Experiences and recommendations of veterinarians for the Dutch disciplinary system-a survey-based study.

Introduction: In the Netherlands, the veterinary disciplinary system is governmentally executed and was originally designed to provide an educational effect on veterinarians as part of maintaining quality standards. Methods: Over 900 veterinarians were questioned (20% of the total number of veterinarians working in veterinary medicine in the Netherlands). It was ascertained whether or not they were aware of the disciplinary system, if it affected their way of working and what impact it had on their way of working after having faced a disciplinary case. Respondents were given the opportunity to express their opinions about the system and possible improvements. Results: The risk of complaints was found to be significantly greater when a veterinarian owned a practice compared to veterinarians who were employees. Veterinarians with their own practice were generally older (male) veterinarians. Whether this was a direct effect or just the effect of having a longer career could not be answered. Multiple disciplinary procedures appeared to have no influence. In 13%, veterinarians indicated that the disciplinary system had led to a more defensive way of practicing medicine to avoid complaints. Discussion and conclusions: Most veterinarians supported a disciplinary system as a tool for maintaining and improving the integrity and reputation of the profession as a whole. Recommendations to improve were (1) shortening the length of the procedure, (2) screening for validity, (3) using online systems for communication with the disciplinary council, (4) the option of mediation before getting involved in a full procedure and (5) instituting a complaint fee.

Intratumoral injection of holmium-166 microspheres as neoadjuvant therapy of soft tissue sarcomas in dogs.

Introduction: Minimally invasive microbrachytherapy is in development to treat solid tumors by intratumoral injection of (radioactive) holmium-166 (166Ho) microspheres (MS). A high local dose can be administered with minimal damage to surrounding tissue because of the short soft tissue penetration depth of 166Ho beta radiation. We aimed to prospectively evaluate the safety and efficacy of 166Ho microbrachytherapy in client-owned canine patients with soft tissue sarcomas (STS). Methods: We included seven dogs with STS not suitable for local excision due to tumor size and/or location. 166HoMS were suspended in a carrier fluid and multiple needle-injections were performed in predetermined tumor segments to maximize tumor coverage. Tumor response was evaluated using 3D caliper and CT measurements. Follow-up further included monitoring for potential side effects and registration of subsequent treatments and survival, until at least two years after treatment. Results: Delivered radioactive doses ranged from 70 to 969 Gy resulting in a mean tumor volume reduction of 49.0 ± 21.3% after 33 ± 25 days. Treatment-related side effects consisted of local necrosis (n = 1) and ulceration of the skin covering the tumor (n = 1), which resolved with basic wound care, and surgical excision of residual tumor, respectively. Residual tumor was surgically resected in six patients after 22-93 days. After a mean follow-up of 1,005 days, four patients were alive, two patients were euthanized because of unrelated causes, and one patient was euthanized because of disease progression after the owner(s) declined subsequent surgical treatment. Conclusion: 166Ho microbrachytherapy was a safe and effective neoadjuvant treatment option for canine patients with STS.

15 years of facts and figures on veterinary disciplinary measures in the Netherlands.

In most countries, a veterinary disciplinary system is in force to ensure the quality of the veterinary profession and to offer an objective platform for complaints. We present an analysis of 15 years of veterinary disciplinary verdicts (2001-2016) to compare facts and figures and identify which factors are of major influence on the outcome of the verdicts. Rulings were collected from both paper files and the digital database of the veterinary disciplinary council (VDC), categorized, and used to create a database that enabled a statistical analysis. The results showed that complaints pertaining to companion animals are filed predominantly by owners, whereas complaints about livestock are mostly filed by the governmental civil servant (CS). CS complaints mostly address compliance issues. For the complaints made by owners (client complaints, CCs), reporting, communication, and veterinary mistakes appeared to be of statistical significance. Further studies are needed to investigate the impact of the complaints on veterinarians in general and how we can improve the veterinary disciplinary system.

Serological Screening for Antibodies against SARS-CoV-2 in Dutch Shelter Cats.

The COVID-19 pandemic raised concerns that companion animals might be infected with, and could become a reservoir of, SARS-CoV-2. As cats are popular pets and susceptible to Coronavirus, we investigated the seroprevalence of SARS-CoV-2 antibodies in shelter cats housed in Dutch animal shelters during the COVID-19 pandemic. In this large-scale cross-sectional study, serum samples of shelter cats were collected during the second wave of human COVID-19 infections in The Netherlands. Seroprevalence was determined by using an indirect protein-based ELISA validated for cats, and a Virus Neutralization Test (VNT) as confirmation. To screen for feline SARS-CoV-2 shedding, oropharyngeal and rectal swabs of cats positive for ELISA and/or VNT were analyzed using PCR tests. In 28 Dutch animal shelters, 240 shelter cats were convenience sampled. Two of these cats (0.8%; CI 95%: 0.1-3.0%) were seropositive, as evidenced by the presence of SARS-CoV-2 neutralizing antibodies. The seropositive animals tested PCR negative for SARS-CoV-2. Based on the results of this study, it is unlikely that shelter cats could be a reservoir of SARS-CoV-2 or pose a (significant) risk to public health.

Case Report: Radioactive Holmium-166 Microspheres for the Intratumoral Treatment of a Canine Pituitary Tumor.

Introduction: In this case study, a client-owned dog with a large pituitary tumor was experimentally treated by intratumoral injection of radioactive holmium-166 microspheres (166HoMS), named 166Ho microbrachytherapy. To our knowledge, this is the first intracranial intratumoral treatment through needle injection of radioactive microspheres. Materials and Methods: A 10-year-old Jack Russell Terrier was referred to the Clinic for Companion Animal Health (Faculty of Veterinary Medicine, Utrecht University, The Netherlands) with behavioral changes, restlessness, stiff gait, and compulsive circling. MRI and CT showed a pituitary tumor with basisphenoid bone invasion and marked mass effect. The tumor measured 8.8 cm3 with a pituitary height-to-brain area (P/B) ratio of 1.86 cm-1 [pituitary height (cm) ×10/brain area (cm2)]. To reduce tumor volume and neurological signs, 166HoMS were administered in the tumor center by transsphenoidal CT-guided needle injections. Results: Two manual CT-guided injections were performed containing 0.6 ml of 166HoMS suspension in total. A total of 1097 MBq was delivered, resulting in a calculated average tumor dose of 1866 Gy. At 138 days after treatment, the tumor volume measured 5.3 cm3 with a P/B ratio of 1.41 cm-1, revealing a total tumor volume reduction of 40%. Debulking surgery was performed five months after 166HoMS treatment due to recurrent neurological signs. The patient was euthanized two weeks later at request of the owners. Histopathological analysis indicated a pituitary adenoma at time of treatment, with more malignant characteristics during debulking surgery. Conclusion: The 40% tumor volume reduction without evident severe periprocedural side effects demonstrated the feasibility of intracranial intratumoral 166HoMS treatment in this single dog.

Molecular markers of prognosis in canine cortisol-secreting adrenocortical tumours.

Hypercortisolism is caused by a cortisol-secreting adrenocortical tumour (ACT) in approximately 15%-20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol-secreting ACTs were included from 40 dogs, of which follow-up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate-high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT-PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour-transforming gene-1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor-1 (SF-1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets.

The Utrecht Score: A novel histopathological scoring system to assess the prognosis of dogs with cortisol-secreting adrenocortical tumours.

A cortisol-secreting adrenocortical tumour (ACT) is the cause of naturally occurring canine hypercortisolism in approximately 15% to 20% of cases. The differentiation between an adrenocortical adenoma and carcinoma is usually based on histopathology. However, histopathological parameters have never been linked to the dogs' survival. Moreover, in human medicine the inter-observer variability of some histopathological parameters that are used for ACTs is high. The objective of this study was to establish a reliable and easy-to-use histopathological scoring system for cortisol-secreting ACTs that can assess the prognosis of dogs after adrenalectomy. Cortisol-secreting ACTs of 50 dogs, collected between 2002 and 2015, were included in this study. Twenty histopathological features were assessed by one veterinary pathologist and one resident in veterinary pathology. In addition, the Ki67 proliferation index was assessed by two observers. Only parameters with intra- and inter-observer agreement scores (intra-class correlation or Cohen's kappa coefficient) of ≥0.40 were included in survival analyses. Use of multivariate forward stepwise regression analysis with associated hazard ratios led us to a scoring system which we call the Utrecht score: the Ki67 proliferation index, +4 if more than 33% of the tumour cells have clear/vacuolated cytoplasm and + 3 if necrosis is present. Using cut-off values of 6 and 11, we could distinguish three groups that had significantly shorter survival times with increasing Utrecht scores. We conclude that the Utrecht score can be used to assess the prognosis of dogs with cortisol-secreting ACTs after adrenalectomy, which can help to select high-risk dogs that might benefit from adjuvant treatment or additional monitoring.

Incidence, possible risk factors and therapies for pseudopregnancy on Dutch dairy goat farms: a cross-sectional study.

Pseudopregnancy is a frequently diagnosed reproductive disorder in (dairy) goats. This cross-sectional study evaluates the incidence, possible risk factors and therapies for pseudopregnancy on Dutch dairy goat farms. Two questionnaires, one for farmers and one for veterinarians, were designed and included questions about general farm demographics, breeding management, hormonal oestrous induction, treatment, measures for reduction and stress moments in dairy goats in the period June 1, 2016-May 31, 2017. In total, 43 farmers (21.5 per cent response rate) and 27 veterinarians (22.5 per cent response rate) completed the questionnaire. The annual incidence of pseudopregnancy varied between 1 and 54 per cent per farm, with a mean annual incidence of 17 per cent (95 per cent CI 0.14 to 0.21). In this study, we found a significant association between incidence of pseudopregnancy and a higher percentage of goats with an extended lactation (p<0.0001) and between incidence of pseudopregnancy and the number of ultrasound examinations per year (p<0.0001). The recommended therapy in literature consists of two administrations of prostaglandins. This was only correctly applied by 10 per cent of the farms. On 52 per cent of the farms, an overdose was used comparing to the recommended dose in literature.

Adipose Tissue-Derived Stem Cell Sheet Application for Tissue Healing In Vivo: A Systematic Review.

Adipose tissue-derived stem cells (ASCs) are known to be tissue-healing promoters due to their cellular plasticity and secretion of paracrine factors. Cultured ASC sheets provide a novel method of ASC application and can retain ASCs at the targeted tissue. The purpose of this systematic review is to evaluate preclinical studies using ASC sheet transplantation therapy for promoting tissue healing. First, we searched databases to identify studies of ASC sheet therapy in different experimental animal models, and then determined the quality score of studies using SYRCLE's risk bias tool. A total of 18 included studies examined the role of ASC sheets on tissue healing and function in models for myocardial infarction, dilated cardiomyopathy, full-thickness skin wounds, hind limb ischemia, esophageal strictures, and oral ulcers. ASC sheet application after myocardial infarction improved survival rate, cardiac function, and capillary density and reduced the extent of fibrosis. Application of ASC sheets to a full-thickness skin wound decreased the wound size and stimulated wound maturation. In the hind limb ischemia model, ASC sheet application improved limb perfusion and capillary density, and decreased the amount of ischemic tissue and inflammation. ASC sheet application to mucosal wounds of the digestive tract accelerated wound healing and decreased the degree of stricture and fibrosis. Taken together, transplanted ASC sheets had a positive effect on tissue healing and reconstruction in these preclinical studies. The reported favorable effects of ASC sheet therapy in various tissue healing applications may be implemented in future translational studies. It is suggested that future preclinical animal model studies of ASC sheet therapy should concern standardization of culture techniques and investigate the mechanisms of action. In addition, clearly indicated experimental setups according to the SYRCLE's guidelines should improve study quality and validity.

Human Mesenchymal Stromal Cell Sheets Induce Macrophages Predominantly to an Anti-Inflammatory Phenotype.

Tissue healing is a highly complex process involving a cascade of biochemical and cellular events. Excessive inflammation can impair the healing response. Previous in vitro studies have shown that mesenchymal stromal cells can modulate macrophage-induced inflammation and, therefore, are promising candidates for cell-based therapies aimed at promoting tissue repair. Recently, cell sheets were introduced as a new method of delivering stromal cells to the repair site. The goal of the current study was to compare the effect of different types of stromal cell sheets on the inflammatory state of macrophages in vitro. We compared the effects of adipose tissue-derived stromal cell (ASC) sheets, bone marrow derived stromal cell (BMSC) sheets, and fibroblast sheets on macrophage functional phenotype using flow cytometric analysis, gene expression, as well as cell sheet protein secretion. This was evaluated with and without inflammatory stimulation. Viability and senescence for the different types of sheet were also evaluated. Macrophages cultured in ASC sheet conditioned medium (CM) displayed a higher fluorescence intensity of the anti-inflammatory CD206 surface marker than when cultured in BMSC sheet CM and expressed more CCL18 and IL1RA than when cultured in fibroblast sheet CM. Moreover, ASC sheets had higher cell viability and less senescent cells than BMSC sheets and fibroblast sheets. Taken together, ASC and BMSC can stimulate the anti-inflammatory macrophage (M2) phenotype to a better extent than fibroblasts. It is suggested that ASC sheets might outperform BMSC sheets in an inflammatory situation since ASC sheet CM induced-macrophages have more M2 characteristics, and ASC in the sheet was more viable.

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

Effects of adipose stem cell sheets on colon anastomotic leakage in an experimental model: Proof of principle.

The most dreaded complication of colorectal surgery is anastomotic leakage. Adipose tissue-derived stem cell sheets (ASC sheets) prepared from temperature-responsive culture surfaces can be easily transplanted onto tissues. These sheets are proposed to improve cell transplant efficiency and enhance wound healing. The aim of this study was to investigate whether application of ASC sheets could prevent leakage of sutured colorectal anastomoses. Insufficient suturing of colorectal anastomoses was performed in Wistar rats to create a colorectal anastomotic leakage model. Rats were randomized to ASC sheet application or control group. Leakage, abscess formation, adhesion formation, anastomotic bursting pressure (ABP), and histology were evaluated on postoperative day 3 or 7. ASC sheet application significantly reduced anastomotic leakage compared to controls, without increased adhesion formation. ASC sheet transplantation resulted in more CD3+ T-cells and CD163+ anti-inflammatory macrophages at the anastomotic site than the control group. ABP, vessel density and collagen deposition were not different between groups. Using cell sheet technology, we generated ASC sheets that prevented disruption of sutured colorectal anastomoses as shown by reduced leakage. Increased numbers of anti-inflammatory macrophages and T-cells might have contributed to this positive effect.

Effect of Cell Seeding Density and Inflammatory Cytokines on Adipose Tissue-Derived Stem Cells: an in Vitro Study.

Adipose tissue-derived stem cells (ASCs) are known to be able to promote repair of injured tissue via paracrine factors. However, the effect of cell density and inflammatory cytokines on the paracrine ability of ASCs remains largely unknown. To investigate these effects, ASCs were cultured in 8000 cells/cm2, 20,000 cells/cm2, 50,000 cells/cm2, and 400,000 cells/cm2 with and without 10 or 20 ng/ml tumor necrosis factor alpha (TNFα) and 25 or 50 ng/ml interferon gamma (IFNγ). ASC-sheets formed at 400,000 cells/cm2 after 48 h of culture. With increasing concentrations of TNFα and IFNγ, ASC-sheets with 400,000 cells/cm2 had increased production of angiogenic factors Vascular Endothelial Growth Factor and Fibroblast Growth Factor and decreased expression of pro-inflammatory genes TNFA and Prostaglandin Synthase 2 (PTGS2) compared to lower density ASCs. Moreover, the conditioned medium of ASC-sheets with 400,000 cells/cm2 stimulated with the low concentration of TNFα and IFNγ enhanced endothelial cell proliferation and fibroblast migration. These results suggest that a high cell density enhances ASC paracrine function might beneficial for wound repair, especially in pro-inflammatory conditions.

Development of the Dutch Johne's disease control program supported by a simulation model.

The development of a simulation model, "JohneSSim", was part of a research program aimed at designing a national Johne's disease control program for The Netherlands. Initially, the focus was mainly directed towards different compulsory "test-and-cull" strategies. However, the results from the JohneSSim model showed that eradication of Johne's disease based on such strategies would not be possible within 20 years and that it was also economically unattractive. However, improved calf management seemed to be more effective in reducing the prevalence within the same time period. Simulation of a strategy using an "ideal test" (80% sensitivity in all infected animals) showed a considerably faster decrease in prevalence. However, this strategy proved to be economically unattractive because of the high culling rate of (young) test-positive animals. The simulation model was also adapted to study beef cow herds. However, the results indicated that none of the strategies were able to reduce the mean true prevalence to almost zero for such herds. Only strategies based on "separation of calves and adult animals" proved to significantly reduce the prevalence but such a strategy is unpractical and uneconomic for Dutch beef cow herds. Due to this finding and the relative low number of Dutch beef cow farms, first priority has been given to the development of a Johne's disease control program for dairy farms. Based on the results of the "JohneSSim" model, the new national voluntary Johne's disease control program for dairy, Paratuberculosis Program Netherlands (PPN), started in September 2000. The PPN is based on a stepwise improvement of calf hygiene, with little dependency on "test and culling". The model results indicated that, if dairy farmers consistently carried out the necessary management adaptations, PPN considerably decreased prevalence and that it was economically more attractive than any previous plans.

A simulation of Johne's disease control.

A dynamic and stochastic simulation model (the "JohneSSim model") was developed to evaluate the economic and epidemiological effects of different strategies for control of paratuberculosis in dairy herds. Animals occupy one of the six defined infection states; the spread of Johne's disease is modeled with five infection routes. Many different dairy farm situations can be simulated. Control strategies that can be simulated are: (1) test-and-cull; (2) calf hygiene management; (3) vaccination and (4) grouping of animals. Losses are caused by: (1) reduced milk production; (2) diagnosis and treatment costs; (3) lower slaughter value of cows and (4) sub-optimal culling. The benefits were calculated as reduction in the losses caused by Johne's disease; the costs of each strategy were calculated on the basis of actual costs of each item; and net present value (NPV) was calculated as benefits minus costs. Herd and prevalence data from The Netherlands and Pennsylvania, USA were used. In both situations, a low true mean prevalence within 20 years could be reached only when all calf management tools were applied. The Dutch control program (PPN) was on average economically attractive (with or without labor costs, the average NPV was Euro 1183 and 12,397, respectively). In Pennsylvania, contract heifer rearing and improved calf hygiene reduced the prevalence effectively and had large economic benefits (US$ 43,917 for 20-year period) if the calves were sent to the heifer facility while very young. Validation with data from 21 infected Dutch dairy farms (as well as face-validation: comparison of the results of the JohneSSim model with experiences of Johne's experts) supported the basic assumptions in the model.

VEGF-PET imaging is a noninvasive biomarker showing differential changes in the tumor during sunitinib treatment.

Non-invasive imaging of angiogenesis could ease the optimization of antiangiogenesis treatments for cancer. In this study, we evaluated the role of VEGF-PET as a biomarker of dynamic angiogenic changes in tumors following treatment with the kinase inhibitor sunitinib. The effects of sunitinib treatment and withdrawal on the tumor was investigated using the new VEGF-PET tracer (89)Zr-ranibizumab as well as (18)F-FDG PET, and (15)O-water PET in mouse xenograft models of human cancer. The obtained imaging results were compared with tumor growth, VEGF plasma levels and immunohistologic analyzes. In contrast to (18)F-FDG and (15)O-water PET, VEGF-PET demonstrated dynamic changes during sunitinib treatment within the tumor with a strong decline in signal in the tumor center and only minimal reduction in tumor rim, with a pronounced rebound after sunitinib discontinuation. VEGF-PET results corresponded with tumor growth and immunohistochemical vascular- and tumor- markers. Our findings highlight the strengths of VEGF-PET imaging to allow serial analysis of angiogenic changes in different areas within a tumor.

Spatiotemporal expression of heme oxygenase-1 detected by in vivo bioluminescence after hepatic ischemia in HO-1/Luc mice.

Upregulation of heme oxygenase-1 (HO-1) has been proposed as a critical mechanism protecting against cellular stress during liver transplantation, providing a potential target for new therapeutic interventions. We investigated the feasibility of in vivo bioluminescence imaging (BLI) to noninvasively quantify the spatiotemporal expression of HO-1 after warm hepatic ischemia in living animals. Luciferase activity was measured by BLI as an index of HO-1 transcription in transgenic reporter mice (Ho1-luc) at standardized time points after 60 minutes of warm hepatic ischemia. HO-1 mRNA levels were measured in postischemic livers of mice sacrificed at the same time points in separate experiments. Bioluminescent signals from postischemic liver lobes were first detected at 3 hours after reperfusion. Peak levels were reached at 9 hours, after which bioluminescent activity declined and returned to baseline values at 48 hours after reperfusion. Upregulation of HO-1 as detected by in vivo BLI was preceded by increased HO-1 mRNA expression and confirmed by enhanced immunohistochemical staining of hepatocytes. In conclusion, this study shows that in vivo BLI allows a sensitive assessment of HO-1 expression after hepatic ischemia in living animals. The capability of whole-body temporal imaging of HO-1 expression provides a valuable tool in the development of novel strategies to modulate HO-1 expression in liver transplantation.