Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.

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.

Nodular gill disease in Northeastern Italy: An investigation on the prevalence of the disease and the risks of introduction in rainbow trout farms.

Nodular Gill Disease (NGD) is an emerging pathogenic condition that causes gill damage and mainly affects farmed freshwater fish, rainbow trout (Oncorhynchus mykiss) in particular, and this inevitably generates noticeable economic losses for the industry. The present study aimed to assess the prevalence of NGD in the Autonomous Province of Trento, a highly productive area located in Northeastern Italy, traditionally suited to rainbow trout production, and to identify possible risk factors for the introduction of this disease in trout farms. The necessary data were obtained through a questionnaire and the collection of fish samples. According to the data analysis, 42% of the investigated farms tested positive for NGD. The two possible risk factors identified for its introduction in farms are the presence of other diseases in the same farm (OR = 17.5; 95% CI = 2.7; 111.5) and having farms located 5 km upstream (OR = 24.8; 95% CI = 2.9; 211.1). These results highlight (i) a possible impairment of the immune system caused by other diseases as a predisposing factor to the manifestation of the pathology and (ii) the role of water in spreading pathogens.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction.

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Cutaneous angiomatosis-like presentation in koi carp (Cyprinus carpio koi): Clinical-pathological investigations.

The skin represents an indicator of an animal's health status. Causes of cutaneous diseases in fish most often trace back to biological agents. However, fish skin diseases can also arise from a complex interaction of infectious and non-infectious causes, making it more difficult to identify a specific aetiology. In the period between April and September of the years 2019-2022, four koi carp (Cyprinus carpio koi) from two European countries presented with multifocal, irregularly round, few mm to 1 cm, variably raised cutaneous reddened areas. The fish displayed good general condition. Cutaneous samples, investigated by microbiological and molecular methods and microscopy, did not indicate a primary pathogenic agent. Gross and histological findings of the cutaneous biopsies were consistent with a multifocal/reactive process centred on dermal vessels. The histological features were reminiscent of angiomatosis, a benign proliferative condition affecting the dermal vessels of mammals, including human patients. The clinical-pathological presentation and the dermatologic condition that affected the koi carp are discussed and compared with the veterinary and human literature.

Pearl formation associated with gymnophallid metacercariae in Mytilus galloprovincialis from the Northwestern Adriatic coast: Preliminary observations.

Gymnophallids of the genus Parvatrema are small trematodes infecting waterbirds in their adult stage. Several species of clams and mussels have been found to act as first and second intermediate hosts, in which the trematode larval stages induce the formation of pearls. In this study, a wild population of Mytilus galloprovincialis was sampled along the Northwestern coast of the Adriatic Sea to evaluate the origin and extent of visible pearls. Parasitological investigations, including morphological and molecular analyses, and histopathology were carried out on a representative sample of mussels (n = 158) from June to September 2021. The overall prevalence of infection reached 75.3 %, and the intensity of infection ranged from a few trematodes to thousands per mussel, mostly occurring in the mantle and surrounded by variable numbers of conchiolin layers. Morphological studies allowed classification of the metacercariae as belonging to the genus Parvatrema, and the pairwise comparison of the obtained sequences, encompassing the internal transcribed spacer (ITS) region, revealed a genetic similarity (96.8 %) to Parvatrema duboisi. However, the phylogenetic analysis demonstrated the independent clustering of the obtained ITS sequences compared to other available Parvatrema species. For the relevant commercial impact that pearl formation may have on farmed mussels, ecological and epidemiological aspects of this infection would deserve further investigation in the area.

Cytologic scoring of equine exercise-induced pulmonary hemorrhage: Performance of human experts and a deep learning-based algorithm.

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses, which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of annotators and to validate a deep learning-based algorithm for the THS. Digitized cytological specimens stained for iron were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotator's and algorithmic performance included a ground truth dataset, the mean annotators' THSs, and chemical iron measurements. Results of the study showed that annotators had marked interobserver variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared with the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or ≥ 75) of 75.7%, whereas, the algorithm had an accuracy of 92.3% with no relevant differences in correlation with chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Antioxidant Responses Induced by PFAS Exposure in Freshwater Fish in the Veneto Region.

In recent decades, the interest in PFAS has grown exponentially around the world, due to the toxic effects induced by these chemical compounds in humans, as well as in other animals and plants. However, current knowledge related to the antistress responses that organisms can express when exposed to these substances is still insufficient and, therefore, requires further investigation. The present study focuses on antioxidant responses in Squalius cephalus and Padogobius bonelli, exposed to significant levels of PFAS in an area of the Veneto Region subjected to a recent relevant pollution case. These two ubiquitous freshwater species were sampled in three rivers characterised by different concentrations of PFAS. Several biomarkers of oxidative stress were evaluated, and the results suggest that PFAS chronic exposure induces some physiological responses in the target species, at both cellular and tissue scales. The risk of oxidative stress seems to be kept under control by the antioxidant system by means of gene activation at the mitochondrial level. Moreover, the histological analysis suggests an interesting protective mechanism against damage to the protein component based on lipid vacuolisation.

Severe gastroenteropathy associated with Clostridium perfringens isolation in starving juvenile sturgeons.

In November 2020 a mortality episode (30%) in juvenile Siberian and Russian sturgeons (Acipenser baerii, Brandt, and A. gueldenstaedtii, Brandt & Ratzeburg) and GUBA hybrid sturgeons (A. gueldenstaedtii × A. baerii) occurred in a hatchery in Northern Italy, associated with severe coelomic distension and abnormal reverse surface swimming. The fish were reared in concrete tanks supplied by well water, fed at 0.4% of body weight (b.w.) per day. Thirty sturgeon specimens were collected for necropsy, histological, bacteriological and virological examination. Macroscopic findings included diffuse and severe bloating of gastrointestinal tracts due to foamy contents with thinning and stretching of the gastrointestinal walls. Histological analysis revealed variable degrees of sloughing and necrosis of the intestinal epithelium, and the presence of bacterial aggregates. Anaerobic Gram-positive bacteria were investigated, and Clostridium perfringens was isolated from the gut. Specific PCRs identified the toxinotype A and the ß2 toxin gene. The daily feed administration was increased to 1.5% b.w. and after 5 days, the mortality ceased. A new animal cohort from the same groups was examined after 12 weeks, showing neither gut alterations nor isolation of C. perfringens. The imbalance of intestinal microbiota, presumably caused by underfeeding, favoured C. perfringens overgrowth and severe gas formation. The diet increase possibly restored the normal microbiota.

The use of Corticotropin-releasing factor (CRF) as a marker of agonic stress in abused dogs: Preliminary results.

In Veterinary Forensic Medicine, determination of the degree of animal suffering is an essential element for the prosecution of perpetrators of animal abuse. The purpose of this study is to find a suitable immunohistochemical marker for the assessment of suffering to be routinely used in Veterinary Forensic Pathology, by analyzing the expression of Corticotropin-releasing factor (CRF) in formalin-fixed brains of dogs as a measurement of the agonic stress. CRF, a key peptide element in exogenous and endogenous stressors adaptation, can regulate endocrine-behavioral responses to stress stimulating pituitary ACTH release and consequent adrenal secretion of glucocorticoids. Since CRF acts in days or weeks, this study investigates its role as a potential distinctive marker between sudden death and death associated with a longer agonic period. The study used immunohistochemistry (IHC) to evaluate the CRF expression in the brain of dogs that suffered sudden death, as compared to dogs that died after long-term agonic stress. IHC labelling analysis was performed with machine-learning-based software and the results were statistically evaluated. Our results demonstrate for the first time that CRF is a promising marker of stress in abused patients also in Veterinary Medicine.

Case Report of a Mare Diagnosed with a Metastatic Mammary Carcinoma after the Excision of a Recurrent Intraocular Neuroepithelial Tumor.

A 24-year-old Irish Cob mare was presented with a peripheral iris mass, which was surgically resected and diagnosed as an undifferentiated neuroepithelial tumor. A few months later, a relapse occurred with histological features characterized by a more solid appearance and squamous differentiation. Subsequently, the mare was presented with rapidly spreading multiple subcutaneous masses and, at the onset of neurological signs, was humanely euthanized and subjected to a complete post mortem examination. The necropsy confirmed the presence of numerous widespread masses in the subcutaneous tissue, several internal organs, and mammary gland. Histological and immunohistochemical (IHC) examinations were performed on all masses, allowing the diagnosis of mammary carcinoma with several visceral and subcutaneous metastases. Considering the post mortem findings, the second intraocular mass was submitted to histological and IHC re-evaluation to differentiate it from an intraocular metastasis of the mammary carcinoma. The results of the histological and IHC analyses confirmed the diagnosis of neuroepithelial tumor relapse. This is the first case of a metastatic mammary carcinoma concurrent with a recurrent intraocular neuroepithelial tumor in a mare. This case was a challenge for both clinicians and pathologists involved and highlighted the importance of post mortem and IHC evaluations.

KIT Somatic Mutations and Immunohistochemical Expression in Canine Oral Melanoma.

Canine oral melanoma (COM) is an aggressive neoplasm with a low response to therapies, sharing similarities with human mucosal melanomas. In the latter, significant alterations of the proto-oncogene KIT have been shown, while in COMs only its exon 11 has been adequately investigated. In this study, 14 formalin-fixed, paraffin-embedded COMs were selected considering the following inclusion criteria: unequivocal diagnosis, presence of healthy tissue, and a known amplification status of the gene KIT (seven samples affected and seven non-affected by amplification). The DNA was extracted and KIT target exons 13, 17, and 18 were amplified by PCR and sequenced. Immunohistochemistry (IHC) for KIT and Ki67 was performed, and a quantitative index was calculated for each protein. PCR amplification and sequencing was successful in 97.62% of cases, and no single nucleotide polymorphism (SNP) was detected in any of the exons examined, similarly to exon 11 in other studies. The immunolabeling of KIT was positive in 84.6% of the samples with a mean value of 3.1 cells in positive cases, yet there was no correlation with aberration status. Our findings confirm the hypothesis that SNPs are not a frequent event in KIT activation in COMs, with the pathway activation relying mainly on amplification.

Pathology of Coronavirus Infections: A Review of Lesions in Animals in the One-Health Perspective.

Coronaviruses (CoVs) are worldwide distributed RNA-viruses affecting several species, including humans, and causing a broad spectrum of diseases. Historically, they have not been considered a severe threat to public health until two outbreaks of COVs-related atypical human pneumonia derived from animal hosts appeared in 2002 and in 2012. The concern related to CoVs infection dramatically rose after the COVID-19 global outbreak, for which a spill-over from wild animals is also most likely. In light of this CoV zoonotic risk, and their ability to adapt to new species and dramatically spread, it appears pivotal to understand the pathophysiology and mechanisms of tissue injury of known CoVs within the "One-Health" concept. This review specifically describes all CoVs diseases in animals, schematically representing the tissue damage and summarizing the major lesions in an attempt to compare and put them in relation, also with human infections. Some information on pathogenesis and genetic diversity is also included. Investigating the lesions and distribution of CoVs can be crucial to understand and monitor the evolution of these viruses as well as of other pathogens and to further deepen the pathogenesis and transmission of this disease to help public health preventive measures and therapies.

Cyclin D1 immunohistochemical expression and somatic mutations in canine oral melanoma.

Canine oral melanoma (COM) is the most frequent tumour with oral localization in dogs. Copy number gains and amplifications of CCND1, a gene coding for Cyclin D1, are the most frequent chromosomal aberrations described in human non-UV induced melanomas. Twenty-eight cases of COM were retrieved from paraffin-blocks archives. A total of 4 µm thick sections were immunostained with an antibody against human Cyclin D1 and Ki-67. Cyclin D1 and Ki-67 expressions were scored through two counting methods. DNA was extracted from 20 µm thick sections of formalin-fixed paraffin-embedded blocks. Pathological and surrounding healthy tissue was extracted independently. Cyclin D1 immunolabelling was detected in 69% (18/26) while Ki-67 was present in 88.5% (23/26) of cases. Statistical analysis revealed correlation between two counting methods for Cyclin D1 (r = 0.54; P = .004) and Ki-67 (r = 0.56; P = .003). The correlation found between Ki-67 and Cyclin D1 indexes in 16/26 cases labelled by both antibodies (r = 0.7947; P = .0002) suggests a possible use of Cyclin D1 index as prognostic marker. Polymerase chain reaction analysis on CCND1 coding sequence revealed the presence of nine somatic mutations in seven samples producing synonymous, missense and stop codons. Since none of the single-nucleotide polymorphisms was found to be recurrent, it is suggested that overexpression of Cyclin D1 may be the consequence of alterations of CCND1 upstream regions or other genetic aberrations not detectable with the methodology used in this study. Future studies are needed to verify the potential use of Cyclin D1 index as prognostic indicator and to highlight the molecular events responsible for Cyclin D1 overexpression in COMs.

Dynamics of blood circulation during diving in the bottlenose dolphin (Tursiops truncatus): the role of the retia mirabilia.

The retia mirabilia are vascular nets composed of small vessels dispersed among numerous veins, allowing blood storage, regulation of flow and pressure damping effects. Here, we investigated their potential role during the diving phase of the bottlenose dolphin (Tursiops truncatus). To this effect, the whole vertebral retia mirabilia of a series of dolphins were removed during post-mortem analysis and examined to assess vessel diameters, and estimate vascular volume and flow rate. We formulated a new hemodynamic model to help clarify vascular dynamics throughout the diving phase, based on the total blood volume of a bottlenose dolphin, and using data available about the perfusion of the main organs and body systems. We computed the minimum blood perfusion necessary to the internal organs, and the stroke volume and cardiac output during the surface state. We then simulated breath-holding conditions and perfusion of the internal organs under the diving-induced bradycardia and reduction of stroke volume and cardiac output, using 10 beats min-1 as the limit for the heart rate for an extended dive of over 3 min. Within these simulated conditions, the retia mirabilia play a vital role as reservoirs of oxygenated blood that permit functional performances and survival of the heart and brain. Our theoretical model, based on the actual blood capacity of the retia mirabilia and available data on organ perfusion, considers the dynamic trend of vasoconstriction during the diving phase and may represent a baseline for future studies on the diving physiology of dolphins and especially for the blood supply to their brain.

Array Comparative Genomic Hybridization Analysis Reveals Significantly Enriched Pathways in Canine Oral Melanoma.

Human Mucosal Melanoma (hMM) is an aggressive neoplasm of neuroectodermal origin with distinctive features from the more common cutaneous form of malignant melanoma (cMM). At the molecular level, hMMs are characterized by large chromosomal aberrations rather than single-nucleotide mutations, typically observed in cMM. Given the scarcity of available cases, there have been many attempts to establish a reliable animal model. In pet dogs, Canine Oral Melanoma (COM) is the most common malignant tumor of the oral cavity, sharing clinical and histological aspects with hMM. To improve the knowledge about COM's genomic DNA alterations, in the present work, formalin-fixed, paraffin-embedded (FFPE) samples of COM from different European archives were collected to set up an array Comparative Genomic Hybridization (aCGH) analysis to estimate recurrent Copy Number Aberrations (CNAs). DNA was extracted in parallel from tumor and healthy fractions and 19 specimens were successfully submitted to labeling and competitive hybridization. Data were statistically analyzed through GISTIC2.0 and a pathway-enrichment analysis was performed with ClueGO. Recurrent gained regions were detected, affecting chromosomes CFA 10, 13 and 30, while lost regions involved chromosomes CFA 10, 11, 22, and 30. In particular, CFA 13 showed a whole-chromosome gain in 37% of the samples, while CFA 22 showed a whole-chromosome loss in 25%. A distinctive sigmoidal trend was observed in CFA 10 and 30 in 25 and 30% of the samples, respectively. Comparative analysis revealed that COM and hMM share common chromosomal changes in 32 regions. MAPK- and PI3K-related genes were the most frequently involved, while pathway analysis revealed statistically significant perturbation of cancer-related biological processes such as immune response, drug metabolism, melanocytes homeostasis, and neo-angiogenesis. The latter is a new evidence of a significant involvement of neovascularization-related pathways in COMs and can provide the rationale for future application in anti-cancer targeted therapies.