PDL1 immunohistochemistry in canine neoplasms: Validation of commercial antibodies, standardization of evaluation, and scoring systems.

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.

Caudal peduncle mineralized lesion resembling calcinosis circumscripta in a wels catfish Silurus glanis.

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.

Red mark syndrome (RMS) in farmed rainbow trout: First report of outbreaks in Bosnia and Herzegovina.

Red mark syndrome (RMS) is a non-lethal inflammatory skin disorder spreading in farmed adult rainbow trout (Oncorhynchus mykiss) and reported worldwide. The aetiology is still uncertain, but positive correlation was found between Midichloria-like organism and RMS-affected fish. Here, we describe the first cases of RMS in Bosnia and Herzegovina. The outbreaks under study occurred in two intensive farms during the late winter and spring of 2020. Affected fish showed signs of disease ascribable to RMS, confirmed by pathological and molecular examination.

Novel insights on microbiome dynamics during a gill disease outbreak in farmed rainbow trout (Oncorhynchus mykiss).

The generic term "Gill disease" refers to a wide range of disorders that affect the gills and severely impact salmonid aquaculture systems worldwide. In rainbow trout freshwater aquaculture, various etiological agents causing gill diseases have been described, particularly Flavobacterium and Amoeba species, but research studies suggest a more complex and multifactorial aetiology. Here, a cohort of rainbow trout affected by gill disease is monitored both through standard laboratory techniques and 16S rRNA Next-Generation Sequencing (NGS) analysis during a natural disease outbreak and subsequent antibiotic treatment with Oxytetracycline. NGS results show a clear clustering of the samples between pre- and post-treatment based on the microbial community of the gills. Interestingly, the three main pathogenic bacteria species in rainbow trout (Yersinia ruckeri, Flavobacterium psychrophilum, and Flavobacterium branchiophilum) appear to be weak descriptors of the diversity between pre-treatment and post-treatment groups. In this study, the dynamics of the gill microbiome during the outbreak and subsequent treatment are far more complex than previously reported in the literature, and environmental factors seem of the utmost importance in determining gill disease. These findings present a potential novel perspective on the diagnosis and management of gill diseases, showing the limitations of conventional laboratory methodologies in elucidating the complexity of this disease in rainbow trout. To the authors' knowledge, this work is the first to describe the microbiome of rainbow trout gills during a natural outbreak and subsequent antibiotic treatment. The results of this study suggest that NGS can play a critical role in the analysis and comprehension of gill pathology. Using NGS in future research is highly recommended to gain deeper insights into such diseases correlating gill's microbiome with other possible cofactors and establish strong prevention guidelines.

Double trouble: Co-infection of Angiostrongylus vasorum and Dirofilaria immitis in golden jackal (Canis aureus) in Friuli Venezia Giulia, Italy.

This study reports the first co-infection of Angiostrongylus vasorum and Dirofilaria immitis, two nematodes affecting canid cardiopulmonary systems, in golden jackals (Canis aureus) in Italy. Data on golden jackal carcasses, recovered in Friuli Venezia Giulia (Italy) from 2020 to 2023, were recorded using InfoFaunaFVG wildlife monitoring network. Out of 60 necropsied golden jackals, 24 tested positive for either pathogen, with 3 animals displaying co-infection. Pathological findings included verminous pneumonia, abdominal and thoracic bleeding, and adult individuals in the heart and pulmonary arteries. The recent expansion of the golden jackal in northern Italy may favour the establishment and spread of these nematodes, posing challenges for disease containment and surveillance. Given the increasing prevalence of angiostrongylosis and cardiopulmonary dirofilariosis in Europe, health monitoring of wild canids is essential to reduce their potential impact.

Clinical and Histological Findings of Male Uterus (Uterus Masculinus) in Three Dogs (2014-2018).

Data from three cases of uterus masculinus were retrieved from 2014 to 2018. Two out of three cases presented clinical signs compatible with systemic infection, as observed in bitches with pyometra. Ultrasound examination revealed a tubular fluid-filled structure with a thin irregular wall located cranially to the prostate and in continuity with the cranial part of the gland. In two cases, two other tubular fluid-filled structures were visualized in the caudal part of the abdominal cavity, ventrally to the prostate gland and urinary bladder. After surgical removal of these, histological examination revealed the presence of a uterine structure morphologically similar to the female counterpart. Various types of epithelial cell lining were found, including simple columnar, simple stratified and squamous epithelium associated with glands in the underlying stroma. Immunohistochemistry to anti-Müllerian hormone (AMH) produced a positive result on glands, while multifocal expression was found in the lining epithelium. AMH seems involved in the pathogenesis of uterus masculinus, but its role is not fully understood. Thorough clinical and ultrasonographical examinations, followed by a histological confirmation, are necessary to properly diagnose uterus masculinus in dogs.

Understanding the Pathogenesis of Red Mark Syndrome in Rainbow Trout (Oncorhynchus mykiss) through an Integrated Morphological and Molecular Approach.

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 ß, 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.

Exploring Immunohistochemistry in Fish: Assessment of Antibody Reactivity by Western Immunoblotting.

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.

Porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus alone or associated are frequent intralesional detected viruses in porcine respiratory disease complex cases in Northern Italy.

Methods: This study aimed to examine the pathological impact of Porcine Circovirus type 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) through histological and immunohistochemical analysis of 79 cases of Porcine Respiratory Disease Complex (PRDC) collected from 22 farms in Northern Italy. Lung tissue and several lymphoid organ samples were deployed to associate PCV2-positive stain with Circovirus-associated Diseases (PCVD). Results: The most common lung lesion observed was interstitial pneumonia, alone or combined with bronchopneumonia. By immunohistochemistry, 44 lungs (55.7%) tested positive for PCV2, 34 (43.0%) for PRRSV, 16 (20.3%) for both viruses and in 17 cases (21.5%) neither virus was detected. Twenty-eight out of 44 (63.6%) PCV2-positive cases had lymphoid depletion or granulomatous inflammation in at least one of the lymphoid tissues examined; thus, they were classified as PCV2 Systemic Diseases (PCV2-SD). In the remaining 16 out of 44 cases (36.4%), PCV2-positive lung lesions were associated with hyperplastic or normal lymphoid tissues, which showed PCV2-positive centrofollicular dendritic cells in at least one of the lymphoid tissues examined. Therefore, these cases were classified as PRDC/PCV2-positive. In the PCV2-positive animals, 42.9% of the PCV2-SD cases (12/28) showed immunohistochemistry (IHC) positivity for PRRSV in the lung tissue, while 25.0% of PRDC/PCV2-positive cases (4/16) showed double positivity for PCV2 and PRRSV. Discussion: In light of the caseload presented in this study, characterized by the high proportion of PCV2-SD cases alongside the overall respiratory symptomatology, it is imperative to emphasize the crucial role of a comprehensive sampling protocol. This is critical to avoid underestimating the harm caused by PCV2 in farms, particularly with respect to the systemic form of the disease. PCV2 and PRRSV remain the primary infections associated with PRDC in Italy that can significantly impact farm health and co-infections in the field can worsen the pathology, thus the selection of appropriate preventive measures is critical.

Discovery of an unrecognized nidovirus associated with granulomatous hepatitis in rainbow trout.

Rainbow trout (Oncorhynchus mykiss) is the principal species of inland-farmed fish in the Western hemisphere. Recently, we diagnosed in farmed rainbow trout a disease in which the hallmark is granulomatous-like hepatitis. No biotic agents could be isolated from lesions. Still, unbiased high-throughput sequencing and bioinformatics analyses revealed the presence of a novel piscine nidovirus that we named "Trout Granulomatous Virus" (TGV). TGV genome (28,767 nucleotides long) is predicted to encode non-structural (1a and 1 ab) and structural (S, M, and N) proteins that resemble proteins of other known piscine nidoviruses. High loads of TGV transcripts were detected by quantitative RT-PCR in diseased fish and visualized in hepatic granulomatous sites by fluorescence in situ hybridization. Transmission electron microscopy (TEM) revealed coronavirus-like particles in these lesions. Together, these analyses corroborated the association of TGV with the lesions. The identification and detection of TGV provide means to control TGV spread in trout populations.

Porcine Lawsonia intracellularis Ileitis in Italy and Its Association with Porcine Circovirus Type 2 (PCV2) Infection.

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.

Histological and Ultrastructural Description of Benign Adipocytic Tumors in Farmed Striped Sea Bream (Lythognathus mormyrus).

Cutaneous neoplasms affecting wild striped bream (Lythognathus mormyrus) have been recorded after their introduction in a marine aquaculture farm in the Adriatic Sea. The tumors were evident on 24% of the reared fish, showing single or multiple nodules, with a diameter ranging between 0.5-4.0 cm. Histologically, all the neoplastic lesions were located in the stratum spongiosum of the dermis and were surrounded by a thin capsule of connective tissue. The tumors were predominantly composed of adipocytes grouped and surrounded by a thin net of fibroblasts and collagen fibers. In some lipomas a mixture of adipocytes and uniform spindle cells were also observed. Fibroblasts and collagen fibers, or spindle cells, showing few mitotic figures were mainly observed in other nodules. Three of the tumors showed bands of cells with elongated nuclei. Five neoplasms differed from the classic spindle cell lipoma due to the presence of scattered giant cells. These cells presented acidophilic abundant cytoplasm with multiple hyperchromatic nuclei showing a concentric "floret-like" arrangement. The tumors were further characterized by ultrastructural observations that allowed ruling out the presence of virus-like particles within the lesions. Histological features of the masses lead to the identification of four prevalent patterns of neoplasms: lipoma, fibrolipoma, spindle cell lipoma (SCL), and atypical spindle cell-like lipoma (ASCL). The different neoplasms could arise from the transformation of mesenchymal cells of dermal origin. To the author's knowledge, this is the first report describing key differential histological and ultrastructural features of these neoplasms in striped sea bream.