Kristiane Barington

Forensic cases of bruises in pigs

Bruises in pigs inflicted by blunt trauma are a significant animal welfare problem, and affected skin and underlying muscle are regularly submitted for forensic investigation. Central to the evaluation is an assessment of the age of the bruises. This paper presents cases of bruises in pigs sent for forensic investigation that were collected retrospectively. Data comprised photographs of the gross lesions, slides for histology, and written reports. The time from collecting the animals at the farms and delivery to the slaughterhouse was recorded together with the time of slaughter. Since 2005 there has been an increase in cases, with a peak in 2008 and 2009 of 40 cases for each year. At gross examination, the pattern of bruises often reflected the type of object which caused them. Histologically, haemorrhage and cellular infiltrations were frequently present. Currently, the age of bruises may be estimated to be more or less than four hours based on a porcine bruise model. In bruises more than four hours old, estimations of two-hour intervals are used based on studies of wound healing. The time from collecting the pigs at the farms until slaughter was between one and four hours in 44.1 per cent of cases, during which time the pigs had been handled by several people. In addition, in 22.0 per cent of cases of bruising an inflammatory response was absent, making it impossible to estimate the age of the bruise.

The apparent prevalence of skin lesions suspected to be human-inflicted in Danish finishing pigs at slaughter

Skin lesions on pigs inflicted by humans compromise animal welfare and are the subject of increased public and political attention in Denmark. Systematic surveillance of such skin lesions was enforced in April 2010 at all Danish pig abattoirs, through the recording of meat inspection Code 904 for the presence of skin lesions suspected to be human inflicted. The objectives of the present study were to (a) estimate the apparent prevalence of Code 904s at the pig and herd owner level; (b) characterise the distribution of deliveries with pigs demonstrating a Code 904; (c) characterise the distribution of herd owners with repeated Code 904 recordings; and (d) determine the developments in Code 904 prevalence over time in Danish finishing pigs in the period from May 1, 2010 to September 30, 2013. Data from the 12 largest finishing pig abattoirs from Denmark were included and recordings were comprised from 65,504,021 pigs from 651,681 deliveries originating from 10,796 herd owners. Overall, 7200 (0.011%) of the pigs were recorded with a Code 904, and 21% of herd owners had a minimum of one Code 904 delivery with at least one pig with skin lesions inflicted by humans. On the pig-level, the apparent prevalence was 0.020% in 2010, which was reduced to 0.008% in 2013. In the first quarter of the study period, 17% of the herd owners had a Code 904 delivery, while 7% had one in the last quarter. Nine per cent of the herds had more than one Code 904 recording, with up to 16 Code 904 deliveries from one herd owner. Most deliveries included one single pig with a Code 904, but up to 102 Code 904 recordings were made in a single delivery. The apparent prevalence at the four smallest and four middle sized abattoirs decreased from the first to the second quarter, while the apparent prevalence decreased more substantially in the largest four abattoirs; with significant decreases from both the first to the second, and from the second to the third quarter. The study showed that recorded skin lesions suspected to be inflicted by humans are prevalent, but the apparent prevalence decreased from 2010 to 2012 and 2013. The development in Code 904 over time could be due to a real decrease or be due to other factors such as changes in the way the lesions were recorded, while both underestimation and overestimation appeared to be present.

Do umbilical outpouchings affect the behaviour or clinical condition of pigs during 6 h housing in a pre-transport pick-up facility?

This study focused on behavioural and clinical effects of umbilical outpouchings (UOs) in pigs. Matched pairs of pigs with UOs (diameter 12 cm; range 4-20; diagnosed p.m. as hernia or non-hernia) and controls (N=28) were compared during a 6-h stay in a pick-up facility. Overall, skin lesion scores were increased after the 6-h stay. Behaviour of the UO-pigs differed from the controls (a shorter latency to lie down (P<0.05) and decreased aggression (P<0.05)). Pigs with umbilical hernia showed e.g. increased sitting (P<0.05) and decreased lying (P<0.05) compared to pigs with non-hernia UOs. No effects of the size of the OUs were found. These results are among the first to establish knowledge about UO-pigs and suggest that a stay in a pick-up facility can be challenging for pig welfare. The behavioural findings suggest that UO-pigs, and especially pigs with hernia, may be less fit for mixing and housing in barren environments.

Intrabronchial Microdialysis: Effects of Probe Localization on Tissue Trauma and Drug Penetration into the Pulmonary Epithelial Lining Fluid.

Recent intrabronchial microdialysis data indicate that the respiratory epithelium is highly permeable to drugs. Of concern is whether intrabronchial microdialysis disrupts the integrity of the respiratory epithelium and thereby alters drug penetration into the pulmonary epithelial lining fluid (PELF). The objective of this study was to investigate the effect of intrabronchial microdialysis on the integrity of the bronchial epithelium. Microdialysis sampling in PELF in proximal (n = 4) and distal bronchi (n = 4) was performed after intravenous inulin and florfenicol administration in anaesthetized pigs. Inulin was used as a marker molecule of permeability of the epithelium, and florfenicol was used as test drug. Bronchial tissue was examined by histopathology (distal and proximal bronchi) and gene expression analysis (RT‐qPCR, proximal bronchi) at the termination of the experiment (6.5 hr). The microdialysis probe caused overt tissue trauma in distal bronchi, whereas no histopathological lesions were observed in proximal bronchi. A moderate up‐regulation of the pro‐inflammatory cytokines IL1B, IL6 and acute‐phase reactant serum amyloid A was seen in proximal bronchi surrounding the microdialysis probes suggesting initiation of an inflammatory response. The observed up‐regulation is considered to have limited impact on drug penetration during short‐term studies. Inulin penetrated the respiratory epithelium in both proximal and distal bronchi without any correlation to histopathological lesions. Likewise, florfenicol penetration into PELF was unaffected by bronchial histopathology. However, this independency of pathology on drug penetration may not be valid for other antibiotics. We conclude that short‐term microdialysis drug quantification can be performed in proximal bronchi without disruption of tissue integrity.

The intensity of the inflammatory response in experimental porcine bruises depends on time, anatomical location and sampling site

The assessment of the age of bruises inflicted on livestock is an important component of veterinary forensic pathology investigations. However, the sampling site within a bruise, the anatomical location and the mass and speed of the object inflicting the blunt trauma might influence the intensity of the inflammatory reaction. In the present study, the variation of the inflammatory reaction within and along experimental porcine bruises was evaluated in order to determine the optimal sampling site. Moreover, we evaluated if a combination of histological characteristics and gene expression signatures was able to differentiate bruises according to anatomical location, age of bruises and the speed and mass of the object used to cause the impact. Twelve experimental slaughter pigs were anesthetized, and on each animal four blunt traumas were inflicted on the back using either a plastic tube or an iron bar, respectively. The pigs were euthanized at 2, 5 or 8 h after infliction. Following gross examination, skin and underlying muscle tissue were sampled from the center and both ends of bruises and evaluated histologically. Subcutaneous fat tissue from the center of the bruises was sampled for quantitative real-time polymerase chain reaction to evaluate mRNA expression of 13 selected genes. Uninjured tissue was sampled from the right thigh of all pigs and served as control tissue. The amount of tissue damage and the intensity of the inflammatory reaction in bruises depended on the sampling site within and along a bruise, the anatomical location and the age of the bruise. The optimal site for sampling, i.e. the most pronounced inflammatory reaction, was at the center of the bruises where the plastic tube or iron bar first struck the skin. Moreover, bruises inflicted in areas with a thin layer of subcutaneous fat tissue showed more damage and inflammation in the underlying muscle tissue compared to bruises inflicted in areas with a thicker layer of subcutaneous fat tissue. In addition, hemorrhage in the muscle tissue was more likely present when bruises were inflicted with an iron bar compared to a plastic tube. Combining histology and mRNA expression of the 13 genes showed that the age of bruises could be determined with a precision of ±2.04 h. Moreover, the age of bruises could be determined with a precision of ±1.84 h based solely on mRNA expression of a selection of four genes.

Grading of shoulder ulcerations in sows by biopsies

Shoulder ulcerations can be graded postmortem from 0 to 4 on a pathoanatomical scale. However, veterinarians and farmers express difficulties evaluating the grade of the lesions antemortem. Accurate grading is needed in order to comply with veterinary instruction in relation to the Danish legislation, stating that sows with shoulder ulcers grade 3 or 4 must be kept loose and have access to soft bedding. Thus, the aim of the present study was to evaluate if biopsies from the center of a shoulder ulcer can be used to point out animals for which an intervention must be initiated. Postmortem, a punch biopsy was sampled from the center of the ulceration or from the tissue overlaying the tuber spina scapula. Afterward, the shoulders were cross-sectioned and evaluated grossly and histologically (“gold standard”). In total, 121 shoulders were included in the study, and the diagnostic value of a punch biopsy in grading shoulder ulcerations was evaluated. The results showed a sensitivity of 0.78, a specificity of 0.98, a positive likelihood ratio of 38.36, and a negative likelihood ratio of 0.22. The agreement between the cross-section evaluation and the punch biopsy was found to be 0.90 by calculating the Cohen kappa value. In conclusion, a single punch biopsy from the center of an ulcer is useful for determining the grade of a shoulder ulcer and can be used to facilitate the identification of sows with ulcers requiring an intervention.

Forensic aspects of incised wounds and bruises in pigs established post-mortem

Recognizing post-mortem (PM) changes is of crucial importance in veterinary forensic pathology. In porcine wounds established PM contradicting observations regarding infiltration of leukocytes have been described. In the present study, skin, subcutis and muscle tissue sampled from experimental pigs with PM incised wounds (n=8), PM bruises (n=8) and no lesions, i.e. controls (n=4), were examined for signs of vitality over time. All tissue samples were subjected to gross and histopathological evaluation. Hemorrhages were present along the edges of PM incised wounds but deposits of fibrin were never observed. PM bruise led to hemorrhage in the subcutis visible on cross section of the skin in 3 out of 8 pigs. Histologically, hemorrhages in the subcutaneous tissue and disrupted muscle fibers were observed in PM bruises and could not be differentiated from similar lesions in ante-mortem (AM) bruises. Vital reactions, i.e. infiltrating leukocytes, hyper-leukocytosis and pavement of leukocytes, were absent in all incised wounds and bruises regardless of the time of sampling after traumatization. In conclusion, a vital reaction was not present in PM incised wounds, regardless of the time of sampling. Moreover, it was found that AM bruises free of leukocyte infiltration cannot be distinguished from PM bruises, an observation which is of crucial importance when timing bruises in forensic cases.

Fluorescence In Situ Hybridization for the Tissue Detection of Bacterial Pathogens Associated with Porcine Infections.

Fluorescence in situ hybridization (FISH) is an efficient technique for the identification of specific bacteria in tissue of both experimental and spontaneous infections. The method detects specific sequences of nucleic acids by hybridization of fluorescently labeled probes to complementary target sequences within intact cells. FISH allows direct histological localization of the bacteria in the tissue and thereby a correlation between the infection and the histopathological changes present. This chapter presents protocols for FISH identification of bacterial pathogens in fixed deparaffinized tissue samples mounted on glass slides. Two different methods are presented: one is illustrated with the use of peptide nucleic acid (PNA) that is carried out directly on glass slides (Method I), whereas the other is exemplified by using a DNA probe in a Shandon rack (Method II). In the two methods, both PNA and DNA probes can be used.

Forensic age determination of human inflicted porcine bruises inflicted within 10 h prior to slaughter by application of gene expression signatures

Prediction-models based on gene expression profiles from experimental bruises are capable of determining the age of bruises with a precision of ±2 h. However, these models have not yet been applied on tissue from pigs in forensic cases requested by the police. We applied two prediction-models, based on mRNA expression of 13 (prediction-model no. 1) and 4 genes (prediction-model no. 2) involved in inflammation, on forensic cases of porcine bruises in order to determine if gene expression profiles can be used for age determination in forensic cases. Subcutaneous fat tissue from bruises notified to the police was sampled: 1) within 6 h after slaughter (group no. I, n = 142), and 2) after freezing the skin for up to 1 year (group no. II, n = 40). qPCR of genes involved in inflammation was performed to predict the bruise age after partial least squares analysis. mRNA expression data were obtained for 52.8% and 7.5% bruises in group nos. I and II, respectively. Prediction-model no. 2, based on the mRNA expression of Selectin E, Selectin P, Interleukin 6 and Nuclear Factor Kappa Beta Subunit1, was most suitable for predicting the age of bruises within 8 h prior to slaughter. In conclusion, mRNA expression profiles can assist in estimating the age of bruises. However, when applying gene expression signatures in forensic cases the age estimate should be interpreted together with histological manifestations. Subcutaneous tissue must be stabilized hours after the bruises are detected in order to obtain mRNA of a sufficient quality.

Histological evaluation of experimental porcine bruises

Age estimation is a crucial part of the forensic investigation of bruises in livestock pigs [1], [2], [3]. Currently, age estimations are based on histological evaluation of the lesions in the skin and underlying muscle tissue [2]. However, the intensity of inflammation and tissue damage depends not only on the age of bruises but also on sampling site, anatomical location and the speed, mass and force used to inflict the lesions [1], [4], [5]. Twelve experimental slaughter pigs were anesthetized and on each animal, four blunt traumas were inflicted on the back (area of impact Nos. 1–4). The pigs were euthanized at 2, 5 or 8 h after infliction. Skin and underlying muscle tissue were sampled from the center (B) and both ends of bruises (A, C) and evaluated histologically. Descriptive statistics were performed on the data obtained and presented in figures and tables. Differences (odds ratios) between sampling sites (A, B and C), object used to inflict bruises (plastic tube or iron bar), anatomical location (area of impact Nos. 1–4) and bruise age (2, 5 and 8 h) were evaluated using the GENMOD procedure in SAS Enterprise Guide 7.1 and presented in tables. In addition, the agreements (estimated as Cohen׳s kappa) between two observers evaluating the histological parameters were calculated and presented. Data have been further analyzed and discussed in a recent paper [1]