Development and validation of a multiplex fluorescent microsphere immunoassay assay for detection of porcine cytokines.

Cytokines are cell signalling proteins that mediate a number of different physiological responses. The accurate measurement of cytokine profiles is important for a variety of diagnostic and prognostic scenarios in relation to animal health and welfare. Simultaneous quantification of cytokine profiles in a single sample is now possible using fluorescent microsphere immunoassays (FMIA). We describe the development and validation of a novel multiplex assay using the Bio-Plex® 200 system to quantify cytokines in five different porcine tissues (brain, placenta, synovial tissue and fluid, plasma). The cytokine profiles are both tissue, and research hypothesis, -dependent but include Interleukin-1beta (IL-1ß), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10) and Tumor necrosis factor (TNF-α). This methods paper is reported in two parts: the development of a FMIA for porcine tissues and validation of pre-treatment for optimal cytokine recovery in porcine brain, placenta, synovial tissue and plasma. Validation steps are critical in ensuring an assay is suitable for novel sample types. This technique advances traditional ELISAs by: •FMIA provides insight into the profiles of multiple porcine cytokines in certain situations (e.g. disease, parturition).•Use of the Bio-Plex® 200 system to investigate novel sample types, including brain, placenta and synovial tissue.•Multiplexing utilises a fraction of the sample volume compared with multiple ELISAs.

Transferability of antimicrobial resistance from multidrug-resistant Escherichia coli isolated from cattle in the USA to E. coli and Salmonella Newport recipients.

OBJECTIVES: This study aimed to evaluate conjugative transfer of cephalosporin resistance among 100 strains of multidrug-resistant Escherichia coli (MDRE) to Salmonella enterica serotype Newport and E. coli DH5α recipients. METHODS: Phenotypic and genotypic profiles were determined for MDRE as well as for Salmonella Newport (trSN) and E. coli DH5α (trDH) transconjugants. RESULTS: Of 95 MDRE donor isolates, 26 (27%) and 27 (28%) transferred resistance to trSN and trDH recipients, respectively. A total of 27 MDRE (27%) were confirmed as extended-spectrum ß-lactamase (ESBL)-producers based on the double-disk synergy assay and whole-genome sequencing (WGS). WGS was performed on 25 of the ESBL-producing isolates, showing that 2 isolates carried blaCTX-M-6, 22 possessed blaCTX-M-32 and 1 was negative for blaCTX-M genes. Fourteen of the ESBLs sequenced were qnrB19. Differential transfer of IncA/C and IncN from MDRE32 was observed between trSN32 and trDH32. IncN-positive trDH32 displayed an ESBL phenotype, whereas IncA/C-positive trSN32 displayed an AmpC phenotype. The rate of ESBL transfer to trSN and trDH recipients was 11% and 96%, respectively. CONCLUSIONS: Twenty-seven MDRE were phenotypically identified as ESBL-producers. WGS of 25 MDRE revealed that 2 and 22 isolates carried blaCTX-M-6 and blaCTX-M-32, respectively. One multidrug-resistant isolate exhibited conversion from an AmpC phenotype to an ESBL phenotype with the transfer of only the IncN plasmid. The rate of resistance transfer to Salmonella or E. coli recipients was nearly identical. However, the ESBL phenotype was transferred with significantly greater prevalence to E. coli compared with Salmonella Newport (96% and 11%, respectively).

Why are most EU pigs tail docked? Economic and ethical analysis of four pig housing and management scenarios in the light of EU legislation and animal welfare outcomes.

To limit tail biting incidence, most pig producers in Europe tail dock their piglets. This is despite EU Council Directive 2008/120/EC banning routine tail docking and allowing it only as a last resort. The paper aims to understand what it takes to fulfil the intentions of the Directive by examining economic results of four management and housing scenarios, and by discussing their consequences for animal welfare in the light of legal and ethical considerations. The four scenarios compared are: 'Standard Docked', a conventional housing scenario with tail docking meeting the recommendations for Danish production (0.7 m2/pig); 'Standard Undocked', which is the same as 'Standard Docked' but with no tail docking, 'Efficient Undocked' and 'Enhanced Undocked', which have increased solid floor area (0.9 and 1.0 m2/pig, respectively) provision of loose manipulable materials (100 and 200 g/straw per pig per day) and no tail docking. A decision tree model based on data from Danish and Finnish pig production suggests that Standard Docked provides the highest economic gross margin with the least tail biting. Given our assumptions, Enhanced Undocked is the least economic, although Efficient Undocked is better economically and both result in a lower incidence of tail biting than Standard Undocked but higher than Standard Docked. For a pig, being bitten is worse for welfare (repeated pain, risk of infections) than being docked, but to compare welfare consequences at a farm level means considering the number of affected pigs. Because of the high levels of biting in Standard Undocked, it has on average inferior welfare to Standard Docked, whereas the comparison of Standard Docked and Enhanced (or Efficient) Undocked is more difficult. In Enhanced (or Efficient) Undocked, more pigs than in Standard Docked suffer from being tail bitten, whereas all the pigs avoid the acute pain of docking endured by the pigs in Standard Docked. We illustrate and discuss this ethical balance using numbers derived from the above-mentioned data. We discuss our results in the light of the EU Directive and its adoption and enforcement by Member States. Widespread use of tail docking seems to be accepted, mainly because the alternative steps that producers are required to take before resorting to it are not specified in detail. By tail docking, producers are acting in their own best interests. We suggest that for the practice of tail docking to be terminated in a way that benefits animal welfare, changes in the way pigs are housed and managed may first be required.

Attitudes of farmers and veterinarians towards pain and the use of pain relief in pigs.

A survey of UK-based pig farmers and veterinarians was conducted, in order to investigate attitudes to pain and the use of pain relief in pigs. Survey respondents were asked to indicate which anti-inflammatory drugs they used or prescribed for pigs, how often these were administered, and the level of pain they associated with particular conditions. The survey found that veterinarians used a range of anti-inflammatory products to treat pigs with lameness. While both farmers and veterinarians gave similar pain scores overall, farmers rated gastrointestinal disease as more painful and conversely veterinarians scored lameness higher. Female and younger respondents gave higher pain scores than males and older respondents. Overall, farmers and veterinarians had a positive attitude towards pain relief in pigs with the majority agreeing that animals recovered more promptly when pain relief was administered. Most farmers agreed that the recognition and management of pain is an important part of pig husbandry, and many expressed an interest in finding out more about identifying pain in this species as well as the treatment options available. The study highlighted potential barriers to the increased application of pain relief in pigs in that almost one-third of veterinarians and two-thirds of farmers did not agree that they discussed pain management with each other, while other respondents indicated that they found it difficult to recognise pain in pigs, and did not know how to treat it appropriately.

Behavioral and physiological responses of primiparous sows to mixing with older, unfamiliar sows.

Pigs are mixed into new social groups at various stages of production, and this study investigated the response of gilts to mixing with older unfamiliar sows. Four groups of 6 gilts and 1 group of 5 gilts were housed in stable groups until the most recently bred female reached d 39 of gestation. Then, stable groups were split such that 15 gilts remained in their home pens (CON) and 14 gilts were mixed (MIX) with 3 multiparous sows for 1 wk (M1), returned to the home pen for 1 wk, and then mixed with 3 different sows for 1 wk (M2). Continuous behavioral observations of gilt location in the pen, posture, aggressive, and other social interactions were made from the start of M1 and M2 for 5 h. Five-minute scans of location, posture, and behavior as well as salivary cortisol measurements were taken before, during, and after each mix week, whereas skin lesions were counted before and after each mix. During the mixed period, MIX gilts spent more (P ≤ 0.009) time in the individual feeding stalls, ventral lying, sitting, involved in aggression, and idle and less (P < 0.001) time in the straw-bedded area, lateral lying, and performing exploratory behavior (chewing, nosing, and rooting) than CON gilts. Additionally, MIX gilts had greater (P < 0.001) salivary cortisol concentrations and accumulated more (P < 0.001) skin lesions during mixing sessions compared to CON gilts. Mixed gilts in M2 spent more (P ≤ 0.013) time in the feeding stalls and ventral lying and less (P ≤ 0.029) time standing and in the dunging passageway compared to M1. Compared to M1, gilts had lower (P < 0.001) salivary cortisol concentrations and fewer (P ≤ 0.027) lesions in front and middle regions of the body during M2. During M2, salivary cortisol concentration was positively correlated with time spent in the dunging passageway (Spearman's rank correlation [rs] = 0.650, P = 0.022) and negatively correlated with time spent in the feeding stalls (rs = -0.762, P = 0.004). Results indicated that gilts adapted their behavior during M2 by using the feeding stalls more, and they became less active, especially when interacting with the sows. Despite this adaptation in M2, lesions and salivary cortisol in M IX gilts were much greater than in CON gilts, highlighting the severity of mixing gilts with multiparous sows.

Development and validation of on-farm behavioural scoring systems to assess birth assistance and lamb vigour.

Lamb mortality remains a significant welfare and economic issue for sheep production. Lamb survival is to a degree dependent upon an easy delivery and the expression of appropriate behaviours from both mother and offspring, such as rapid standing, udder seeking and sucking by the lamb. Genetic solutions have the potential to improve birth assistance and lamb behaviour but large amounts of data are needed. Therefore, to achieve this objective, simple, proxy methods (scoring systems) were developed to quantify the level of birth difficulties and lamb vigour on farm. In the first study, detailed historical behavioural data from 1156 lambs (Scottish Blackface and Suffolk (S)) were analysed to develop criteria for 3 scores: birth assistance, lamb vigour and sucking assistance. The birth assistance score was developed by analysing the relationships between birth presentation and intervention levels, and intervention level and labour length. Lambs with abnormal birth positions required more assistance than normally presented lambs and lambs with long labours required more and greater assistance than those with short labours. Lamb vigour score was developed by analysing the latencies for the lamb to first perform specific behaviours; more vigorous lambs reach landmark behaviours faster than low vigour lambs. The sucking assistance score was developed from the relationship between the latency to suck successfully and assistance level, where lambs that were slow to suck required more assistance than lambs that were quick to suck. In the second study, the behaviour scoring systems (5-point categorical scales) were validated using a commercial flock of 80 twin-bearing crossbred ewes mated with either Texel (T) or S sires by simultaneously recording scores and the latency to perform specific landmark behaviours (i.e. to stand, seek the udder and suck). The vigour scores (recorded at 5 min of age) were compared with the latency from birth to standing and showed that lambs with lower (better) vigour scores were faster to stand after birth than those with higher scores. The sucking assistance scores were compared with the latency from birth to sucking, and showed that lambs with lower sucking assistance scores are quicker to suck than those with high scores. These results showed that the scoring systems could provide a practical and reliable assessment of birth assistance and lamb behaviour on farm and were sufficiently sensitive to discriminate vigour levels between lambs sired by either S or T rams.

Pigs' aggressive temperament affects pre-slaughter mixing aggression, stress and meat quality.

Pre-slaughter stress has a negative impact on animal welfare and on meat quality. Aggressive behaviour when pigs are mixed together for transportation to, or on arrival at, the abattoir is an important factor in pre-slaughter stress. Aggressiveness of pigs varies between individuals in the population, and this study investigated its effects on stress and meat quality at slaughter. We mixed pigs at a young age to identify individuals of high (H) or low (L) aggressive temperament using the previously validated approach of lesion scoring. To contrast extremes of social stress single-sex groups of eight pigs were mixed according to their aggressiveness in HH, HL or LL combinations or left unmixed (U) prior to transport and slaughter (n = 271). Each treatment was replicated in at least two groups in each of four slaughter batches. Mixing per se had little effect, but mixed groups composed of aggressive pigs (HH) had more carcass skin lesions and higher levels of plasma cortisol at slaughter and had loin muscle samples with higher pH at 24 h, and lower redness (a*) and yellowness (b*) compared to the other treatments. Females had higher levels of plasma cortisol at slaughter, a more rapid decline in pH post-slaughter and greater lean content of meat. Lactate and creatine kinase (CK) levels and meat pH were affected by the interaction of sex and treatment. Genetic factors, dam and sire line composition, and halothane locus (ryanodine receptor 1, RYR1) genotype, also affected a number of production and meat quality parameters as expected. Additionally, 'commercially normal' levels of social stress were studied in four further slaughter batches with no manipulation of group composition (n = 313). In these pigs, the proportion of unfamiliar pigs and group size of lairage groups explained limited variation in lesion scores at slaughter, but earlier aggressiveness did not. High numbers of skin lesions on the carcass were associated with high levels of cortisol and lactate and low glucose at slaughter, but not with meat quality measures. When stress and meat quality measures were compared for all pigs, high lactate was associated with low early pH and high drip loss, while high cortisol and CK were associated with high pH at 24 h and changes in meat colour. In conclusion, mixing pigs of above average aggressiveness resulted in greater aggression and stress, and changes in meat quality parameters, consistent with the effects of pre-slaughter stress on muscle chemistry.

Genetic validation of postmixing skin injuries in pigs as an indicator of aggressiveness and the relationship with injuries under more stable social conditions.

The objective of the study was to estimate genetic correlations between skin lesions and aggressive behavior postmixing and under more stable social conditions as a potential means of selecting against pig aggressiveness. Postmixing aggression in commercial pig production is common, compromises welfare and profitability, and cannot be significantly reduced by low-cost changes to the environment. A genetic component to individual aggressiveness has been described in pigs and other species. Selective breeding against aggressiveness ought to be possible if an easily measured indicator trait can be shown to be genetically associated with aggressive behavior. Aggressive behavior was recorded continuously for 24 h after mixing, and a count of skin lesions (lesion count, LC) was recorded at 24 h and 3 wk postmixing on 1,663 pigs. Two behavioral traits were found to have a moderate to high heritability similar to that of growth traits; duration of involvement in reciprocal fighting (0.43 +/- 0.04) and delivery of nonreciprocal aggression (NRA; 0.31 +/- 0.04), whereas receipt of NRA had a lower heritability (0.08 +/- 0.03). Genetic correlations (r(g)) suggested that lesions to the anterior region of the body 24 h after mixing were associated with reciprocal fighting (r(g) = 0.67 +/- 0.04), receipt of NRA (r(g) = 0.70 +/- 0.11), and to a lesser extent, delivery of NRA (r(g) = 0.31 +/- 0.06). Lesions to the center and rear were primarily genetically associated with receipt of NRA (r(g) = 0.80 +/- 0.05, 0.79 +/- 0.05). Genetic correlations indicated that pigs that engaged in reciprocal fighting delivered NRA to other animals (r(g) = 0.84 +/- 0.04) but were less likely to receive NRA themselves (r(g) = -0.41 +/- 0.14). A genetic merit index using lesions to the anterior region as one trait and those to the center or rear or both as a second trait should allow selection against animals involved in reciprocal fighting and the delivery of NRA. Positive correlations between LC 24 h and 3 wk after mixing were found, especially for lesions to the center and rear of the body, indicating that postmixing lesions are predictive of those received under more stable group conditions. As well as reducing immediate aggression at mixing, selection on postmixing LC is expected to have a long-term impact on injuries from aggression, even after dominance relationships are established.

Genetics of animal temperament: aggressive behaviour at mixing is genetically associated with the response to handling in pigs.

Aggression when pigs are mixed into new social groups has negative impacts on welfare and production. Aggressive behaviour is moderately heritable and could be reduced by genetic selection. The possible wider impacts of selection for reduced aggressiveness on handling traits and activity in the home pen were investigated using 1663 male and female pedigree pigs (898 purebred Yorkshire and 765 Yorkshire × Landrace). Aggressive behaviour was observed over 24 h after pigs were mixed at 10 weeks of age into groups balanced for unfamiliarity and weight. Aggression was highly heritable (duration of involvement in reciprocal fighting h2 = 0.47 ± 0.03, and duration of delivering one-sided aggression h2 = 0.34 ± 0.03). Three weeks after mixing, home pen inactivity (indicated by the frequency of lying) was observed over 24 h. Inactivity was weakly heritable (h2 = 0.05 ± 0.01) but showed no significant genetic association with aggression. Pigs' behaviour during handling by humans was assessed on entry to, whilst inside and on exit from a weigh crate at both mixing and end of test at 22 weeks. Pigs were generally easy to handle, moving easily into and out of the crate. Scores indicating 'very difficult to move' were rare. Handling scores at weighing were weakly heritable (h2 = 0.03 to 0.17), and moderately correlated across the two weighings (rg = 0.28 to 0.76). Aggressive behaviour at mixing was genetically associated with handling at the end of test weighing: pigs that fought and delivered one-sided aggression had handling scores indicating more active behaviour at weighing (e.g. moving quickly into the crate v. fighting rg = 0.41 ± 0.05 and v. bullying rg = 0.60 ± 0.04). Also, there was a genetic association between receiving one-side aggression at mixing and producing high-pitched vocalisations in the weigh crate (rg = 0.78 ± 0.08). Correlated behavioural responses occurring across different challenging situations (e.g. social mixing and human handling) have been described by the concept of animal temperament (also known as coping styles, personality or behavioural syndromes), but this has rarely been demonstrated at the genetic level in farm animals. These findings may have practical implications for the development of breeding programmes aimed at altering animal temperament. Breeding to reduce aggression could result in some reduction in activity at weighing. This would have consequences for animal production, because pigs which are inactive at weighing take longer to move into and out of the weigh crate, and perhaps also for animal welfare.

The effect of late pregnancy supplementation of ewes with vitamin E on lamb vigour.

The experiment measured lamb responses to supplementation of the pregnant ewe diet with vitamin E above requirement. Crossbred ewes were mated with either Suffolk or Texel rams. Twin-bearing ewes were randomly allocated (approximately 21 months of age at allocation) to one of four treatment groups (20 ewes per group, 10 mated with Suffolk and 10 with Texel rams). Treatments imposed were 50, 100, 150 or 250 IU supplementary vitamin E per ewe per day to give a four treatment by two sire-type factorial experimental design. Ewes were fed concentrates to meet energy requirements for stage of pregnancy and hay ad libitum. Diets were introduced approximately 6 weeks before lambing. Blood samples were obtained prior to introduction of diets, 17 days after introduction of diets and within 24 h of lambing from a subset of eight ewes per treatment (32 total). Colostrum samples were obtained from 10 ewes per treatment, 12 h after birth of the first lamb. All births were observed and a lamb vigour score was assigned to each lamb 5 min after birth. At 1 and 12 h after birth, rectal temperature, and at 12 h after birth, sex, crown-rump length and BW of each lamb were recorded. Mean ewe plasma α-tocopherol concentration prior to introduction of the diets was 1.5 µg/ml (s.e.m. 0.09) and did not differ between groups. There were positive linear (P < 0.001) effects of dietary vitamin E on plasma (17 days after introduction of diets) and colostrum (12 h after birth) α-tocopherol concentrations. Lamb vigour scores were superior (P < 0.001) for lambs sired by Texel rather than Suffolk rams but there were no differences as a result of vitamin E supplementation. Lamb mortality was low and unrelated to either sire or supplementary vitamin E. Lamb birth and weaning weights were also unaffected by vitamin E supplementation. Supplementing the ewe with vitamin E therefore had no effect on any lamb measurements.