Residues of veterinary antibiotics in manures from pig and chicken farms in a context of antimicrobial use reduction by implementation of health and welfare plans.

The use of antibiotics in food-producing animals can induce the presence of residual substances in manure, which are then released into the environment and may contribute to soil and groundwater contamination. During the on-farm implementation of strategies to improve animal health and welfare in chicken and pig farms, the consequences of antibiotic use were evaluated in terms of the occurrence and levels of antibiotic residues in manure. A set of 35 broiler farms from Cyprus, Greece, the Netherlands and 40 pig farms from France and Italy provided a total of 350 manure samples. The primary objective was to develop a specific LC/MS/MS method capable of quantifying antibiotic residues in both types of manure. The method was able to detect fifteen antibiotics belonging to nine classes, with validated limits of quantification of 10-20 µg/kg, and accuracies ranging from 81% to 138%. With the exception of amoxicillin, which was never detected in any manure, all antibiotics used were detected in manure from treated animals with typical concentrations ranging from 10 to 99198 µg/kg for both chickens and pigs. The occurrence of residual antibiotics was higher in chicken than in pig manure, especially for fluoroquinolones and doxycycline which were detected in 89% and 100% of the chicken manure, respectively, and in 28% of the pig manure. The impact of the health plans on the antibiotic load manure was assessed by measuring for each farm the ratio of the sum of all antibiotic concentrations measured after and before the implementation of the plan. The results showed that, in addition to the frequency of treatments, the class of antibiotic used is an important factor to consider as it strongly influences the stability/instability of the compounds, i.e. their ability to persist in the manure of food-producing animals.

Analysis of Antimicrobials in Muscle and Drinking Water in Terms of Reducing the Need of Antimicrobial Use by Increasing the Health and Welfare of Pig and Broiler.

Antimicrobial residues may pose harmful effects on the health of consumers. At the same time, an adequate quality of drinking water for animals is one of the important element to ensure animal welfare and food without antibacterials. The presented study is aimed at estimating the residue levels of antibacterial compounds, such as penicillins, cephalosporin, macrolides, tetracyclines, quinolones, sulphonamides, aminoglycosides, diaminopirymidines, pleuromutilines and lincosamides in meat and on-farm drinking water samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS), as a part of a surveillance system on pig and broiler farms within the project Healthy Livestock. A total of 870 samples of muscle from pig and broiler, as well as 229 water samples were analysed for antibiotic residues. Samples were collected from farms in EU countries in two steps, before and after implementation of a tailor-made health plan. In muscle samples, the detected concentrations of doxycycline in the post-intervention step (15.9-70.8 µg/kg) were lower than concentrations in the pre-intervention step (20.6-100 µg/kg). In water samples, doxycycline in an average concentration of 119 µg/L in the pre- and 23.1 µg/L in the post-intervention step, as well as enrofloxacin at concentrations of 170 µg/L in the pre- and 1.72 µg/L in the post-intervention step were quantified. Amoxicillin was only present before intervention. The obtained results confirm the effectiveness of the intervention actions. The concentrations of antibiotics in muscles and water were lower after implementation of a health plan on the farms.

Development and implementation of a risk assessment tool for broiler farm biosecurity and a health intervention plan in the Netherlands, Greece, and Cyprus.

Preventing pathogens from entering and spreading on farms is the first step in reducing health problems. For this study a BiosEcurity Assessment Tool was developed to identify strengths and weaknesses in biosecurity on broiler farms, which was used as a starting point to formulate tailor-made health plans to improve broiler health and reduce antimicrobial use. Farms were divided into 3 separate areas according to associated biosecurity risk; high disease risk external areas (red zone), medium risk service areas (orange zone), and the clean and highly secure access-restricted green zone. In the Netherlands, Cyprus, and Greece, 13, 15, and 7 broiler houses were monitored for 4 production cycles (2 preintervention and 2 postintervention cycles). At the start of the study the BiosEcurity Assessment Tool assessment was performed and a health plan was made in consultation with the veterinarian. After the second cycle a start was made with the implementation of the health plan. Overall, the biosecurity level in the green and orange zones were significantly higher in the Netherlands compared to Greece and Cyprus, but there was no difference for the red zone or the transition zones between the countries. The interventions in the health plans were mostly directed towards those measures that could be implemented in the short term and with low costs in the green zone. In Cyprus a decrease in antimicrobial use was found postintervention. This was not the case in Greece and the Netherlands. In Cyprus and Greece footpad lesion improved after interventions were implemented, although this may have been an effect of season. In Dutch farms no improvement was detected, but both antimicrobial use and footpad lesions were lower at the start of the study compared to Cypriot and Greek farms. In conclusion, the BEAT shows to be a promising tool to assess biosecurity risks on broiler farms. The biosecurity assessment in combination with the farm specific health plans could contribute to antimicrobial reduction on broiler farms.

Longitudinal study on the prevalence of extended spectrum cephalosporins-resistant Escherichia coli colonization in Dutch veal farms.

A longitudinal study was performed to investigate the prevalence of Extended-Spectrum Cephalosporin-Resistant (ESC-R) Escherichia coli colonization in Dutch veal farms. Rectal swabs from 683 calves born in 13 Dutch dairy farms were collected one day prior to transportation to the veal farm at 14 or 28 days of age, and at 5 different time points 8 Dutch veal farms. In addition, characteristics of the calf, cows, and farm management were collected. Rectal swabs were selectively cultured for ESC-R E. coli. In total, 1202 ESC-R E. coli isolates were recovered. Overall, the prevalence of ESC-R E. coli increased from 24.4 % at one day prior to transportation to 57.3 % in week two after arrival of calves at the veal farm. No associations were found between the presence of ESC-R E. coli at the dairy or veal farm and age of transportation, sex and breed. The presence of ESC-R E. coli in week 6, 10, and 18 at the veal farm was positively associated with the presence of ESC-R E. coli in week 10, 18, and 24, respectively (p < 0.05). Individual antibiotic treatments applied before week 2 and 6 upon arrival to the veal farms tended to increase the ESC-R E. coli colonization frequency. Our results indicate that ESC-R E. coli colonization frequency substantially increases after arrival of calves on the veal farm. In addition to individual antibiotic treatments, it is considered likely that frequently applied batch antibiotic treatments are also implicated in the ESC-R E. coli colonization frequency.

Effects of pre-transport diet, transport duration and transport condition on immune cell subsets, haptoglobin, cortisol and bilirubin in young veal calves.

The aim of this study was to investigate effects of pre-transport diets, transport durations and transport conditions on immune cell subsets, haptoglobin, cortisol and bilirubin of young calves upon arrival at the veal farm. An experiment was conducted with a 2 × 2 × 2 factorial arrangement with 3 factors: 1) provision of rearing milk or electrolytes at the collection center (CC); 2) transport duration (6 or 18 hours) and 3) transport condition (open truck or conditioned truck). Holstein-Friesian and cross-bred calves were used (N = 368; 18 ± 4 days; 45.3 ± 3.3 kg). Blood samples were collected from calves (N = 128) at the collection center, immediately post-transport (T0) and 4, 24, 48 hours, week 1, 3 and 5 post-transport. Blood was analyzed for cortisol, bilirubin, haptoglobin, IgG and IgM. Moreover, cell counts of neutrophils, lymphocytes, monocytes, basophils and eosinophils were measured in blood samples taken at the collection center and T0. In these same blood samples, different lymphocyte populations were characterized by flow cytometry, including CD14+ cells, NK cells, δγ+ T cells, CD8+ cells, CD4+ cells and CD21+ cells. Calves transported in the conditioned truck had higher amounts of white blood cell count (WBC) (Δ = 1.39 × 109/l; P = 0.01), monocytes (Δ = 0.21 × 109/l; P = 0.04), neutrophils (Δ = 0.93 × 109/l; P = 0.003), than calves transported in the open truck regardless, of pre-transport diet or transport duration. The study showed that transport condition and duration influenced parts of the innate immune system of young veal calves. Cortisol, bilirubin and WBC seemed to be connected by similar underlying mechanisms in relation to transport conditions. However, it is unclear which specific pathways in the immune system of young calves are affected by different transport conditions (e.g. temperature, humidity, draught).

Transport of Young Veal Calves: Effects of Pre-transport Diet, Transport Duration and Type of Vehicle on Health, Behavior, Use of Medicines, and Slaughter Characteristics.

The aim of this study was to investigate effects of different early life transport-related factors on health, behavior, use of medicines and slaughter characteristics of veal calves. An experiment was conducted with a 2 × 2 × 2 factorial arrangement with 3 factors: (1) provision of rearing milk or electrolytes before transport, (2) transport duration (6 or 18 h), and (3) type of vehicle (open truck or conditioned truck). The study included male Holstein-Friesian and cross-bred calves (N = 368; 18 ± 4 days; 45.3 ± 3.3 kg). Data on health status of calves were collected at the collection center and at the veal farm until week 27 post-transport. Behavior of calves was recorded during transport and at the veal farm until week 13 post-transport. Use of herd and individual medical treatments was recorded at the veal farm. The prevalence of loose or liquid manure at the veal farm from day 1 until week 3 post-transport was lower in electrolyte-fed calves transported in the conditioned truck compared to electrolytes-fed calves transported in the open truck or milk-fed calves transported in both the conditioned and open truck (Δ = 11% on average; P = 0.02). In comparison with the open truck, calves transported in the conditioned truck had lower prevalence of navel inflammation in the first 3 weeks post-transport (Δ = 3 %; P = 0.05). More milk-fed calves received individual antibiotic treatments compared to electrolyte-fed calves at the veal farm (P = 0.05). In conclusion, the transport-related factors examined in the present study affected health and behavior of calves in the short-term, but there was no evidence for long-term effects. It remains unknown why no long-term effects were found in this study. Perhaps this absence of transport-related effects was due to multiple use of medical treatments in the first weeks at the veal farm. Alternatively, it might be that the collective effects of the transition from the dairy farm to the veal farm, and of the husbandry conditions during the subsequent rearing period, on the adaptive capacity of calves were so large that effects of individual transport-related factors were overruled.

Response to LPS in female offspring from sows treated with cortisol during pregnancy.

Prenatal stress has been shown to program responsiveness of the hypothalamus-pituitary-adrenal axis (HPA-axis) and behavior in offspring. In pig husbandry, sows are exposed to stressful conditions during gestation. Previously, cortisol treatment of pregnant sows has been shown to alter stress responsiveness and immunological parameters in their piglets. In the present study, we explored whether cortisol treatment of pregnant sows affects the offspring's response to an inflammatory stimulus. Sows were treated orally with cortisol either during the first, second, or third period of gestation, or received a placebo during this period. At 8 weeks of age, female offspring were injected intravenously with lipopolysaccharide (LPS). Offspring of sows that received cortisol during the first and third period of gestation showed a higher fever response to LPS. Cortisol treatment of sows during gestation did not affect offspring's response to LPS with regard to their cortisol response. LPS-induced sickness behavior, which was measured as the latency time in a human approach test, appeared to recover more quickly in offspring from sows that received cortisol during the second period of gestation. These results suggest that prenatal cortisol exposure programs responsiveness to inflammatory stimuli in female piglets.

Cortisol administration to pregnant sows affects novelty-induced locomotion, aggressive behaviour, and blunts gender differences in their offspring.

Several behavioural effects of prenatal stress are reported in literature, and these seem to depend, among other factors, on the gender studied and the period of gestation in which prenatal stress is applied. In the present study, oral administration of hydrocortisone-acetate (HCA) to 41 pregnant sows was used as a model for prenatal stress, since corticosteroids are considered a key mediator in the effects of prenatal stress. HCA was orally administered to pregnant sows during three periods of gestation: 21-50 (period 1, P1, n = 10), 51-80 (period 2, P2, n = 10) and 81-110 (period 3, P3, n = 10) days after insemination (term 115 days). Control sows (n = 11) received vehicle from 21 to 110 days after insemination. Between days 9 and 48 after birth, treatment effects on male and female piglet behaviour were determined in the home pen and in four different behavioural tests. During the backtest, no gender differences were observed in vocalisations in HCA-piglets, while control males vocalised more than control females. In the home pen at 14 days of age, HCA-piglets spent less time in social interactions than control piglets. During the novel environment test, P1- and P3-piglets walked more than control piglets, but this was not observed during the novel object test, four days later. At weaning, P2- and P3-piglets performed less individual play. Prior to mixing with an unfamiliar piglet (male piglets only), HCA-piglets had lower salivary cortisol concentrations than control piglets, but no difference was observed after mixing. P1-, P2-, and P3- piglets had fewer non-aggressive encounters, and P2-piglets continued fighting longer than control piglets. The present study demonstrates that elevated maternal cortisol concentrations during gestation affect piglet behaviour, and effects do differ between male and female piglets. In addition, effects depend on the period of cortisol administration.

Lower birth weight and attenuated adrenocortical response to ACTH in offspring from sows that orally received cortisol during gestation.

Prenatal stress is known to affect several offspring characteristics, but its effects depend among other factors on the period of gestation in which it is applied. In the present study, oral administration of hydrocortisone-acetate (HCA) was used to elevate cortisol concentrations in pregnant sows to levels also observed after psychological stress. HCA was administered during three different periods of gestation (115 days in pigs): period 1: 21-50 (P1, n = 10), period 2: 51-80 (P2, n = 10) and period 3: 81-110 (P3, n = 10) days after insemination. Control sows (n = 11) received vehicle from 21-110 days after insemination. When P1-, P2- and P3-sows did not receive HCA, they also received vehicle. During gestation, weekly saliva samples were taken from the sows to determine salivary cortisol concentrations. Treatment effects on sow, litter and piglet characteristics were determined. In addition, two female piglets per litter were subjected to an ACTH-challenge test at 6 weeks of age to determine the adrenocortical response to ACTH. Pigs were slaughtered at 6 months of age and slaughter weight, back fat thickness and percentage of lean meat were analysed. During the period of treatment with HCA, salivary cortisol concentrations were increased in P1-, P2- and P3-sows compared to control sows (P < 0.01). The total number of piglets born per litter did not differ among treatment groups (P > 0.30), but pooled HCA-litters had a higher percentage of live born piglets (P < 0.05) and fewer mummies than control litters (P < 0.05). Gestation length did not differ among treatment groups (P = 0.21), but did affect treatment effects on birth weight. Overall, HCA-piglets weighed less at birth, and remained lighter until weaning (P < 0.05). The salivary cortisol concentrations after i.m. injection of ACTH (2.5 IU/kg) were lower in P1- and P3-piglets compared to control piglets. At slaughter, HCA-treatment indirectly decreased lean meat percentage and increased back fat thickness. In conclusion, elevated peripheral cortisol concentrations in pregnant sows affect both litter characteristics and piglet physiology, the latter depending on the period of gestation during which concentrations were elevated. Underlying mechanisms require further investigation.

Evaluation of oral administration of cortisol as a model for prenatal stress in pregnant sows.

OBJECTIVE: To design a treatment that increases plasma corticosteroid concentrations to mimic prenatal stress in pregnant sows. ANIMALS: 24 pregnant sows. PROCEDURE: Sows were assigned to 1 of 4 treatment groups; treatment consisted of twice-daily oral administration of a placebo or 20, 60, or 180 mg of hydrocortisone acetate (HCA)/sow from 7 to 11 weeks of gestation. Blood and saliva samples for determination of cortisol concentrations were obtained hourly on treatment days 3 and 25 and twice weekly for the remainder of the treatment period. The WBC, neutrophil, and lymphocyte counts and concentrations of interleukin (IL)-2 and IL-4 were determined on 4 days during treatment. Litter characteristics were recorded. RESULTS: Plasma and salivary cortisol concentrations were significantly increased in sows that received 60 or 180 mg of HCA (0.30 to 0.37 mg/kg and 0.95 to 1.15 mg/kg, respectively), compared with control sows. Except for the second day of treatment, the number of WBCs and the IL-2:IL-4 ratio did not differ among treatment groups. The neutrophil-to-lymphocyte ratio was significantly higher in sows that received 180 mg of HCA. Gestation duration was significantly shorter in sows that received 180 mg of HCA. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of 60 mg of HCA is suitable to increase plasma and salivary cortisol concentrations in pregnant sows in a controlled manner to concentrations comparable to concentrations detected after psychologic stressors. This model seems to be suitable to study the effects of increased maternal corticosteroid concentrations on young pig behavior, physiologic variables, and development.