Multiple myeloma in dogs: Use of the cell block technique as a new diagnostic tool.

BACKGROUND: The diagnosis of multiple myeloma (MM) in dogs may be challenging and complex. The cell blocks are a diagnostic technique that allows the characterization of neoplastic cells and, therefore, might help in the diagnosis of atypical MM. OBJECTIVE: The objective of the present work is to describe three clinical cases in which the cell blocks and immunohistochemistry contributed to the definitive diagnosis of canine MM. METHODS: Three dogs, one female and two males, with different clinical signs, were presented for consultation with anemia, hyperproteinemia with monoclonal gammopathy, and the presence of plasmacytosis in the bone marrow. Cytologic analysis of the spleen was performed in two dogs and was suggestive of the presence of lymphocytes or plasma cells of a neoplastic nature in one of the cases and plasma cell hyperplasia associated with extramedullary hematopoiesis in the other. Given the hypotheses of lymphoid neoplasms with a plasma cell phenotype, cell blocks from aspiration punctures were performed for immunohistochemical analysis with anti-CD3, CD20, CD79αcy, PAX5, and MUM1 antibodies. RESULTS: The results revealed positive staining for MUM1 in 80% of the cells in the spleen cell block and for CD20 and MUM1 in 70% of the cells in the bone marrow cell blocks, with negative staining for the other antibodies. The immunophenotyping results allowed the diagnosis of MM in the three cases and excluded other lymphoid neoplasms. CONCLUSIONS: This work reinforces the importance of using cell blocks in the diagnosis of neoplasms by demonstrating their potential to aid the diagnosis of MM.

Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

BACKGROUND: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. RESULTS: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. CONCLUSION: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

A novel and simple heat-based method eliminates the highly detrimental effect of xylene deparaffinization on acid-fast stains.

OBJECTIVES: Histopathology is an important method for diagnosing extrapulmonary tuberculosis, yet tissue sections are often negative for mycobacteria after use of acid-fast stain (AFS). This study investigated the mechanism of AFS use and the detrimental effect of histologic processing-in particular, xylene deparaffinization-on AFS and mycobacterial detection. METHODS: The target of the fluorescent Auramine O (AuO) AFS was investigated using triple staining with DNA- and RNA-specific dyes. The effect of xylene deparaffinization on the acid fastness of mycobacteria in cultures or tissue sections was studied using AuO fluorescence as a quantitative marker. The xylene method was compared with a novel, solvent-free projected-hot-air deparaffinization (PHAD). RESULTS: Co-localization of AuO with DNA/RNA stains suggests that intracellular nucleic acids are the true target of AFS, producing highly specific patterns. Xylene reduces mycobacterial fluorescence significantly (P < .0001; moderate effect size, r = 0.33). The PHAD process yielded significantly higher fluorescence than xylene deparaffinization in tissues (P < .0001; large effect size, r = 0.85). CONCLUSIONS: Auramine O can be applied for nucleic acid staining of mycobacteria in tissues producing typical beaded patterns. Acid-fast staining depends heavily on the integrity of the mycobacterial cell wall, which xylene appears to damage. A solvent-free tissue deparaffinization method has the potential to increase mycobacterial detection significantly.

Co-Expression of T- and B-Cell Markers in a Canine Intestinal Lymphoma: A Case Report.

An 8-year-old female neutered Labrador retriever was presented for a second opinion consultation due to vomiting and lethargy, having failed to respond to symptomatic therapy. Blood analysis revealed hyperbilirubinemia and hypoalbuminemia, associated with hypocobalaminemia. An abdominal ultrasound identified diffused bowel thickening and hypoechoic hepatomegaly. An ultrasound-guided liver fine-needle aspiration was performed for cytology and also for cell block immunocytochemistry. Gastric and duodenal biopsies were collected by gastroduodenoscopy. Liver cytology showed numerous lymphocytes, suggesting lymphoma at the hepatic infiltration stage, and immunocytochemistry in the cell block of the hepatic aspirate indicated co-expression of CD3 and CD20 in the lymphoid cells present. The histopathology of gastric and duodenal biopsies supported the hypothesis of gastrointestinal lymphoma due to heavy lymphoid infiltration of the gastric epithelium and intestinal mucosa, including the villi. Concurrent immunohistochemistry was performed using CD3, CD20, PAX5, and CD79αcy antibodies. Immunomarking was positive for CD3 and CD20, which overlapped populations of lymphoid cells, and was negative for all other antibodies. In the clonality test, lymphocyte co-expression of CD3 and CD20 was confirmed by monoclonal rearrangement of T-cell gamma receptors. The final diagnosis was type 2 enteropathy-associated T-cell lymphoma with hepatic infiltration. Co-expression was examined in conjunction with the PARR result in the presence of T-cell monoclonal rearrangement.

Cost-Effective Mechanical Aggregation of Cardiac Progenitors and Encapsulation in Matrigel Support Self-Organization in a Dynamic Culture Environment.

Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling.

Vet-OncoNet: Malignancy Analysis of Neoplasms in Dogs and Cats.

Analysis of canine and feline tumor malignancy data can help clinicians identify high-risk patients and make more accurate decisions. Based on a sample of 16,272 cancer records, including 3266 cats and 13,006 dogs, collected from January 2019 to December 2021 in the Vet-OncoNet Network database, this study aimed to compare the tumor malignancy profile between cats and dogs, considering animal-related factors (sex, age, and breed), topography, and geographic location using a mixed-effects logistic regression model. Cats had a higher proportion of malignant tumors (78.7%) than dogs (46.2%), and the malignancy profile was very different regarding tumors' topographies. The mean age of malignant tumors occurred eight months later than benign ones (9.1, SD = 3.4; 9.8, SD = 3.2), in general. Species (OR = 3.96, 95%CI 3.57: 4.39) and topography (MOR = 4.10) were the two most important determinants of malignancy risk. Female dogs had a higher risk than male dogs (OR = 1.19, 95%CI 1.08: 1.31), which does not appear to be the case in cats (OR = 0.98, 95%CI 0.77: 1.23). Breed contributed significantly to differences in malignancy risk in dogs (MOR = 1.56), particularly in pit bulls and boxers. District of residence was not so relevant in predicting malignancy risk (MOR = 1.14). In both species, the risk of malignancy increased by approximately 20% every three years. It could be hypothesized that species differences in genetic structure may contribute to tumor malignancy.

Cross Species Analysis and Comparison of Tumors in Dogs and Cats, by Age, Sex, Topography and Main Morphologies. Data from Vet-OncoNet.

The animal cancer burden is essential for the translational value of companion animals in comparative oncology. The present work aims to describe, analyze, and compare frequencies and associations of tumors in dogs and cats based on the Animal Cancer Registry created by Vet-OncoNet. With 9079 registries, regarding 2019 and 2020, 81% (n = 7355) belonged to dogs. In comparison, cats have a general one-year right advance in the mean age of cancer diagnosis compared to dogs. The multivariate topography group analysis shows a distinct pattern between the two species: dogs have higher odds of cancer in the genito-urinary system, spleen, soft tissue tumors and skin, while cats show higher odds for tumors in the eyes, digestive organs, nasal cavity, lymph nodes, bones and mammary glands. Regarding morphologies, dogs are overrepresented in mast cell tumors (MCT), melanomas, and hemangiosarcomas. While cats are overrepresented in fibrosarcomas, lymphomas (T and B-cell), in malignant mammary tumors, and squamous cell carcinoma (SCC). Females have greater odds only in the mammary gland, with males having greater odds in six of twelve topographies. This study is the first outcome of continuous animal cancer registration studies in Portugal.

Evaluation of Commercial Myxomatosis Vaccines against Recombinant Myxoma Virus (ha-MYXV) in Iberian Hare and Wild Rabbit.

The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus granatensis) has resulted in numerous outbreaks with high mortality leading to the reduction, or even the disappearance, of many local populations of this wild species in the Iberian Peninsula. Currently, the available vaccines that prevent myxomatosis in domestic rabbits caused by classic strains of myxoma virus have not been assessed for use in Iberian hares. The main objective of this study was to evaluate the efficacy of commercial rabbit vaccines in Iberian hares and wild rabbits against the natural recombinant myxoma virus (ha-MYXV), bearing in mind its application in specific scenarios where capture is possible, such as genetic reserves. The study used a limited number of animals (pilot study), 15 Iberian hares and 10 wild rabbits. Hares were vaccinated with Mixohipra-FSA vaccine (Hipra) and Mixohipra-H vaccine (Hipra) using two different doses, and rabbits were vaccinated with the Mixohipra-H vaccine or the Nobivac Myxo-RHD PLUS (MSD Animal Health) using the recommended doses for domestic rabbits. After the vaccination trials, the animals were challenged with a wild type strain of ha-MYXV. The results showed that no protection to ha-MYXV challenge was afforded when a commercial dose of Mixohipra-FSA or Mixohipra-H vaccine was used in hares. However, the application of a higher dose of Mixohipra-FSA vaccine may induce protection and could possibly be used to counteract the accelerated decrease of wild hare populations due to ha-MYXV emergence. The two commercial vaccines (Mixohipra-H and Nobivac Myxo-RHD PLUS) tested in wild rabbits were fully protective against ha-MYXV infection. This knowledge gives more insights into ha-MYXV management in hares and rabbits and emphasises the importance of developing a vaccine capable of protecting wild populations of Iberian hare and wild rabbit towards MYXV and ha-MYXV strains.

A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids.

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies.

In late 2019, the first herpesvirus in the genus Lepus, named leporid gammaherpesvirus 5 (LeHV-5) was described. At the time, herpetic typical lesions were observed in hares infected by the myxoma virus, which is known to induce immunosuppression. Though the real impact of LeHV-5 is still poorly understood, since it affects reproduction, it poses an additional threat to the already fragile populations of Iberian hare, demanding prevalence investigations. In this article, we describe the first quantitative molecular method for LeHV-5 detection, using either Taqman or the EvaGreen systems. This method has excellent sensitivity and specificity, it is able to detect 2.1 copies of LeHV-5 DNA and was validated with an internal control targeting the 18S rRNA gene, allowing monitoring extraction and PCR amplification efficiencies.

Recombinant myxoma virus infection associated with high mortality in rabbit farming (Oryctolagus cuniculus).

Myxomatosis is an emergent disease in the Iberian hare, having been considered a rabbit disease for decades. Genome sequencing of the strains obtained from Iberian hares with myxomatosis showed these to be distinct from the classical ones that circulated in rabbits since the virus introduction in Europe, in 1952. The main genomic difference in this natural recombinant hare myxoma virus (ha-MYXV) is the presence of an additional 2.8 kb region disrupting the M009L gene and adding a set of genes homologous to the myxoma virus (MYXV) genes M060R, M061R, M064R, M065R and M066R originated in Poxviruses. After the emergence of this recombinant virus (ha-MYXV) in hares, in the summer of 2019, the ha-MYXV was not detected in rabbit surveys, suggesting an apparent species segregation with the MYXV classic strains persistently circulating in rabbits. Recently, a group of six unvaccinated European rabbits (Oryctolagus cuniculus cuniculus) from a backyard rabbitry in South Portugal developed signs of myxomatosis (anorexia, dyspnoea, oedema of eyelids, head, ears, external genitals and anus, and skin myxomas in the base of the ears). Five of them died within 24-48 hr of symptom onset. Molecular analysis revealed that only the recombinant MYXV was present. This is the first documented report of a recombinant hare myxoma virus in farm rabbits associated with high mortality, which increases the concern for the future of both the Iberian hare and wild rabbits and questions the safety of the rabbit industry. This highlights the urgent need to evaluate the efficacy of available vaccines against this new MYXV.

A Potential Atypical Case of Rabbit Haemorrhagic Disease in a Dwarf Rabbit.

Rabbit haemorrhagic disease (RHD) is a highly contagious infectious disease of European wild and domestic rabbits. Rabbit haemorrhagic disease virus (RHDV, GI.1) emerged in 1986 in Europe, rapidly spreading all over the world. Several genotypes of RHDV have been recognised over time, but in 2010, a new virus (RHDV2/RHDVb, GI.2) emerged and progressively replaced the previous RHDV strains, due to the lack of cross-immunity conferred between RHDV and RHDV2. RHDV2 has a high mutation rate, similarly to the other calivirus and recombines with strains of RHDV and non-pathogenic calicivirus (GI.4), ensuring the continuous emergence of new field strains. Although this poses a threat to the already endangered European rabbit species, the available vaccines against RHDV2 and the compliance of biosafety measures seem to be controlling the infection in the rabbit industry Pet rabbits, especially when kept indoor, are considered at lower risk of infections, although RHDV2 and myxoma virus (MYXV) constitute a permanent threat due to transmission via insects. Vaccination against these viruses is therefore recommended every 6 months (myxomatosis) or annually (rabbit haemorrhagic disease). The combined immunization for myxomatosis and RHDV through a commercially available bivalent vaccine with RHDV antigen has been extensively used (Nobivac® Myxo-RHD, MSD, Kenilworth, NJ, USA). This vaccine however does not confer proper protection against the RHDV2, thus the need for a rabbit clinical vaccination protocol update. Here we report a clinical case of hepatitis and alteration of coagulation in a pet rabbit that had been vaccinated with the commercially available bivalent vaccine against RHDV and tested positive to RHDV2 after death. The animal developed a prolonged and atypical disease, compatible with RHD. The virus was identified to be an RHDV2 recombinant strain, with the structural backbone of RHDV2 (GI.2) and the non-structural genes of non-pathogenic-A1 strains (RCV-A1, GI.4). Although confirmation of the etiological agent was only made after death, the clinical signs and analytic data were very suggestive of RHD.

Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus).

In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. A recombinant Myxoma virus (strains MYXV-Tol and ha-MYXV) was rapidly identified, harbouring a 2.8 kbp insertion containing evolved duplicates of M060L, M061L, M064L, and M065L genes from myxoma virus (MYXV) or other Poxviruses. Since 2017, 1616 rabbits and 125 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ha-MYXV strains. A subset of the positive samples (20%) from both species was tested for the insert with MYXV being detected in rabbits and the recombinant MYXV in hares. Recently, three wild rabbits were found dead South of mainland Portugal, showing skin oedema and pulmonary lesions that tested positive for the 2.8 kbp insert. Sequencing analysis showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs and eyelids of rabbits by electron microscopy, and isolation in RK13 cells attested virus infectivity. Despite that the analysis of complete genomes may predict the recombinant MYXV strains' ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, raising serious concerns for the future of the Iberian wild leporids while emphasises the need for the continuous monitoring of MYXV and recombinant MYXV in both species.

Establishing Animal Welfare Rules of Conduct for the Portuguese Veterinary Profession-Results from a Policy Delphi with Vignettes.

Promoting animal welfare is one of the basic tenets of the veterinary profession and, in doing so, veterinarians are expected to abide to the highest legal and professional standards. However, the Portuguese veterinary code of conduct, established in 1994, largely overlooks animal welfare and fails to address issues such as the euthanasia or humane killing of animals. As part of a wider research aiming to revise the Portuguese veterinary code of conduct, a Policy Delphi study was conducted in late 2018, using a pre-validated three-round structure and vignette methodology, to explore the range of opinions and the level of agreement on end-of-life dilemmas and animal welfare rules of conduct of a purposeful sample of forty-one (out of seventy) Portuguese veterinarians. When faced with ethical vignettes involving end-of-life dilemmas, veterinarians were shown to privilege personal moral agency over legal obligations in order to defend the interests of stakeholders, namely of the animals. Most participants agreed that the suggested animal welfare rules of conduct reflected their own views on the subject (88%), in addition to representing a significant improvement in terms of regulatory standards (93%). We expect that this study will support regulation and policy-making by the Portuguese Veterinary Order and by veterinary representative bodies elsewhere.

Opinions of Portuguese Veterinarians on Telemedicine-A Policy Delphi Study.

Telemedicine has only received limited attention by veterinary professional regulatory bodies, particularly in Europe. In Portugal, telemedicine is currently outside what is considered acceptable practice by the regulator, the Portuguese Veterinary Order (Ordem dos Médicos Veterinários). As part of a wider research aimed at gathering evidence for developing a new veterinary Code of Professional Conduct, this study describes the use of the Policy Delphi technique to gather the views and perceptions of a purposeful sample of 41 Portuguese veterinarians regarding telemedicine. Four main issues were addressed using mixed research methods: teleconsultation, teleconsulting, teleadvice, and the regulator's role. Responses highlight participants' perception of both the relevance of medical digital technologies in improving healthcare and their limitations. Overall opinion was that, although restrictions to remote veterinary practice should be reduced, improved guidance and regulation are warranted. Eighty percent of participants considered that limits to the use of veterinary telemedicine should be imposed and two thirds considered that a remote consultation must always be preceded by a face-to-face consultation. While most respondents thought that vet-to-vet teleconsulting using social media (namely Facebook) should not be banned, 83% recognized that it should be regulated by ethical standards. Participants' concerns with telemedicine had mostly to do with reputational risk for the veterinary profession, while overlooking privacy or confidentiality issues. A consultative group should be established to ensure that telemedicine providers comply with professional requirements. It is expected that these results will support policy-making by the Portuguese Veterinary Order and by veterinary regulators at other jurisdictions.

Immunohistochemical Studies of Cytokeratins and Differentiation Markers in Bovine Ocular Squamous Cell Carcinoma.

Bovine Ocular Squamous Cell Carcinoma is considered the most common bovine tumour, causing significant economic losses, mainly by abattoir condemnations. To obtain a better insight into the genesis and neoplastic transformation, 19 samples collected at slaughter from Holstein Friesian cattle and diagnosed as Ocular Squamous Cell Carcinoma were studied. Tumours were histologically classified into three categories: poorly (26.3%), moderately (26.3%), and well differentiated (47.4%). Expression of keratins (MNF116 and LP34) and of cornified envelope precursors (involucrin and profilaggrin) was studied. Expression of MNF116 was observed in all carcinomas. LP34 immunostaining was seen in all but three carcinomas, one from each degree. Involucrin immunoreaction was observed in all but one poorly differentiated carcinoma. Profilaggrin was present in only two moderately differentiated carcinomas, in all but one well differentiated, and in all but one poorly differentiated. MNF116 is a useful marker to confirm the epithelial origin of the tumour and stain most neoplastic cells in these tumours. The expression of involucrin and LP34 demonstrates that, in all tumours, cells have reached the final program of differentiation, regardless of the grade. The expression of profilaggrin could indicate molecular changes during malignant transformation but their expression does not seem to be of diagnostic value.

Pexiganan in Combination with Nisin to Control Polymicrobial Diabetic Foot Infections.

Diabetic foot ulcers (DFUs) are major complications of Diabetes mellitus being responsible for significant morbidity and mortality. DFUs frequently become chronically infected by a complex community of bacteria, including multidrug-resistant and biofilm-producing strains of Staphylococcus aureus and Pseudomonas aeruginosa. Diabetic foot infections (DFI) are often recalcitrant to conventional antibiotics and alternative treatment strategies are urgently needed. Antimicrobial Peptides (AMPs), such as pexiganan and nisin, have been increasingly investigated and reported as effective antimicrobial agents. Here, we evaluated the antibacterial potential of pexiganan and nisin used in combination (dual-AMP) to control the growth of planktonic and biofilm co-cultures of S. aureus and P. aeruginosa clinical strains, co-isolated from a DFU. A DFU collagen three-dimensional (3D) model was used to evaluate the distribution and efficacy of AMPs locally delivered into the model. The concentration of pexiganan required to inhibit and eradicate both planktonic and biofilm-based bacterial cells was substantially reduced when used in combination with nisin. Moreover, incorporation of both AMPs in a guar gum delivery system (dual-AMP biogel) did not affect the dual-AMP antimicrobial activity. Importantly, the application of the dual-AMP biogel resulted in the eradication of the S. aureus strain from the model. In conclusion, data suggest that the local application of the dual-AMPs biogel constitutes a potential complementary therapy for the treatment of infected DFU.

Surgical Excision of an Adrenal Neuroblastoma in a Dog.

A 11-month-old, intact male, Rhodesian Ridgeback was presented to the Veterinary Teaching Hospital with signs of inappetence, lethargy, and abdominal pain for 3 days. A large and well-defined abdominal retroperitoneal mass, related with the left kidney, at the expected location of the adrenal gland, was revealed by radiography, ultrasound, and computed tomography. The mass extended caudally to the iliac artery bifurcation, compressing the aorta, caudal vena cava, and both kidneys. Cytology findings were compatible with a malignant round cell tumor. The most probable diagnosis was neuroblastoma. Following a comprehensive discussion with the owners about a treatment plan, surgical excision was performed. Because a wide excision would compromise major vessels, excision was performed after careful dissection of the aorta and vena cava. The left kidney was removed because the proximal ureter could not be separated from the tumor. The animal recovered successfully. Diagnosis was confirmed by histopathology and immunohistochemistry, but the owners decided not to pursue any further treatment. Clinical signs of abdominal pain recurred within 1 month following surgery. Therefore, the animal was euthanized upon the owners' request. This report describes the diagnosis, surgical treatment, and follow-up of a dog with an abdominal peripheral neuroblastoma.

First description of a fatal equine infection with Halicephalobus gingivalis in Portugal. Relevance for public health.

Halicephalobus gingivalis is a small saprophytic rhabditid nematode, represented only by females with a typical rhabditoid oesophagus and one egg in the uterus, capable of infecting vertebrates. This opportunistic parasite present in the soil, manure and decaying humus, is thought to penetrate through previous injuries to the mouth, eyes and skin of horses and migrate to various organs. The brain is one such organ, where the females lay their eggs, leading to malacia and causing a sudden onset of neurological signs, such as anorexia, ataxia, urinary incontinence, blindness, decreased menace and tonal reflexes, tremors and aggressiveness. The disease is invariably fatal whenever brain lesions are present, and the diagnosis usually achieved only post-mortem. The present work aims to describe the first case of infection by H. gingivalis ever reported in Portugal. An 8-year old warmblood horse presented with an 8-day history of progressive blindness involving the left eye, initially with normal pupillary reflexes, advancing to bilateral blindness and increasing deterioration in clinical condition. After euthanasia, the animal was submitted for necropsy. Organ samples were collected and fixed in 10% neutral buffered formalin for routine histopathology. A large mass was found in the left kidney corresponding to fibrous tissue heavily infiltrated with inflammatory cells and numerous nematodes. In the brain, multiple, bilateral and asymmetrical foci of malacia containing several rhabditoid nematodes, larvae and zygotes, and high numbers of inflammatory cells were found. The nematodes were identified as H. gingivalis. The clinical history, necropsy and histological findings presented constitute a typical case of H. gingivalis infection in a horse, never previously described in Portugal to the authors' best knowledge. Humans can be infected by contact with contaminated manure, which makes this nematode a public health concern, especially for people living and/or working in close proximity to horses.

Establishment of a bioluminescent canine B-cell lymphoma xenograft model for monitoring tumor progression and treatment response in preclinical studies.

Canine diffuse large B-cell lymphoma (DLBCL) is one of the most common cancers in dogs which shares remarkable similarities with its human counterpart, making the dog an excellent model for the investigation of novel therapeutic agents. However, the integration of canine lymphoma in comparative studies has been limited due in part to the lack of suitable xenograft mouse models for preclinical studies. To overcome these limitations, we established and characterized a localized subcutaneous bioluminescent canine DLBCL xenograft mouse model. The canine CLBL-1 cell line stably expressing the luciferase and green fluorescent protein reporters was generated and used to establish the xenograft tumor model. A pilot study was first conducted with three different cell densities (0.1×10(6), 0.5×10(6) and 1×10(6) cells) in SCID mice. All mice presented homogeneous tumor induction within eight days after subcutaneous injection, with a 100% engraftment efficiency and no significant differences were observed among groups. The tumors were highly aggressive and localized at the site of inoculation and reproduced histological features and immunophenotype consistent with canine DLBCL. Importantly, xenograft tumors were detected and quantified by bioluminescent imaging. To assess response to therapy, a therapeutic study with a histone deacetylase inhibitor, panobinostat, was performed. The results demonstrated that panobinostat (20 mg/kg) efficiently inhibited tumor growth and that bioluminescent imaging allowed the monitorization and quantification of tumor response to therapy. In summary, this study provides a bioluminescence canine DLBCL model that offers high engraftment efficiency, preservation of tumor features, and noninvasive monitoring of tumor progression, validating the model as a promising preclinical tool for both veterinary and human medicine.