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.

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.

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.

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.

Post-weaning diarrhea in pigs weaned without medicinal zinc: risk factors, pathogen dynamics, and association to growth rate.

BACKGROUND: Porcine post-weaning diarrhea (PWD) has reemerged as an important topic in pig production, as common control strategies based on prophylactic use of antimicrobials and zinc oxide have been deemed unsustainable. The objectives of this study were to estimate the cumulative incidence of porcine post-weaning diarrhea with different etiologies in production systems weaning without zinc oxide and prophylactic antimicrobials, to assess risk factors for post-weaning diarrhea, and to estimate the impact of post-weaning diarrhea on growth rate. A cohort study was conducted at two commercial indoor producers weaning without medicinal zinc oxide and prophylactic antimicrobials. RESULTS: Piglets were included at birth (n = 300) and 272 survived until weaning. After insertion to the nursery units, the piglets were clinically examined every day for 14 days, and rectal swabs were collected and analyzed for enterotoxigenic Escherichia coli (ETEC) and rotavirus A. The cumulative incidences of PWD the first 14 days after insertion to the nursery units were 41.8% (CI 33.6, 50.4) and 51.1% (CI 42.3, 60.0) at the two producers, respectively. We found a low incidence of cases associated to ETEC, and detected a substantial proportion of cases associated to rotavirus. We observed a biphasic pattern in the assumed etiology with rotavirus occurring first, and then a shift towards cases associated to ETEC/non-ETEC hemolytic E. coli. Being offspring of older sows was a protective factor for the development of PWD (Hazard ratio = 0.88 [CI 0.78, 0.99] per unit increase in parity of the dam). Low birth weight reduced the post-weaning growth rate (- 5.2 g/day [CI - 7.5, - 2.9] per 100 g decrease in birthweight) and increased the hazard of developing PWD (Hazard ratio for birthweight below 1100 g: 2.30 [CI 1.41-3.74]). The combined effect of having diarrhea for 2 days or more and receiving antimicrobial treatment was associated with an increased average daily weight gain. CONCLUSIONS: This study suggests novel insights regarding pathogen dynamics and risk factors for PWD in productions not using prophylactic antimicrobials and medicinal zinc. The findings may have important implications for both antimicrobial usage and prevention strategies.

In Vivo Gentamicin Susceptibility Test for Prevention of Bacterial Biofilms in Bone Tissue and on Implants.

The objective of this study was to set up an in vivo gentamicin susceptibility test for biofilm prevention in bone tissue and on implants. Twenty-five pigs were allocated to six groups. Pigs in group A (n = 6) were inoculated with saline. Pigs in groups B (n = 6), C (n = 3), D (n = 3), E (n = 3), and F (n = 4) were inoculated with 10 µl saline containing 104 CFU of Staphylococcus aureus Different concentrations based on the MIC of gentamicin for the specific strain were added to the 10-µl inoculum for groups C (160× MIC), D (1,600× MIC), E (16,000× MIC), and F (160,000× MIC). The inocula were injected into a predrilled tibial implant cavity, followed by insertion of a steel implant (2 by 15 mm). The pigs were euthanized after 5 days. In vitro, all the doses used were found to be bactericidal after up to 6 h. All implant cavities of pigs inoculated with bacteria and bacteria plus 160× MIC or 1,600× MIC of gentamicin were positive for S. aureus In animals in each of groups E (16,000× MIC) and F (160,000× MIC), 2/3 and 1/4 of the implant cavities were S. aureus positive, respectively. By grouping groups C and D (<10,000× MIC) and groups E and F (>10,000× MIC), a significant decrease in the number of implant-attached bacteria was seen only between the high-MIC-value group and group B. Histologically, it was demonstrated that 1,600×, 16,000×, and 160,000× MIC resulted in a peri-implant tissue reaction comparable to that in saline-inoculated animals. In vivo, the antimicrobial tolerance of the inoculated planktonic bacteria was increased by in vivo-specific factors of acute inflammation. This resulted in bacterial aggregation and biofilm formation, which further increased the gentamicin tolerance. Thus, susceptibility patterns in vitro might not reflect the actual in vivo susceptibility locally within a developing infectious area.