3% vs. 80%), which could have important consequences for contamination of retail meat.

Distribution of trematodes in snails in ponds at integrated small-scale aquaculture farms.

In integrated small-scale aquaculture farming, animal and human excreta maybe used as fish feed and pond fertilizer, thereby enhancing transmission of fish-borne zoonotic trematodes (FZTs) from final hosts, like humans, pigs and chickens, to snails. Areas within a pond could vary in trematode egg-load due to the immediate bordering land, and this might provide implications for control of these trematodes or sampling in field studies measuring FZT prevalence in snails. We therefore estimated the effect of bordering land use on prevalence and FZT burden in snails in different areas within small-scale aquaculture ponds. Nine sampling areas within a pond were assigned in six ponds. For each sampling area, about 120 Melanoides tuberculata snails were collected. Based on land use bordering a sampling area, these were categorized in 5 risk-categories: low-risk (road, rice planted in pond, agriculture, or middle of pond), human access point to pond, livestock sty (pigs or poultry), both human access point and livestock sty, and water connection to canal. In total, 5392 snails were collected. Percentages of snails with parapleurolophocercous cercariae varied between 6% in areas categorized as low-risk and areas with livestock sty only to 15% in areas with both human access point and livestock sty; only this 15% was significantly different from the prevalence in the low-risk category. Percentages of snails with xiphidio cercariae did not differ between risk-categories and varied between 5% and 10%. Mean snail size was 15.2mm, and was significantly associated with both the probability of infection as well as parasite burden. Very small differences in parasite burden were found at different land use areas; the maximum difference was about 11 cercariae. This study demonstrated only small differences between areas surrounding a pond on risk of snails to be infected with fish-borne trematodes within different pond areas. In field studies on FZTs in M. tuberculata snails in ponds, sampling from ponds can therefore be done without considering areas within ponds.

Quantification of transmission of livestock-associated methicillin resistant Staphylococcus aureus in pigs.

Antimicrobial resistance in pigs becomes a public health issue when resistant organisms transfer from pigs to humans. Pigs are a large reservoir for livestock-associated (LA-)MRSA and people in contact with pigs are at risk for infection with LA-MRSA. Transmission and persistence of LA-MRSA within a pig population contributes to the maintenance of this zoonotic reservoir. Current knowledge on colonization and transmission of LA-MRSA in pigs is limited and mainly based on observational field surveys. Two experiments were performed to colonize pigs and quantify transmission of LA-MRSA between pigs. In the first experiment, colonization of six-week old piglets failed after intranasal inoculation, confirming the complexity of MRSA-colonization. In the second experiment, naive pigs got colonized after exposure to orally inoculated pigs. Subsequently, these contact-infected pigs transmitted MRSA to a new group of naive pigs. The reproduction ratio, R(0), was estimated with a SIS-model to quantify transmission between the first and second contact pigs as this resembles more the natural transmission. Two scenarios were evaluated, with different assumptions regarding infection status of individual pigs. R(0) varied between 3.7 and 4.3 and was significantly above 1, indicating a high probability of persistence of LA-MRSA, even without antimicrobial use.

Transmission of methicillin resistant Staphylococcus aureus among pigs during transportation from farm to abattoir.

The prevalence of methicillin resistant Staphylococcus aureus (MRSA) in pigs at abattoirs is higher than in pigs sampled on farms. This study investigated whether MRSA negative pigs can become MRSA positive during transportation from the farm to the abattoir after exposure to other pigs and environmental sources of MRSA. Nasal swabs were collected from four batches of pigs during loading at the farm, on arrival at the abattoir and after stunning. Environmental wipes were taken from lorries after transporting pigs and from lairages after holding pigs. All pigs (n=117) tested MRSA negative before transportation. On arrival at the abattoir, 12/117 (10.3%) pigs in two batches tested MRSA positive. In lorries that tested positive after transportation, the prevalence of MRSA positive pigs was 21.1%, whereas no MRSA was detected in pigs that had been transported in lorries that tested negative after transportation. At stunning, all batches and 70/117 (59.8%) pigs tested MRSA positive. Pigs can become MRSA positive in the short period of time during transportation from the farm to stunning at the abattoir.