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Grading of bacterial aggregates and distribution of bacterial species detected in equine experimental excisional limb wounds inoculated or not inoculated with S. aureus and P. aeruginosa on day 4
Source publication
Objective: Relevant animal models to study effects of bacterial aggregates on wound healing are lacking. We aimed at establishing an equine wound model with bacterial aggregates to investigate the impact of bacterial inoculation on normal (thorax) and impaired (limb) wound healing.
Approach: Wounds were created on three limbs and both thorax sides...
Contexts in source publication
Context 1
... aggregates were detected in most in- oculated limb wounds on day 7 (91%), 14 (100%), and 21 (100%), and in fewer wounds (40%) on day 27 (Table 2 and Fig. 5). Bacterial aggregates were detected in some noninoculated limb wounds on day 7 (80%), 14 (50%), 21 (67%), and 27 (33%). ...Context 2
... are reported as mean -SD of having bacterial aggregates present than the noninoculated limb wounds (odds ratio 7.1, 2.4- 21.0, p = 0.0086). Furthermore, inoculated limb wounds had significantly higher bacterial aggre- gate grades on day 14 ( p = 0.031) and day 21 ( p = 0.0087) compared with noninoculated limb wounds (Table 2). In thorax wounds, bacterial ag- gregates were found in inoculated wounds only, and only on day 7 (83%), 14 (50%), and 21 (20%) ( Table 3). ...Citations
... Our results showed a 100-and 20-fold increase in the MIC of ciprofloxacin and gentamicin for bacteria forming biofilms compared to planktonic, as described for chronic wound biofilms 45,46 , indicating that the proposed in vitro model is viable and functional. Moreover, in vivo results confirmed that ciprofloxacin therapy prevents septicemia, since bacterial inoculation without treatment resulted in 100% mortality of mice, an outcome previously reported in mice and horse models 36,38,47 . Despite the daily dose of ciprofloxacin 10 mg/ kg, biofilm implantation generated a persistent infection, with a detrimental effect over animal welfare as seen in higher health score, permanent weight loss and initial febrile state, resembling the severely affected life quality of chronic wound patients 5 . ...
... Regarding the morphology at the wound site, histologic analysis indicated two types of cellular interactions: at the wound edge, there was an influx of PMN cells as an inflammatory response against biofilm, whereas in the wound bed the low count of cells indicated inhibition of cellularization and epithelialization. Consistent with this observation, a qualitative histological analysis shows the lack of myofibroblasts, cells in a proliferative state and endothelial cells are linked to a reduced capacity for regeneration, suggesting that biofilm infection seriously impairs wound healing, as previously described in other animal models 34,35,38 . ...
Chronic wounds cannot heal due to impairment of regeneration, mainly caused by the persistent infection of multispecies biofilms. Still, the effects of biofilm wound infection and its interaction with the host are not fully described. We aimed to study functional biofilms in physiological conditions in vitro , and their potential effects in health and regeneration in vivo . Therefore, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis were seeded in collagen-based scaffolds for dermal regeneration. After 24 h, scaffolds had bacterial loads depending on the initial inoculum, containing viable biofilms with antibiotic tolerance. Afterwards, scaffolds were implanted onto full skin wounds in mice, together with daily supervision and antibiotic treatment. Although all mice survived their health was affected, displaying fever and weight loss. After ten days, histomorphology of scaffolds showed high heterogeneity in samples and within groups. Wounds were strongly, mildly, or not infected according to colony forming units, and P. aeruginosa had higher identification frequency. Biofilm infection induced leucocyte infiltration and elevated interferon-γ and interleukin-10 in scaffolds, increase of size and weight of spleen and high systemic pro-calcitonin concentrations. This functional and implantable 3D biofilm model allows to study host response during infection, providing a useful tool for infected wounds therapy development.
... Second intention healing is prone to be impaired for equine limb wounds. As described below (Knottenbelt 1997;Wilmink et al. 2002), this impaired limb wound healing pattern is remarkably different from equine body wounds that normally heal well (Jacobs et al. 1984;Wilmink et al. 1999a;Jørgensen et al. 2019). Thus, especially limb wounds pose a substantial economic burden on the equine industry besides the obvious welfare issues Anonymous 2016;Perkins et al. 2005). ...
... How bioburden and potential presence of bacterial biofilms affect healing in equine traumatic wounds is unknown, though a negative influence is expected (Westgate et al. 2010). In our recent experimental equine wound study, biofilms seemed to impair limb wound healing further, while not affecting healing of body wounds (Jørgensen et al. 2019), as described below. ...
... Next, we established an equine biofilm-infected wound model (Jørgensen et al. 2019). Full-thickness wounds were created on the limbs and bodies of six horses and were bandaged and left to heal by second intention. ...
Horses frequently sustain wounds due to their “fight and flight” nature. Equine body wounds heal well whereas limb wounds often display impaired healing. This disparity in healing patterns has been attributed to factors like hypoxia and prolonged chronic inflammation in equine limb wounds. Recent research suggests biofilm infections to also be part of the pathogenesis in equine chronic limb wounds, as biofilms are present in equine limb wounds and further impair the healing, whereas biofilms are seldom and do not affect healing in body wounds. However, specific veterinary research, horses included, within wound biofilm infections is scarce and just emerging. In human wounds, biofilms have been studied for a longer period and are considered one of the causes of the healing pattern in chronic wounds like diabetic foot ulcers and venous leg ulcers. As human chronic wounds and equine limb wounds share many similarities, such as chronicity, weak inflammation, hypoxia, biofilms, and increased pH, the horse might be a relevant animal model species to study interactions between host and biofilms and to investigate future treatments strategies.
... Horses appear to be valid model animals for wound healing in humans because of their similar skin architecture and the relative role of contraction and epithelialization in healing [12,13]. Because of their surroundings, horses' wounds have a significant risk of infection, as the infected wounds house a variety of microorganisms. ...
Various herbal compounds are used for medical purposes due to their safety, as there are no or minimal side effects. This study was performed to assess the wound healing and antioxidant activities of ethyl acetate (EtOAc) and methanolic extract (MeoH) of Solenostemma arghel (S. arghel). Their antifungal activities were also evaluated against isolated swabs of equine wounds. They underwent GC-MS analysis for the characterization of both extracts. For wound healing evaluation, forty-five male albino rats were divided into three groups; the control group was treated with normal saline, and the other two groups were treated with S. arghel EtOAc and MeoH extract gels, respectively. The wounds were examined clinicopathologically and immunohistochemistry on the 3rd, 7th, and 14th days post-wounding. GC-Ms analysis of S. arghel recorded fifty-one volatile organic compounds (VOCs) within EtOAc extraction and thirty VOCs in MeoH extract. VOCs represented in EtOAc extract showed higher antioxidant activity and better and faster wound healing than VOCs of MeOH extract. The treated groups showed improved wound healing clinically and pathologically in comparison with the control group as they decreased the wound surface area (WSA) and percent (WSA%) and increased the wound contraction percent (WC%), epithelization, fibroblast proliferation with neovascularization, and reduced the inflammatory reaction. Moreover, the treated groups showed higher expression of vascular endothelial growth factor (VEGF) compared with the control. The EtOAc extract showed higher antifungal activity against Penicillium funiculosum, P. jensenii, M. cinctum, and Candida albicans, which were isolated from infected clinical equine wounds, than MeOH extract. The treated groups showed improved wound healing clinically and pathologically in comparison with the control group as they decreased the wound surface area (WSA) and percent (WSA%) and increased the wound contraction percent (WC%), epithelization, fibroblast proliferation with neovascularization, and reduced the inflammatory reaction. Moreover, the treated groups showed higher expression of vascular endothelial growth factor (VEGF) compared with the control. Additionally, the two extract gels showed promising healing of equine wounds. In conclusion, the study recommended the use of S. arghel EtOAc extract as it was proven to promote wound healing compared with MeoH extract. View Full-Text
... In addition to continuous motion, the presence of foreign bodies, necrotic tissue, bone sequestra and dirt can induce and maintain EGT (Wilmink and Van Weeren, 2004). Impaired wound healing can further be complicated by microorganisms (Freeman et al., 2009;Metcalf and Bowler, 2013;Jørgensen et al., 2017Jørgensen et al., , 2019a. Despite using sterile bandaging material, biofilms were more prevalent in bandaged limb wounds with EGT formation. ...
Exuberant granulation tissue (EGT) is often observed during second intention wound healing in horses. Despite its impact on wound care, the basic mechanisms leading to EGT are still unclear and effective strategies to prevent and/or treat EGT are lacking. The development of EGT is a poorly understood, multifactorial process involving hyperproliferating fibroblasts and malfunctional differentiation of keratinocytes, suboptimal wound contraction, dysfunctional vascularisation, and chronic inflammation. To consolidate and describe basic and clinical research literature on EGT and to identify knowledge gaps and opportunities for future research, a search was systematically conducted using predefined search terms. Subsequently, a scoping review was conducted using specific criteria to select the peer-reviewed literature that described methods to treat and/or prevent EGT. Proposed mechanisms of effects as well as results and main conclusions were extracted and tabulated. The systematic search resulted in 1062 publications in PubMed and 767 in Web of Science. Twenty additional studies were later included. Of these, 327 studies were reviewed for the narrative review on basic research and 35 controlled clinical trials were eligible for the scoping review. All 35 studies were conducted in university hospitals, and all but one involved surgically induced non-infected wounds. The study population was predominantly horses (n = 230) with a small number of ponies (n = 18) and donkeys (n = 14). In conclusion, there remains a strong need for evidence-based recommendations on EGT treatment, preferably using multi-centre studies that represent the general population of horses, include higher numbers of animals, and are performed in naturally occurring wounds. This narrative and scoping review also emphasises the importance of incorporating basic research knowledge in the study design of clinical trials.
... Pseudomonas aeruginosa (gramnegative) was chosen to demonstrate the specific mechanism of IA and its efficiency on both gram-positive and gram-negative bacteria. It is noteworthy that, both these species, are commonly associated with tissue infections [21]. The results showed that IA was able to completely suppress the bacterial growth on all the tested microorganisms. ...
The presence of bacteria of various origins on horse hoofs enables the onset of infections following trauma or even post-surgical wounds. Thus, the analysis of new antibacterial substances is of fundamental importance. In this study, the antibacterial efficacy of Iron Animals (IA), a stable colloidal suspension of iron oxide, organic acids, and detergents, was tested in vitro and in vivo. In vitro assays were performed to test the unspecific inhibitory effect of IA on both gram-positive and gram-negative bacteria monitoring the microorganism growth by spectrophotometry (optical density OD600) at 37 °C for 24 h. In vivo test consists on the quantification of the bacterial load in colony forming units per gram (CFU/g) of specimens collected from the frog region of the anterior hooves of 11 horses. Sampling followed the application of four disinfectant protocols consisting of two consecutive 3 min scrubs with 50 mL of 10% Povidone-iodine (PI) or 4% Chlorhexidine (CHx), with or without an additional application for 15 min of 10 mL of Iron Animals (PI+IA and CHx+IA). In vitro, IA completely suppressed the bacterial growth of all the tested microorganisms, resulting in effectiveness also against CHx-resistant bacteria, such as Staphylococcus aureus. In vivo, PI emerged as an ineffective protocol; CHx was effective in 18% of cases, but with the addition of IA (CHx + IA) its use emerged as the best disinfectant protocol for horse hoof, achieving the lowest bacterial load in 55% of cases. The addition of IA, after PI or CHx, improves the effectiveness of both disinfectants leading to the highest bactericidal activity in 82% of cases.
... 14 Evidence is increasing that such biofilms play a role in impaired limb wound healing in horses as well. 9,15 In a recent experiment, 16 we created excisional dermal limb and body wounds in horses and inoculated these wounds with Staphylococcus aureus and Pseudomonas aeruginosa on day 4 after wound creation to induce biofilm formation. Clinical findings and wound scores were similar for inoculated and noninoculated limb wounds, but from day 10 after wound creation onward, inoculated limb wounds healed slower (had larger wound areas) than did noninoculated limb wounds. ...
... Furthermore, biofilms were more often detected in inoculated limb wounds, and the majority (70% to 100%) of limb wound biofilms consisted of S aureus. 16 On the other hand, in body wounds, the bioburden was quickly cleared and bacterial inoculation had no effect on healing. 16 The purpose of the study reported here was to explore the mechanisms underlying impaired wound healing in horses after inoculation of wounds with S aureus and P aeruginosa. ...
... 16 On the other hand, in body wounds, the bioburden was quickly cleared and bacterial inoculation had no effect on healing. 16 The purpose of the study reported here was to explore the mechanisms underlying impaired wound healing in horses after inoculation of wounds with S aureus and P aeruginosa. To do this, we used biopsy specimens from our previous study 16 and assessed expression of several genes involved in healing and histologic features to further characterize the observed impaired healing. ...
Objective:
To evaluate histologic changes and gene expression patterns in body and limb wounds in horses in response to bacterial inoculation.
Sample:
Wound biopsy specimens from 6 horses collected on days 7, 14, 21, and 27 after excisional wounds (20 wounds/horse) were created over the metacarpal and metatarsal region and lateral thoracic region (body) and then inoculated or not inoculated on day 4 with Staphylococcus aureus and Pseudomonas aeruginosa.
Procedures:
Specimens were histologically scored for the amount of inflammation, edema, angiogenesis, fibrosis organization, and epithelialization. Quantitative PCR assays were performed to quantify gene expression of 10 inflammatory, proteolytic, fibrotic, and hypoxia-related markers involved in wound healing.
Results:
Except for gene expression of interleukin-6 on day 27 and tumor necrosis factor-α on day 14, bacterial inoculation had no significant effect on histologic scores and gene expression. Gene expression of interleukin-1β and -6, serum amyloid A, and matrix metalloproteinase-9 was higher in limb wounds versus body wounds by day 27. Gene expression of cellular communication network factor 1 was higher in limb wounds versus body wounds throughout the observation period.
Conclusions and clinical relevance:
The lack of clear markers of wound infection in this study reflected well-known difficulties in detecting wound infections in horses. Changes consistent with protracted inflammation were evident in limb wounds, and gene expression patterns of limb wounds shared similarities with those of chronic wounds in humans. Cellular communication network factor warrants further investigation and may be useful in elucidating the mechanisms underlying poor limb wound healing in horses.
... Biopsies were available from a study involving experimental excisional dermal wounds in six adult horses. 31 Horses were clinically healthy and were without any signs or history of dermatological diseases or injuries. They were housed in box stalls and were handwalked for 20 min twice daily. ...
... The experimental procedure is described in detail elsewhere. 31 In brief, the horses had four wounds (2 9 2 cm) created on the lateral aspect of the metatarsus/-carpus (limb) and on the lateral thoracic wall (body). The horses were sedated and received local analgesia and NSAIDs before wound creation; the horses further received sedation and local analgesia when skin biopsies were obtained. ...
Background
The re‐epithelialization process in equine wound healing is incompletely described. For epithelial cells to migrate during embryogenesis they undergo epithelial‐to‐mesenchymal transition (EMT); this phenotypic transition occurs during wound healing in humans and rodents, but it has not been investigated in horses.
Hypothesis/objectives
To investigate keratinocyte differentiation and EMT in equine experimental excisional limb and body wounds healing by second intention.
Animals
Six adult research horses.
Methods and materials
Immunohistochemical analysis was used to detect expression of the differentiation markers cytokeratin (CK)10, CK14, loricrin and peroxisome proliferator‐activated receptor alpha (PPAR‐α), and of the EMT markers E‐cadherin and N‐cadherin in normal limb and body skin, and biopsies from limb and body wounds.
Results
Loricrin and CK10 were expressed in normal skin and periwound skin but not in migrating epithelium of body and limb wounds. However, they reappeared at the migrating epithelial tip of body wounds only. CK14 and PPAR‐α had uniform distribution throughout the migrating epithelium. N‐cadherin was not expressed in normal unwounded skin but was detected in periwound skin adjacent to the wound margin. E‐cadherin expression decreased at the wound margin.
Conclusions and clinical importance
Presence of N‐cadherin suggests that cadherin switching occurred during wound healing, this may be an indication that EMT occurs in horses. To the best of the authors’ knowledge, this has never been described in horses before and warrants further investigation to assess the clinical implications. The tip of the migrating epithelium in body wounds appeared more differentiated than limb wounds, which could be part of the explanation for the superior healing of body wounds.
Introduction
Chronic wounds are a major drain on healthcare resources and can lead to substantial reductions in quality of life for those affected. Moreover, they often precede serious events such as limb amputations and premature death. In the long run, this burden is likely to escalate with an ageing population and lifestyle diseases such as obesity. Thus far, the identification of beneficial therapeutics against chronic wounds have been hindered by the lack of an ideal chronic wound animal model. Although animal models of delayed healing have been developed, none of these models fully recapitulate the complexity of the human chronic wound condition. Furthermore, most animals do not develop chronic wounds. Only the thoroughbred racehorse develops chronic ulcers.
Areas covered
In this review, the different characteristics of chronic wounds that highlight its complexity are described. In addition, currently available models reflecting different aspects of chronic wound pathology and their relevance to human chronic wounds are discussed. This article concludes by listing relevant features representative of an ideal chronic wound model. Additionally, alternative approaches for the development of chronic wound models are discussed.
Expert opinion
Delayed models of healing, including the streptozotocin diabetic model, skin flap model and magnet-induced IR models have emerged. While these models have been widely adopted for preclinical therapeutic testing, their relevance towards human chronic wounds remains debatable. In particular, current delayed healing models often fail to fully incorporate the key characteristics of chronic ulcers. Ultimately, more representative models are required to expedite the advancement of novel therapeutics to the clinic.
Microbial biofilms defined as extremely complex ecosystems are considered clinically important for humans. However, the concept and significant roles of microbial biofilms in the progression of disease has seriously lagged in veterinary medicine, when compared with human medicine. Although the research on biofilms in animals is just beginning to emerge, limited studies have paid attention that microbial biofilms are clinically important in the field of veterinary medicine, especially the livestock industry, and lead to serious economic losses. In this review, the importance of microbial biofilms causing high economic losses in the livestock industry has been highlighted. Besides, the concept of microbial biofilm, their role in the pathogenesis of the animal disease, as well as diagnosis approaches and possible therapeutic strategies needed to overcome their detrimental effects in veterinary medicine, has been discussed.
In chronic wounds in humans, biofilm formation and wound chronicity are linked, as biofilms contribute to chronic inflammation and delayed healing. Biofilms are aggregates of bacteria, and living as biofilms is the default mode of bacterial life; within these aggregates, the bacteria are protected from both antimicrobial substances and the immune response of the host. In horses, delayed healing is more commonly seen in limb wounds than body wounds. Chronic inflammation and hypoxia are the main characteristics of delayed wound healing in equine limbs, and biofilms might also contribute to this healing pattern in horses. However, biofilm formation in equine wounds has been studied to a very limited degree. Biofilms have been detected in equine traumatic wounds, and recent experimental models have shown that biofilms protract the healing of equine limb wounds. Detection of biofilms within wounds necessitates advanced techniques that are not available in routine diagnostic yet. However, infections with biofilm should be suspected in equine limb wounds not healing as expected, as they are in human wounds. Treatment should be based on repeated debridement and application of topical antimicrobial therapy.
