Massimo Orioles

Histological classification of mammary proliferative lesions in dogs and cats is essential for prognostic and predictive studies, as well as for comparative oncology; however, standardization of diagnoses is highly affected by substantial interobserver variability. In this study, a structured diagnostic algorithm based on the Davis–Thompson Foundation classification was assessed for the ability to improve interobserver agreement in the morphological diagnoses of mammary gland lesions in dogs and cats. Consensus diagnoses of 114 selected mammary lesions (91 canine, 23 feline) made by 16 veterinary pathologists with mammary tumor expertise were used as the gold standard, and 79 veterinary pathologists from 21 countries evaluated digitized whole slides in 2 rounds: a first reading without the algorithm and a second reading with the algorithm. Interobserver concordance was measured using unweighted kappa statistics for lesion category (hyperplastic/dysplastic, benign, malignant) and histological subtype. Across all lesions, agreement was higher for lesion category than for histological subtype. A slight improvement in concordance was observed when applying the algorithm, both for category (k = 0.72, before, and k = 0.73, after) and histotype classifications (k = 0.40, before to k = 0.42, after). Also, by using the algorithm, 73/79 raters (92%) modified at least 1 diagnosis and, among these, approximately two-thirds reached agreement with the gold standard in at least 1 case. Considering histotypes, malignant tumors with mixed epithelial and myoepithelial components showed persistently low agreement. Overall, the algorithm provided a reproducible diagnostic framework that may support training and improve standardization of canine and feline mammary tumor classification.

Columnaris‐causing bacteria (CCB) represent a group of four Flavobacterium species, previously classified under Flavobacterium columnaris , causing a threatening condition in salmonid farming characterised by cutaneous and gill lesions, commonly referred to as ‘saddleback disease’. A peracute skin disease outbreak with high mortality in rainbow trout ( Oncorhynchus mykiss ) farms in Northern Italy was investigated. The disease presented with skin discoloration and scale lifting without internal organ abnormalities, leading to a weekly cumulative mortality of up to 80%. The disease was successfully managed with Oxytetracycline treatment, with no relapses observed. Conventional investigation methods produced inconsistent results, prompting additional analyses. Metagenomic sequencing of the 16S rRNA identified Flavobacterium species differing from the classical CCB based on the alignment of the V3 and V4 regions, with best matches to Flavobacterium bernardetii , Flavobacterium aquicola , and Flavobacterium hiemivividum . Histopathology and SEM confirmed epidermal necrosis and bacterial infiltration in the dermis, with filamentous bacteria resembling Flavobacterium morphology yet differing from classical CCB lesions. These findings point to a previously undescribed Flavobacterium ‐related skin disease with significant economic implications, supporting the value of metagenomic in investigating microbial dynamics in aquaculture diseases, especially in sites exposed to external environments. Further research is required to clarify the pathogenic mechanisms and guide effective management strategies for future outbreaks.

Sturgeon species are well-suited for aquaculture because of their favourable characteristics, including robustness, suitability for farming in facilities unsuitable for other fish species, and adaptability to diverse farming conditions. The Russian sturgeon (Acipenser gueldenstaedtii, Brandt and Ratzeburg 1833) is one of the most prominent farmed species; however, like other aquaculture species, it is susceptible to significant losses from bacterial and viral diseases. Beyond infectious causes, there are few reports documenting conditions that produce cutaneous masses in Russian sturgeons. This study presents a multidisciplinary investigation of six farmed Russian sturgeons exhibiting discrete, multiple cutaneous masses. Bacteriological analysis of tissue samples revealed the presence of Morganella morganii and Aeromonas veronii biovar sobria, identified as opportunistic bacteria. Virological assays targeting the principal viruses affecting sturgeon, Acipenser iridovirus and Acipenser herpesvirus, yielded negative results. Ultrastructural analysis with direct negative staining revealed no evidence of biological agents. Histologically, the dermal masses were well-demarcated, expansile, and moderately cellular, consisting of spindle-to-stellate neoplastic cells that were multifocally periodic acid–Schiff-positive and embedded in abundant alcianophilic ground substance. Immunohistochemistry with the S-100 antibody confirmed cytoplasmic staining of the neoplastic cells. A final diagnosis of cutaneous nerve sheath tumour with myxoid differentiation was made, replicating findings from a similar tumour in rainbow trout. To the best of our knowledge, this represents the first description of multiple cutaneous nerve sheath tumours in sturgeon species. The potential factors contributing to the development of this neoplastic condition are discussed.

Immuno-oncology research has brought to light the paradoxical role of immune cells in the induction and elimination of cancer. Programmed cell death protein 1 (PD1), expressed by tumor-infiltrating lymphocytes, and programmed cell death ligand 1 (PDL1), expressed by tumor cells, are immune checkpoint proteins that regulate the antitumor adaptive immune response. This study aimed to validate commercially available PDL1 antibodies in canine tissue and then, applying standardized methods and scoring systems used in human pathology, evaluate PDL1 immunopositivity in different types of canine tumors. To demonstrate cross-reactivity, a monoclonal antibody (22C3) and polyclonal antibody (cod. A1645) were tested by western blot. Cross-reactivity in canine tissue cell extracts was observed for both antibodies; however, the polyclonal antibody (cod. A1645) demonstrated higher signal specificity. Canine tumor histotypes were selected based on the human counterparts known to express PDL1. Immunohistochemistry was performed on 168 tumors with the polyclonal anti-PDL1 antibody. Only membranous labeling was considered positive. PDL1 labeling was detected both in neoplastic and infiltrating immune cells. The following tumors were immunopositive: melanomas (17 of 17; 100%), renal cell carcinomas (4 of 17; 24%), squamous cell carcinomas (3 of 17; 18%), lymphomas (2 of 14; 14%), urothelial carcinomas (2 of 18; 11%), pulmonary carcinomas (2 of 20; 10%), and mammary carcinomas (1 of 31; 3%). Gastric (0 of 10; 0%) and intestinal carcinomas (0 of 24; 0%) were negative. The findings of this study suggest that PDL1 is expressed in some canine tumors, with high prevalence in melanomas.

This report presents an intriguing case of a mineralized lesion resembling calcinosis circumscripta observed in the caudal peduncle of a wels catfish, housed in a public aquarium in northern Italy. The investigation encompasses a comprehensive analysis of various aspects, including clinical presentation, diagnostic imaging, as well as gross and microscopic pathology, and immunohistochemistry. Histopathology, in particular, highlighted a severe focally extensive granulomatous reaction, confirmed by dense histiocytic inflammatory infiltrates and the massive presence of multinucleated foreign body type giant cells localized around lakes and aggregates of mineralized material. Moreover, the usefulness and limitations of immunohistochemistry and special stains in characterizing fish tissues and cell types are highlighted. Although reported in elasmobranchs and sturgeons, to our knowledge, this is the first description of calcinosis circumscripta in teleost fish and it offers valuable insights into the understanding of similar pathologies in aquatic organisms.

In recent years, research on fish has seen remarkable advancements, especially in aquaculture, ornamental fish industry, and biomedical studies. Immunohistochemistry has become crucial in fish research, aiding in physiological and pathological investigations. However, the use of antibodies originally developed for mammals has raised concerns about their cross-reactivity and specificity in fish. This study systematically evaluated the reactivity of commonly used antibodies for diagnostic purposes, especially in fish pathology, including pan-cytokeratin, vimentin, S-100, glial fibrillary acidic protein, and desmin in the tissue of Sparus aurata, Dicentrarchus labrax, Oncorhynchus mykiss, and Carassius auratus. Western immunoblotting was employed to assess antibody specificity. The results revealed that the pan-cytokeratin and glial fibrillary acidic protein antibodies cross-react with all tested fish species, while S-100 demonstrated specific staining in sea bream, goldfish, and rainbow trout tissues. Conversely, vimentin and desmin antibodies displayed no reactivity. In conclusion, the anti-cytokeratin clone AE1/AE3 and the polyclonal rabbit anti-glial fibrillary acidic protein antibody, which are extensively used in mammals, were validated for fish immunohistochemical studies. Regrettably, D33 anti-desmin and V9 anti-vimentin clones are unsuitable for immunohistochemistry in the tested fish. These findings underscore the need for species-specific antibodies and proper validation for accurate immunohistochemistry analyses in fish research.

The Department of Agricultural, Food, Environmental and Animal Sciences (DI4A) at the University of Udine, in collaboration with Friuli Venezia Giulia regional authorities, within northeastern Italy, set up a wildlife monitoring and surveillance regional network, named InfoFaunaFVG. Here we describe the development and application of this data repository system based on a novel progressive web application, and report the data gathered in the first two and a half years of its use. InfoFaunaFVG is made of a Web Database and an integrated WebGIS system. In particular, the following open source softwares are used: Apache HTTP Server, Oracle MySQL, Symfony, Apache Tomcat, GeoServer, OpenLayers. The web app can be accessed from any web browser or by installing the progressive web application in the desktop or mobile devices. In short, operating from November 2019, InfoFaunaFVG currently (April 2022) contains a total of 40,175 records, from 300 different users, from 16 institutions. Among all species recorded, mammals were 40% (16,018) of the total, whereas avian species represented 59% (23,741), and others (reptiles and amphibians) 1% (416), respectively. Two hundred twenty-six different species (175 avian and 51 mammals) were recorded. Details about causes of death and live animal rescue were reported. To date, InfoFaunaFVG has proven to be a successful wildlife data repository system providing high quality consistent, accurate and traceable data. These had a considerable impact on regional wildlife governance. In the authors’ knowledge, InfoFaunaFVG is the first example described in literature of such a progressive web application, coordinated on an institutional level, and not based on voluntary-citizen observations. InfoFaunaFVG has the potential to become the largest wildlife monitoring and surveillance data repository system on a national level.

The objective of this study was to employ a diagnostic algorithm, which involves detecting positive farms by stool PCR followed by PCR and histology/immunohistochemistry on ileum samples, for diagnosing Lawsonia intracellularis proliferative enteritis in Northern Italy. The primary aim was to examine the relationship between the gold standard of L. intracellularis diagnostics, namely histology and immunohistochemistry, and PCR in acute and chronic cases of L. intracellularis enteritides. An additional goal was to investigate the coinfection of L. intracellularis with porcine circovirus type 2 (PCV2). Twenty-eight ileum samples, including four from acute cases and 24 from chronic cases, were collected. PCR yielded positive results in 19 cases (four acute and 15 chronic cases). In comparison, immunohistochemistry was positive in 16 cases (four acute and 12 chronic cases), with an observed agreement of 89%. The findings suggest that performing the two tests in series can increase the specificity of the causal diagnosis. PCR may be used as a screening tool to identify the presence of the microorganism, and only positive cases will be examined by histology and immunohistochemistry to confirm the causative role of L. intracellularis. Co-infection with PCV2 was demonstrate in two out of four acute cases and in two out of 24 chronic cases, providing further evidence to support the hypothesis that when the infection starts with ubiquitous pathogens such as L. intracellularis, it may boost the possibility of PCV2 replication, especially in acute cases. As a result, this may trigger a transition from subclinical to clinical forms of PCV2 disease.

Red mark syndrome (RMS) is a widespread skin disorder of rainbow trout in freshwater aquaculture, believed to be caused by a Midichloria-like organism (MLO). Here, we aimed to study the pathologic mechanisms at the origin of RMS by analyzing field samples from a recent outbreak through gene expression, MLO PCR, quantitative PCR, and a histopathological scoring system proposed for RMS lesions. Statistical analyses included a One-Way Analysis of Variance (ANOVA) with a Dunnett’s multiple comparisons test to assess differences among gene expression groups and a nonparametric Spearman correlation between various categories of skin lesions and PCR results. In short, the results confirmed the presence of a high quantity of 16S gene copy numbers of Midichloria-like organisms in diseased skin tissues. However, the number of Midichloria-like organisms detected was not correlated to the degree of severity of skin disease. Midichloria-like organism DNA was found in the spleen and head kidney. The spleen showed pathologic changes mainly of hyperplastic type, reflecting its direct involvement during infection. The most severe skin lesions were characterized by a high level of inflammatory cytokines sustaining and modulating the severe inflammatory process. IL-1 β, IL-6, IL-10, MHC-II, and TCR were upregulated in severe skin lesions, while IL-10 was highly expressed in moderate to severe ones. In the moderate form, the response was driven to produce immunoglobulins, which appeared crucial in controlling the skin disease’s severity. Altogether our results illustrated a complex immune interaction between the host and Midichloria-like organism.