Immunohistochemical study of CD31 and α-SMA expression for age estimation of porcine skin wounds.

Age estimation of wounds in veterinary forensic investigations is based on the presence and amount of granulation tissue. However, accurate age assessment is challenging and new time-dependent markers are warranted to support and improve the current procedure. The objective of this study was to evaluate the expression of CD31-positive blood vessels and α-smooth muscle action (α-SMA)-positive myofibroblasts in granulation tissue in order to evaluate their value as markers for porcine wound age estimation in a veterinary forensic context. Immunohistochemical expression of CD31 and α-SMA in 14 experimental porcine skin wounds of different ages (4, 6, 8, 10, 18, 27 and 35 days) and 11 forensic porcine wound specimens (of unknown age) were evaluated. CD31-positive blood vessels and α-SMA-positive myofibroblasts were present in the granulation tissue in the experimental wounds at all time points. A significant decrease in the mean blood vessel counts was found in wounds aged 18, 27 and 35 days compared with wounds aged 6 days (P < 0.001), when assessing both the superficial and deep part of the wound bed. α-SMA expression was lower at 27 and 35 days post wounding compared with 6-18 days post wounding. Combined assessment of three parameters (mean blood vessel counts in the superficial and deep wound beds and α-SMA expression) could approximately specify the age of the wounds as either 6-18 days or ≥27 days. In two of the forensic cases a combination of the three parameters yielded results that were similar to the experimental wounds, indicating a wound age of 6-18 days or ≥27 days, respectively. In the remaining forensic cases a combination of the three parameters did not show the same expression pattern as in the experimental wounds. The results indicate that in some forensic cases the application of CD31 and α-SMA markers appeared to support the current procedure for porcine wound age estimation, but this must be combined with pathological characteristics.

Gastro-intestinal lesions are not relatable to diarrhoea or specific pathogens in post-weaning diarrhoea (PWD) in pigs.

BACKGROUND: Post-weaning diarrhoea (PWD) is a multifactorial condition and the most well documented infectious cause is enterotoxigenic Escherichia coli. The objective of the study was to investigate possible associations between pathological manifestations and pathogens in pigs with and without PWD. The study was conducted as a case-control study and included a total of 173 pigs from 9 different commercial intensive indoor production herds in eastern Denmark. RESULTS: Based on clinical examination, a total of 89 piglets with PWD (cases) and 84 piglets without PWD (controls) were included. Most of the pigs (n = 105/173) presented gastric lesions, which were more frequently observed in the control group. The odds of gastric ulcers were lower among pigs with PWD compared to pigs without PWD with an odds ratio (OR) of 0.2 (0.0; 0.7). Abnormal content in the colon was associated with PWD, with an OR of 6.5 (3.2; 14.3). No apparent association was found between lesions and the various pathogens or a combination of these. The odds of neutrophilic granulocyte infiltration were lower in the jejunum among pigs with PWD (OR 0.3 [0.1; 0.6]) compared to pigs without PWD. The association between neutrophilic granulocyte infiltration in jejunum and PWD differed between the herds (P = 0.03). Furthermore, the associations between PWD and hyperleukocytosis (P = 0.04) or infiltration of eosinophilic granulocytes (P = 0.04) in ileum were also herd dependent. Histopathology revealed several lesions not relatable to PWD. CONCLUSION: The association between lesions and specific pathogens or PWD is more complex than anticipated.

An observational field study of porcine post-weaning diarrhea: clinical and microbiological findings, and fecal pH-measurements as a potential diagnostic tool.

BACKGROUND: Recently, in-feed medicinal zinc has been phased out in pig production in the European Union. This makes updated knowledge about porcine post-weaning diarrhea (PWD) crucial. The objectives of the present study were to investigate (i) the clinical presentation of PWD in pigs housed in Danish herds that did not use medicinal zinc, specifically the prevalence of diarrhea and whether PWD was associated to clinical signs of dehydration or altered body temperature; (ii) which microorganism are associated to PWD; and iii) whether measurements of the fecal pH have a potential to be used diagnostically to differentiate between infectious etiologies in cases of PWD. RESULTS: The prevalence of diarrhea varied considerably between the outbreaks in the nine studied herds (median = 0.58, range = 0.10; 0.94). In a cross-sectional design (n = 923), diarrhea was associated with reduced rectal temperature and alkaline feces. Diarrhea was also associated with observably reduced skin elasticity, possibly indicating dehydration. In both diarrheic case pigs (n = 87) and control pigs (n = 86), the presence of Brachyspira pilosicoli, Clostridium perfringens, Cryptosporidium spp., Cystoisopora suis, enterotoxigenic Escherichia coli, Lawsonia intracellularis, porcine circovirus types 2 and 3, rotavirus A, B, C, and H, Samonella enterica spp. enterica, and Trichuris suis was described. PWD was associated with high levels of enterotoxigenic E. coli shedding (odds ratio versus no E. coli detection = 4.79 [CI 1.14; 12.62]). Diarrhea was associated with high levels of rotavirus A shedding (odds ratio versus no/low rotavirus A = 3.80 [CI 1.33; 7.97]). The association between microbiological findings in diarrheic pigs and fecal pH was negligible. CONCLUSIONS: Enterotoxigenic E. coli was confirmed to be a cause of PWD; however, cases of PWD where enterotoxigenic E. coli was not detected in high levels occurred commonly, and this adds to the increasing evidence suggesting that PWD is not necessarily a result of enteric colibacillosis. Rotaviral enteritis might be a differential diagnosis of PWD. pH-measurements cannot be used to differentiate between differential diagnoses for PWD.

Lesions and pathogens found in pigs that died during the nursery period in five Danish farms.

BACKGROUND: Diagnosing and treatment of diseases in pigs are important to maintain animal welfare, food safety and productivity. At the same time antimicrobial resistance is increasing, and therefore, antibiotic treatment should be reserved for individuals with a bacterial infection. The aim of the study was to investigate gross and histological lesions and related pathogens in pigs that died during the nursery period in five Danish farms. In addition, high throughput, real-time qPCR monitoring of specific porcine pathogens in fecal sock and oral fluid samples were carried out to investigate the between-farm and between-batch variation in the occurrence of pathogens. RESULTS: Twenty-five batches of nursery pigs from five intensive, indoor herds were followed from weaning (approximately four weeks) to the end of nursery (seven to eight weeks post weaning). Gross and histological evaluation of 238 dead and 30 euthanized pigs showed the highest prevalence of lesions in the skin, respiratory system, gastrointestinal tract, and joints. Gross and histological diagnoses of lung and joint lesions agreed in 46.5% and 62.2% of selected pigs, respectively. Bacteriological detection of Escherichia coli, Streptococcus suis or Staphylococcus aureus infections in joints, lungs and livers was confirmed as genuine infection on immunohistochemical staining in 11 out of 70 tissue sections. The real-time qPCR analysis of pooled samples showed that most pathogens detected in feces and in oral fluid in general followed the same shedding patterns in consecutive batches within herds. CONCLUSIONS: Gross assessment should be supplemented with a histopathological assessment especially when diagnosing lesions in the lungs and joints. Moreover, microbiological detection of pathogens should optimally be followed up by in situ identification to confirm causality. Furthermore, routine necropsies can reveal gastric lesions that may warrant a change in management. Real-time qPCR testing of fecal sock samples and oral fluid samples may be used to monitor the infections in the individual herd and testing one batch seems to have a good predictive value for subsequent batches within a herd. Overall, optimal diagnostic protocols will provide a more substantiated prescription of antibiotics.

Forensic necropsies of cattle: a study of Danish cases from 2010 to 2021 and a guideline for forensic examination of cattle.

Forensic post-mortem examinations of animals are carried out on suspicion of violation of European and national legislation. In Denmark, and probably also in other countries with large-scale cattle production, cattle are regularly submitted for forensic assessment. Unfortunately, only few studies of forensic pathology in cattle are available. This paper presents a retrospective study of forensic case files on Danish cattle from January 2010 to December 2021. The case files were characterized with respect to types of lesion, age assessments of lesions and other parameters such as age and sex. A total of 118 forensic case files had been archived and related to 132 cattle (14 weeks-20 years of age; 68% female, 30% male and 2% unknown sex) with 228 lesions. Locomotor disorders constituted the majority of lesions. However, cachexia/emaciation, skin ulcerations and overgrowth of cornual horn were also frequent. Most lesions were chronic (91%) and age assessments for more than 2 weeks were stated for 79% of the lesions. This indicates that in Denmark at least, there is a need to consider how cattle with locomotor disorders are treated in a timely manner in order to avoid prolonged futile treatment and, thereby, suffering. Grossly visible reparative granulation tissue and new bone formation were present in lesions of 1-2 weeks and longer duration. However, all age assessments were stated in broad time intervals due to the lack of scientifically based forensic studies of age assessments of lesions in cattle. Therefore, to improve age assessments in forensic cattle cases, studies concerning the chronology of tissue reparation in cattle are warranted. We also present a guideline for the forensic examination of cattle.

Optimising the sampling procedure for forensic investigation of bruises on pigs.

BACKGROUND: Human-inflicted bruises on pigs are a violation of the law and affected tissue is regularly sent for forensic investigation. The authors aimed to evaluate the variation in inflammation within and between human-inflicted porcine bruises in order to determine the optimal sampling procedure. METHODS: Skin and muscle tissues from the centre and ends of 21 bruises were evaluated histologically. Moreover, RNA was extracted from the subcutaneous fat tissue. The bruises were detected during meat inspection at the slaughter line, and all carcases were kept at 5°C for 12-24 hours before sampling. RESULTS: The sampling site with the most infiltration of neutrophils and macrophages differed between bruises inflicted on the same pig and between bruises inflicted on more pigs within the same delivery. The extracted RNA had RIN (RNA integrity number) values from 3 to 6.5. CONCLUSIONS: Tissue samples should always be taken from both skin and underlying muscle tissue. Samples should be collected from several sites along each bruise, and all bruises should be sampled in order to include the site of maximum tissue damage and inflammation. Moreover, RNA of sufficient quality for quantitative PCR and subsequent age estimation cannot be obtained from carcases kept for 12-24 hours at 5°C.

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].

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.

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.

A porcine model for pathomorphological age assessment of surgically excised skin wounds.

A porcine model of second intention wound healing was established and gross and histological changes needed for accurate assessing the age of wounds were determined. Twenty-five pigs were anesthetized before incision of four wounds on each. The wounds were left to heal from 1 h to 35 days when the pigs were euthanized. In 14 pigs, biopsies were sampled from two wounds between days 2 and 18. By histological evaluation the following characteristics were found useful for determining the age of wounds: neutrophil:macrophage ratio, angiogenesis, hyperplasia of fibroblasts, presence of hemosiderophages and granulation tissue. The latter was present from day 4 (n = 8 wounds, 100%), but by gross evaluation it was not recognized until day 5 (n = 4 wounds, 100%). From day 4 to 10, the thickness of granulation tissue increased by 1.2 ± 2.4 mm/day. The thickness of collagen fibers within granulation tissue increased throughout the study period, and complete epithelization was reached from day 18.

Forensic aspects of gene expression signatures for age determination in bruises as evaluated in an experimental porcine model.

Determining the age of bruises and the force used to inflict the trauma is of crucial importance in both human and veterinary forensic pathology. In the present study, the expression of more than 50 different genes in subcutaneous fat and muscle tissue from experimental bruises in pigs was investigated. The aim was to evaluate if expression signatures of selected genes were capable of determining bruises according to age and the force of impact. Eighteen experimental pigs were anesthetized, and on each animal four blunt traumas were inflicted on the back with a low, moderate or high force. The pigs were euthanized from 1 to 10 h after infliction of the trauma and subcutaneous fat and muscle tissues were sampled. As control, subcutaneous fat and muscle tissues were sampled from two un-injured pigs. Quantitative real-time polymerase chain reaction was performed to evaluate mRNA expression of genes involved in inflammation, tissue damage and repair. Expression signatures of thirteen selected genes in subcutaneous fat but not in muscle tissue reflected the age of bruises with a precision of approximately ±2 h. Moreover, the gene expression signature in the subcutaneous fat was to some extend able to separate bruises inflicted with different forces. Expression signatures of selected genes in the subcutaneous fat will increase the precision of the age determination of bruises in pigs. Further, due to the similarity of porcine and human skin physiology and immunity, these results might also provide valuable information in human forensic science.

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.

Gross and histopathological evaluation of human inflicted bruises in Danish slaughter pigs.

BACKGROUND: Human inflicted bruises in slaughter pigs are hampering animal welfare, are an infringement of the animal protection act, and are a focus of public attention. The aim of the present study was to evaluate the gross appearance of human inflicted bruises in slaughter pigs and to compare the inflammatory changes in two lesions as a basis for estimating the age of lesions in the same pig. Pigs with human inflicted bruises slaughtered at two major slaughterhouses in Denmark from November 2013 to May 2014 were evaluated. After slaughter, the bruises were examined grossly and skin and underlying muscle tissue from two similar but separate bruises (a and b) on each pig were sampled for histology. RESULTS: Skin and muscle tissue from 101 slaughter pigs were subjected to gross evaluation. Eighty-one of these were also subjected to histological evaluation. Most frequently (51 out of 101 pigs, 50 %), bruises had a tram-line pattern due to blunt trauma inflicted with long objects such as sticks. Other bruises reflected the use of tattoo-hammers, plastic paddles, double U profiles and chains. Histological evaluation of two bruises from a pig with multiple lesions was found insufficient to assess the overall age of the lesions as substantial variation in the inflammatory response between bruises was present. CONCLUSIONS: Grossly, the pattern of bruises often reflected the shape of the object used for inflicting the lesions. When determining the age of multiple bruises on a pig more than two lesions should be evaluated histologically.

A retrospective study of forensic cases of skin ulcerations in Danish pigs from 2000 to 2014.

BACKGROUND: Ulcerations in pigs, as in other farm animals, are considered to be painful and therefore hampering the welfare. Farmers are obliged to provide an intervention to protect animals against unnecessary suffering and failure to do so is considered negligence. Moreover, animals with severe open wounds are considered unfit for transportation and so are pigs with ulcerations located on hernias. This paper presents a retrospective study of forensic case files concerning ulcerations in Danish pigs from 2000 to 2014. The aim of the study was to clarify the number of cases, the number of pigs, the anatomical localization and size of ulcerations, evaluate changes during years and the age of the lesions. RESULTS: A total of 209 case files concerning 283 pigs with 459 ulcerations were included. In 2004, 2005, 2007-2009 and 2011, sows with shoulder ulcerations were the most frequently submitted, while in 2014 pigs with ulcerations on umbilical outpouchings dominated. The change in pattern on body location most likely reflects specific national regulations enforced from 2003 to 2009. The ulcerations were estimated to be from 4 h to several months old and the median diameter of ulcerations was 4 cm. CONCLUSIONS: Since 2004, the number of cases per year has declined. However, the number of affected pigs has remained almost constant from 2004 to 2014 (23.8 ± 8.5 pigs per year). The change in pattern on body parts with ulcerations likely reflected specific national regulations.

The impact of force on the timing of bruises evaluated in a porcine model.

In animal models developed in order to estimate the age of bruises, focus has been on the changes over time and not considering the force used to inflict the trauma. In the present study, gross and histological changes in 2, 4, 6 and 8 h old bruises which were inflicted with a low, moderate and high force were compared. Twelve experimental pigs were randomly assigned to three groups of force (low, moderate and high force). All pigs were anesthetized, and on each animal four blunt traumas were inflicted on the back with the low, moderate or high force according to the groups. The pigs were kept in anesthesia for 2, 4, 6 or 8 h, after which they were euthanized, and skin and muscle tissues were sampled for histology. As control, two pigs were included. The gross appearance of bruises developed similarly until 0.5 h after infliction at which time the visibility of the bruises depended on the force. The infiltration of subcutaneous neutrophils depended on the time and force used which was confirmed by both manual evaluation and image analysis of immunostained skin sections. In the muscle tissue, the number of macrophages was found useful for age determination in bruises inflicted with the highest force. Therefore, when evaluating forensic cases of bruises in both human and veterinary pathology the impact of force and not only the timing should be taken into consideration.

A novel, comprehensive, and reproducible porcine model for determining the timing of bruises in forensic pathology.

PURPOSE: Calculating the timing of bruises is crucial in forensic pathology but is a challenging discipline in both human and veterinary medicine. A mechanical device for inflicting bruises in pigs was developed and validated, and the pathological reactions in the bruises were studied over time in order to identify gross and histological parameters that may be useful in determining the age of a bruise. METHODS: The mechanical device was able to apply a single reproducible stroke with a plastic tube that was equivalent to being struck by a man. In each of 10 anesthetized pigs, four strokes that resulted in bruises were inflicted on the back. In addition, 2 control pigs were included in the study. The pigs were euthanized consecutively from 1 to 10 h after the infliction of bruises. Following gross evaluation, skin, and muscle tissues were sampled for histology. RESULTS: Grossly, the bruises appeared uniform and identical to the tramline bruises seen in humans and pigs subjected to blunt trauma. Histologically, the number of neutrophils in the subcutis, the number of macrophages in the muscle tissue, and the localization of neutrophils and macrophages in muscle tissue showed a time-dependent response. Combining these parameters, bruises could be grouped as being either less than 4 h old or between 4 and 10 h of age. Gross lesions and changes in the epidermis and dermis were inconclusive with respect to time determination. CONCLUSIONS: The model was reproducible and resembled forensic cases of bruises in pigs and humans. Therefore, the histological parameters are suitable for age determination of bruises in pigs and likely also in humans.

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.

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.

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.