Morphologic, phenotypic, and genotypic similarities between primary tumors and corresponding 3D cell cultures grown in a repeatable system-preliminary results.

BACKGROUND: Three-dimensional (3D) cell cultures are the new frontier for reproducing the tumor micro-environment in vitro. The aims of the study were (1) to establish primary 3D cell cultures from canine spontaneous neoplasms and (2) to demonstrate the morphological, phenotypic and genotypic similarities between the primary canine neoplasms and the corresponding 3D cultures, through the expression of tumor differentiation markers. RESULTS: Seven primary tumors were collected, including 4 carcinomas and 3 soft tissue sarcomas. 3D cell cultures reproduced the morphological features of the primary tumors and showed an overlapping immunophenotype of the primary epithelial tumors. Immunohistochemistry demonstrated the growth of stromal cells and macrophages admixed with the neoplastic epithelial component, reproducing the tumor microenvironment. Mesenchymal 3D cultures reproduced the immunophenotype of the primary tumor completely in 2 out of 3 examined cases while a discordant expression was documented for a single marker in one case. No single nucleotide variants or small indel were detected in TP53 or MDM2 genes, both in primary tumors and in 3D cell cultures specimens. In one sample, MDM2 amplicons were preferentially increased in number compared to TP53 ones, indicating amplification of MDM2, detectable both in the primary tumor and in the corresponding cell culture specimen. CONCLUSION: Here we demonstrate a good cell morphology, phenotype and genetic profile overlap between primary tumors and the corresponding 3D cultures grown in a repeatable system.

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.

Systemic Candida Infection and Pulmonary Aspergillosis in an Alpaca (Vicugna pacos): A Case Report.

This study reports a peculiar case of systemic candidiasis infection associated with pulmonary aspergillosis in an apparently immunocompetent alpaca. A captive 7-year-old female alpaca exhibited respiratory symptoms, underwent treatment with benzylpenicillin and dexamethasone, and succumbed to the infection 40 days later. During the post-mortem examination, subcutaneous emphysema, widespread pneumonia with multiple suppurative foci, scattered necro-suppurative lesions throughout the renal and hepatic parenchyma were evident. Histopathological analysis of the collected tissues revealed multifocal mild lymphoplasmacytic chronic interstitial nephritis, necro-suppurative pneumonia with the presence of fungal hyphae, multifocal foci of mineralization, and fibrosis in the liver. Fungal cultures confirmed the growth of Aspergillus fumigatus from the lungs, and Candida albicans from the liver, kidney, and heart. The only recognizable risk factor for candidiasis and pulmonary aspergillosis in this case was prior corticosteroid and antibiotic therapy. Nevertheless, it is crucial to consider systemic candidosis and pulmonary aspergillosis as potential differential diagnoses in respiratory infections among camelids. Prolonged treatment with glucocorticoids and antibiotics should be avoided as it could represent a risk factor for the onset of pathologies caused by opportunistic fungi such as Candida spp. and Aspergillus spp.

Diagnostic Approach to Enteric Disorders in Pigs.

The diagnosis of enteric disorders in pigs is extremely challenging, at any age. Outbreaks of enteric disease in pigs are frequently multifactorial and multiple microorganisms can co-exist and interact. Furthermore, several pathogens, such as Clostridium perfrigens type A, Rotavirus and Lawsonia intracellularis, may be present in the gut in the absence of clinical signs. Thus, diagnosis must be based on a differential approach in order to develop a tailored control strategy, considering that treatment and control programs for enteric diseases are pathogen-specific. Correct sampling for laboratory analyses is fundamental for the diagnostic work-up of enteric disease in pigs. For example, histology is the diagnostic gold standard for several enteric disorders, and sampling must ensure the collection of representative and optimal intestinal samples. The aim of this paper is to focus on the diagnostic approach, from sampling to the aetiological diagnosis, of enteric disorders in pigs due to different pathogens during the different phases of production.

Multiorgan metastases with massive bone involvement of a medullary thyroid carcinoma in a dog.

A 10-year-old mixed-breed male dog was referred for a subcutaneous mass on the ventral neck. Based on total-body computed tomography (TBCT), the mass was located in the left thyroid lobe. Further alterations included enlargement of the ipsilateral mandibular and prescapular lymph nodes (LNs). Surgical excision of the mass and enlarged LNs was performed. Histopathology and immunohistochemistry were consistent with a medullary (C-cell) thyroid carcinoma, with no evidence of nodal metastases. Surgery was considered curative, and no medical treatment was provided. Periodic follow-up rechecks were unremarkable. After 18 months, the dog exhibited lethargy, vomiting, anorexia, and hind leg stiffness. TBCT revealed polyostotic osteopathy, and cytology suggested a metastatic endocrine carcinoma. Due to the dog's poor clinical condition and prognosis, the owner elected euthanasia, and a necropsy was performed. Based on gross pathology, histopathology, and immunohistochemistry, multiple metastases of the previous thyroid carcinoma were diagnosed, involving the occipital bone, multiple vertebrae, left sacral wing, fourth right rib, left scapula, left humerus, intrathoracic LNs, lung, spleen, and adrenal glands. This report describes a case of medullary thyroid carcinoma with distant multiorgan metastases and massive bone involvement after a disease-free interval of 18 months.

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.

Loggerhead Sea Turtle as Possible Source of Transmission for Zoonotic Listeriosis in the Marine Environment.

Listeria monocytogenes is an ubiquitous pathogen isolated from different host species including fish, crustaceans, and molluscs, but it is rarely a pathogenic microorganism to marine reptiles. In particular, only two cases of fatal disseminated listeriosis have been described in the loggerhead sea turtle (Caretta caretta). In this study, we describe a lethal case of L. monocytogenes infection in a loggerhead sea turtle. The turtle was found alive, stranded on a beach in North-eastern Italy, but perished soon after being rescued. The autoptic examination revealed that heart, lung, liver, spleen, and urinary bladder were disseminated with multiple, firm, 0.1-0.5 mm sized, nodular, white-green lesions. Microscopically, these lesions corresponded with heterophilic granulomas with Gram+ bacteria within the necrotic center. Furthermore, the Ziehl-Neelsen stain was negative for acid-fast organisms. Colonies isolated from heart and liver were tested through MALDI-TOF for species identification, revealing the presence of L. monocytogenes. Whole Genome Sequencing on L. monocytogenes isolates was performed and the subsequent in silico genotyping revealed the belonging to Sequence Type 6 (ST 6); the virulence profile was evaluated, showing the presence of pathogenicity islands commonly observed in ST 6. Our results further confirm that L. monocytogenes should be posed in differential diagnosis in case of nodular lesions of loggerhead sea turtles; thus, given the zoonotic potential of the microorganism, animals should be treated with particular caution. In addition, wildlife animals can play an active role as carriers of possibly pathogenetic and virulent strains and contribute to the distribution of L. monocytogenes in the environment.

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.

Diffuse Large B-Cell Lymphoma (DLBCL) in an Aged Raccoon (Procyon lotor).

A 15-years-old, captive, female raccoon (Procyon lotor) was necropsied after a one-week history of apathy and self-isolation. Gross changes consisted of the severe enlargement of the mesenteric lymph node; hepatosplenomegaly with multifocal to coalescing, white tan nodules in the spleen and liver,; and pale kidneys. Histologically, neoplastic CD79α-positive lymphocytes effaced the mesenteric lymph node and multifocally infiltrated the spleen, liver, and kidneys, and focally infiltrated the heart. Based on pathological and immunohistochemical findings, as well as the canine-adapted World Health Organization (WHO) diagnostic criteria, a diagnosis of diffuse large B-cell lymphoma (DLBCL) was made.

Airway Remodeling in Feline Lungs.

Airway remodeling encompass structural changes that occur as the result of chronic injury and lead to persistently altered airway structure and function. Although this process is known in several human respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD), airway remodeling is poorly characterized in the feline counterpart. In this study, we describe the spontaneous pulmonary changes in 3 cats paralleling the airway remodeling reported in humans. We observed airway smooth muscle cells (ASMCs) hyperplasia (peribronchial and interstitial), airway subepithelial and interstitial fibrosis, and vascular remodeling by increased number of vessels in the bronchial submucosa. The hyperplastic ASMCs co-expressed α-SMA, vimentin and desmin suggesting that vimentin, which is not normally expressed by ASMCs, may play a role in airway thickening, and remodeling. ASMCs had strong cytoplasmic expression of TGFß-1, which is known to contribute to tissue remodeling in asthma and in various bronchial and interstitial lung diseases, suggesting its involvement in the pathogenesis of ASMCs hyperplasia. Our findings provide histologic evidence of airway remodeling in cats. Further studies on larger caseloads are needed to support our conclusions on the value of this feline condition as an animal model for nonspecific airway remodeling in humans.

Pathological and Tissue-Based Molecular Investigation of Granulomas in Cichlids Reared as Ornamental Fish.

Cichlids include hundreds of species with a high economic value for aquaculture. These fish are subjected to intensive trade and farming that expose them to the risk of infectious diseases. This work focuses on ornamental cichlids held in an aquarium commercial facility presenting emaciation, in order to evaluate the presence of lesions in fish skin and organs. The fish were sampled during routine management activities and subjected to pathological and molecular investigations. The presence of lymphocystis disease virus, typically associated with cutaneous nodular disease, was ruled out. Histologically, they presented granulomas in the spleen, sometimes extending to the other visceral organs. Bacterial heat-shock protein 65 PCR products were detected in tissues associated, in the majority of cases, with granulomas; molecular investigation identified Mycobacterium spp. in two cases and Cutibacterium acnes in seven cases. Immunoreactivity to anti-Mycobacterium and anti-C. acnes antibodies was detected within granulomas. The presence of C. acnes within granuloma is elucidated for the first time in fish; however, similarly to what is found in humans, this bacterium could be harmless in normal conditions, whereas other contributing factors would be required to trigger a granulomatogenous response. Further confirmation by bacterial culture, as well as using large-scale studies in more controlled situations, is needed.

The Role of Pathology in the Diagnosis of Swine Respiratory Disease.

The definition "porcine respiratory disease complex" (PRDC) is used to indicate the current approach for presenting respiratory pathology in modern pig farming. PRDC includes pneumonias with variable pictures, mixed with both aerogenous and hematogenous forms with variable etiology, often multimicrobial, and influenced by environmental and management factors. The notion that many etiological agents of swine respiratory pathology are ubiquitous in the airways is commonly understood; however, their isolation or identification is not always associable with the current pathology. In this complex context, lung lesions registered at slaughterhouse or during necropsy, and supplemented by histological investigations, must be considered as powerful tools for assigning a prominent role to etiologic agents. In recent years, the goal of colocalizing causative agents with the lesions they produce has been frequently applied, and valid examples in routine diagnostics are those that indicate pulmonary involvement during porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) infections.

Dataset of immune responses induced in swine by an inactivated Porcine Circovirus 2b vaccine.

A whole virus, inactivated, Porcine Circovirus 2b (PCV2b) vaccine was submitted to a quantal assay of potency, as explained in detail in our companion paper [1]. To this purpose, twenty, 45-day old piglets, checked for maternally-derived antibody (MDA), were allocated to four groups of 5 animals each; these were vaccinated with 800/266/88/0 nanograms, respectively, of an inactivated PCV2b strain, consisting of two distinct virion populations. Twenty-six days later, all the pigs were challenged intranasally with the homologous PCV2b strain. In the presence of a clear dose-dependent protection in terms of viremia, no such effect was observed in terms of weight gain after challenge. The 800 and 266-ng payloads were associated with neutralizing antibody titers above the MDA levels in oral fluids. Higher levels of viremia in control and 88-ng groups [1] coincided with a higher Natural Killer activity of tracheobronchial lymph node cells from PCV2-infected pigs. The PCV2 ORF2-specific ELISPOT assay for IFN-g- secreting cells showed very few (2-4) ORF2-specific cells/105 peripheral blood mononuclear cells beyond the basal levels under our experimental conditions (non-significant differences among groups). Also, no significant differences were observed in the degree of lymphoid tissue hyperplasia among the different groups.

Protective immunity in swine induced by Porcine Circovirus 2b inactivated vaccines with different antigen payload.

Porcine Circovirus 2 (PCV2) vaccines are poorly standardized in terms of antigen payload and correlates of protection. Therefore, twenty, 45-day old piglets were divided into four groups of 5 animals each and vaccinated with 800 / 266 / 88 / 0 nanograms, respectively, of an inactivated PCV2b strain formulated in the same oil adjuvant. Twenty-six days later, all the pigs were challenged intranasally with the homologous PCV2b strain. No clinical signs were observed in the pigs under study. Viremia was observed after challenge in all the control pigs, as well as in 3 pigs of the 266 and 88-ng groups (one and two, respectively). No pigs of the 800-ng group developed viremia. On the basis of post challenge viremia, the PCV2b vaccine under study had a titer of 11 Protective Doses (PD) 50 %, and 1 PD50 amounted to 74 ng of PCV2b Ag. Neutralizing and ELISA Ab titers showed no obvious correlation with protection in the single animals, even though the 800-ng group developed a significantly higher mean Ab response. All the pigs with a PCV2-specific, IFN-gamma response at 3 weeks after vaccination in whole blood samples were protected against viremia. In lymphoid tissues (mainly tonsils and ileum) the presence of sparse reactive histiocytes and multinucleated giant cells was the only PCV2-associated feature and, by immunohistochemistry, only 3 out of 20 subjects showed a low viral load.

Non-Assembled ORF2 Capsid Protein of Porcine Circovirus 2b Does Not Confer Protective Immunity.

Porcine Circovirus 2 (PCV2) vaccines are based on either inactivated whole virion, or recombinant ORF2 capsid protein assembled into Virus-like Particles (VLPs). No data are available about the immunizing properties of free, non-assembled capsid protein. To investigate this issue, ORF2 of a reference PCV2b strain was expressed in a Baculovirus-based expression system without assembly into VLPs. The free purified protein was formulated into an oil vaccine at three distinct Ag payloads: 10.8/3.6/1.2 micrograms/dose. Each dose was injected intramuscularly into five, 37-day old piglets, carefully matched for maternally-derived antibody. Five control piglets were injected with sterile PBS in oil adjuvant. Twenty-eight days later, all the pigs were challenged intranasally with 105.3 TCID50 of PCV2b strain DV6503. After challenge infection, all the pigs remained in good clinical conditions. The recombinant vaccine did not induce significant antibody and PCV2-specific IFN-γ responses. ELISPOT and lymphocyte proliferation data confirmed poor induction of cell-mediated immunity. In terms of PCV2 viremia, there was no significant difference between vaccinated and control animals. The histological data indicated the absence of a detectable viral load and of PCVAD lesions in both vaccinated and control animals, as well as of histiocytes and multi-nucleated giant cells. We conclude that free, non-assembled ORF2 capsid protein does not induce protective immunity.