Canine and feline in situ mammary carcinoma: A comparative review.

Carcinoma in situ of the breast is a well-known entity in humans. In veterinary medicine, particularly in canine and feline mammary literature, there is no agreement whether the term in situ should be used to indicate a specific carcinoma histotype or the noninvasive status of a carcinoma of any histotype. Moreover, in the most recent histologic classification of mammary tumors published by the Davis-Thompson Foundation, it is suggested to abandon the term carcinoma in situ given the lack of standardized criteria defining this entity, replacing it with epitheliosis or ductal/lobular hyperplasia with severe atypia. This publication presents a critical review of the term in situ in human and veterinary medicine considering the evolution of the term over the years and its heterogeneous use by different authors, including variations in immunohistochemical markers for classification. This review aims to point out the lack of uniformity in the nomenclature and classification issues in veterinary medicine regarding the use of the term in situ, laying the ground for a process of standardization in future publications.

Mediterranean Fin Whales (Balaenoptera physalus) Threatened by Dolphin MorbilliVirus.

During 2011-2013, dolphin morbillivirus was molecularly identified in 4 stranded fin whales from the Mediterranean Sea. Nucleoprotein, phosphoprotein, and hemagglutinin gene sequences of the identified strain were highly homologous with those of a morbillivirus that caused a 2006-2007 epidemic in the Mediterranean. Dolphin morbillivirus represents a serious threat for fin whales.

Cetacean strandings in Italy: an unusual mortality event along the Tyrrhenian Sea coast in 2013.

An unusual mortality event involving cetaceans, mainly striped dolphins Stenella coeruleoalba (Meyen, 1833), occurred along the Tyrrhenian Sea coast of Italy during the first 3 mo of 2013. Based on post-mortem analyses carried out according to body condition on 66 dolphins (54% of stranded animals), several hypotheses to explain the causes of this mortality event were proposed. Although no definitive conclusions can be drawn, dolphin morbillivirus was deemed the most likely cause, although other infectious agents (including Photobacterium damselae damselae and herpesvirus) or environmental factors may also have contributed to this recent mortality event.

Histiocytic Sarcoma in a Captive Hybrid Orangutan (Pongo sp.): Morphological and Immunohistochemical Features.

Histiocytic sarcoma (HS), an infrequent highly aggressive hematopoietic tumor, has been observed in diverse animal species, with isolated occurrences in non-human primates. This study describes the first case of disseminated HS in a 45-year-old female hybrid captive orangutan. The clinical profile mirrored symptoms seen in human HS cases, encompassing anorexia and ascites. Detailed histopathological examination demonstrated characteristic features of this tumor and immunohistochemistry, using markers such as Iba-1 and HLA-DR, confirmed the diagnosis. Significantly, the absence of CD163 and CD204 expression challenges their diagnostic utility in non-human primates. This investigation enhances our understanding of HS diagnosis in non-human primates, underscoring the necessity for standardized markers and diagnostic protocols.

Are we even close? Five years marine litter ingestion monitoring in loggerhead turtles along Italian coast reveals how far we are from the Good Environmental Status.

The loggerhead sea turtle Caretta caretta has been chosen as bioindicator to monitor the amount of litter ingested by marine animals within the European Marine Strategy Framework Directive and the Barcelona Regional Sea Convention. European Member States and Contracting Parties are committed to achieve the Good Environmental Status (GES), which is reached when the quantity of ingested litter does not adversely affect the health of the species concerned. Although the monitoring strategy has been outlined for more than a decade, to date no threshold values have been adopted to verify GES achievement. After five years of extensive monitoring along the Italian coasts, this study evaluates the suitability of five different GES scenarios and proposes a new threshold value (i.e., "there should be less than 33% of sea turtles having more than 0.05 g of ingested plastic in the GI") for its implementation in the European seas and the Mediterranean basin.

First Record of Cetacean Killed in an Artisanal Fish Aggregating Device in the Mediterranean Sea.

Fish Aggregating Devices (FADs) are anchored floating structures often made with cheap scrapped materials and used to aggregate pelagic fish species under their artificial shadows. Globally, the dangerous impact of FADs is well known. They pose a severe threat as a source of bycatch, as a danger to navigation, and with their high potential to become marine litter. Unintended entanglement and consequent mortality in FADs of vulnerable (e.g., sharks, sea turtles, and cetaceans) and commercial species is a serious concern for several international inter-governmental bodies (e.g., EU, GFCM, and IWC). This work describes the first case of a cetacean, a striped dolphin (Stenella coeruleoalba), entangled in a FAD in the Mediterranean Sea. A young male of striped dolphins was found dead along the coast of Lazio (central Tyrrhenian Sea) with its peduncle entangled in typical debris from illegal/artisanal FADs (i.e., a nylon rope, teared gardening plastic sheets, bush branches, and scrapped empty plastic bottles). Although this is the first confirmed case of a cetacean entangled in a FAD in Mediterranean waters, given the extent of the deployment of anchored FADs, the scale of this type of interaction with protected species might be seriously underestimated. Therefore, actions and monitoring need to be implemented urgently to effectively protect and conserve marine biodiversity.

Preliminary Assessment of Tumor-Associated Tissue Eosinophilia (TATE) in Canine Mast Cell Tumors: Prevalence and Prognostic Relevance and Its Association with Neoangiogenesis.

Mast cell tumor (MCT) is the most common malignant skin tumor in dogs. In order to gain more information on the prognostic markers in MCT, the role of the eosinophil granulocytes infiltrates was investigated and assessed by the evaluation of tumor-associated tissue eosinophilia (TATE) in 87 canine cutaneous MCTs. In human medicine, high TATE are often described in highly angiogenic tumors: we therefore assessed the vascular endothelial growth factor (VEGF) expression in neoplastic mast cells. TATE and VEGF expression were compared between themselves, with histological grading, immunohistochemical expression of KIT and Ki-67, and with the recurrence. We found a statistically significant correlation between TATE and Patnaik grading (p = 0.041), Kiupel grading (p = 0.022), immunohistochemical KIT expression (p = 0.015), and tumor recurrence (p = 0.000). No associations were observed with Ki-67 and VEGF expression. This is the first evaluation of TATE and its prognostic value in canine MCTs in veterinary oncology. This study suggest that this investigation could be an important source of information for this tumor and for other neoplasms.

Dietary Preferences of Loggerhead Sea Turtles (Caretta caretta) in Two Mediterranean Feeding Grounds: Does Prey Selection Change with Habitat Use throughout Their Life Cycle?

According to their life stage, the loggerhead sea turtle (Caretta caretta) is found in a wide range of habitats, from neritic to more oceanic areas. Their feeding habits are expected to change as they develop, along with habitat use. Juvenile sea turtles are hypothesized to feed on pelagic species in oceanic areas, shifting to more benthic prey during the subadult and adult stages. We analyzed the gastrointestinal content from 150 loggerhead sea turtles stranded and/or bycaught along the Adriatic coast of the Abruzzo and Molise regions (n = 89) and the Tyrrhenian coast of the Lazio and Campania regions (n = 61) from 2018 to 2021. Food items were identified to the lowest taxonomic level, and the frequency of occurrence was calculated for each taxon and most recurrent species to assess changes in prey selection during the development. The marine litter was categorized, and the frequency of occurrence was calculated for the ingestion of litter. The most recurrent taxonomic prey group recorded in the Adriatic sample was Arthropoda (94%), followed by Mollusca (63%) and Chordata (34%). In the Tyrrhenian sample, loggerhead sea turtles fed mostly on Mollusca (84%), Arthropoda (38%), and Chordata (26%). Surprisingly, the Adriatic-Tyrrhenian sample groups showed similar feeding behavior between juveniles, subadults, and adults. A similar correlation has been observed concerning the ingestion of litter. Moreover, this study confirms the opportunistic feeding behavior of loggerhead sea turtles and their high adaptability.

Articular Cartilage Regeneration by Hyaline Chondrocytes: A Case Study in Equine Model and Outcomes.

Cartilage injury defects in animals and humans result in the development of osteoarthritis and the progression of joint deterioration. Cell isolation from equine hyaline cartilage and evaluation of their ability to repair equine joint cartilage injuries establish a new experimental protocol for an alternative approach to osteochondral lesions treatment. Chondrocytes (CCs), isolated from the autologous cartilage of the trachea, grown in the laboratory, and subsequently arthroscopically implanted into the lesion site, were used to regenerate a chondral lesion of the carpal joint of a horse. Biopsies of the treated cartilage taken after 8 and 13 months of implantation for histological and immunohistochemical evaluation of the tissue demonstrate that the tissue was still immature 8 months after implantation, while at 13 months it was organized almost similarly to the original hyaline cartilage. Finally, a tissue perfectly comparable to native articular cartilage was detected 24 months after implantation. Histological investigations demonstrate the progressive maturation of the hyaline cartilage at the site of the lesion. The hyaline type of tracheal cartilage, used as a source of CCs, allows for the repair of joint cartilage injuries through the neosynthesis of hyaline cartilage that presents characteristics identical to the articular cartilage of the original tissue.

Genomics insights into a Mycobacterium pinnipedii isolate causing tuberculosis in a captive South American sea lion (Otaria flavescens) from Italy.

Tuberculosis (TB) affects humans and other animals, and it is caused by bacteria within the Mycobacterium tuberculosis complex (MTBC). In this study, we report the characterisation of Mycobacterium pinnipedii that caused a TB case in a sea lion (Otaria flavescens) kept in an Italian zoo. The animal died due to severe, progressive disorders involving the respiratory and gastro-enteric systems and the skin. At necropsy, typical gross lesions referable to a TB generalised form were found. In particular, nodular granulomatous lesions were detected in the lungs and several lymph nodes, and colonies referable to Mycobacterium spp. were isolated from lung, mesenteric, and mediastinal lymph nodes. The isolate was identified by PCR as a MTBC, had a spoligotype SB 1480 ("seal lineage"), and was characterised and characterised by whole-genome sequencing analysis confirming that the MTBC involved was M. pinnipedii. The analysis of the resistome and virulome indicated the presence of macrolide and aminoglycoside resistance genes intrinsic in M. tuberculosis [erm-37 and aac(2')-Ic] and confirmed the presence of the region of difference 1 (RD1), harbouring the esxA and esxB virulence genes, differently from its closest taxon, M. microti. As for other MTCB members, M. pinnipedii infection can spill over into non-pinniped mammalian species; therefore, zoological gardens, veterinary practitioners, and public health officers should be aware of the hazard posed by tuberculosis from marine mammals. Since the isolate under study, as well as all available genomes of M. pinnipedii investigated in this study retains almost all the M. tuberculosis virulence genes, it could indeed cause infection, lesions, and disease in other animal species, including humans.

Brucella ceti Infection in Striped Dolphins from Italian Seas: Associated Lesions and Epidemiological Data.

Brucella ceti infections have been increasingly reported in cetaceans. In this study, we analyzed all cases of B. ceti infection detected in striped dolphins stranded along the Italian coastline between 2012 and 2021 (N = 24). We focused on the pathogenic role of B. ceti through detailed pathological studies, and ad hoc microbiological, biomolecular, and serological investigations, coupled with a comparative genomic analysis of the strains. Neurobrucellosis was observed in 20 animals. The primary histopathologic features included non-suppurative meningoencephalitis (N = 9), meningitis (N = 6), and meningoencephalomyelitis (N = 5), which was also associated with typical lesions in other tissues (N = 8). Co-infections were detected in more than half of the cases, mostly involving Cetacean Morbillivirus (CeMV). The 24 B. ceti isolates were assigned primarily to sequence type 26 (ST26) (N = 21) and, in a few cases, ST49 (N = 3). The multilocus sequence typing (cgMLST) based on whole genome sequencing (WGS) data showed that strains from Italy clustered into four genetically distinct clades. Plotting these clades onto a geographic map suggests a link between their phylogeny and the topographical distribution. These results support the role of B. ceti as a primary neurotropic pathogen for striped dolphins and highlight the utility of WGS data in understanding the evolution of this emerging pathogen.

Reproducibility and Feasibility of Classification and National Guidelines for Histological Diagnosis of Canine Mammary Gland Tumours: A Multi-Institutional Ring Study.

Histological diagnosis of Canine Mammary Tumours (CMTs) provides the basis for proper treatment and follow-up. Nowadays, its accuracy is poorly understood and variable interpretation of histological criteria leads to a lack of standardisation and impossibility to compare studies. This study aimed to quantify the reproducibility of histological diagnosis and grading in CMTs. A blinded ring test on 36 CMTs was performed by 15 veterinary pathologists with different levels of education, after discussion of critical points on the Davis-Thompson Foundation Classification and providing consensus guidelines. Kappa statistics were used to compare the interobserver variability. The overall concordance rate of diagnostic interpretations of WP on identification of hyperplasia-dysplasia/benign/malignant lesions showed a substantial agreement (average k ranging from 0.66 to 0.82, with a k-combined of 0.76). Instead, outcomes on ICD-O-3.2 morphological code /diagnosis of histotype had only a moderate agreement (average k ranging from 0.44 and 0.64, with a k-combined of 0.54). The results demonstrated that standardised classification and consensus guidelines can produce moderate to substantial agreement; however, further efforts are needed to increase this agreement in distinguishing benign versus malignant lesions and in histological grading.

Polymer composition assessment suggests prevalence of single-use plastics among items ingested by loggerhead sea turtles in the western mediterranean sub-region.

The ingestion of plastic is becoming a major concern for various species and particularly for marine turtles across the globe. The loggerhead sea turtle (Caretta caretta) was recently chosen by the European Commission as a bio-indicator for plastic pollution within the Mediterranean basin. We further investigated which items this key species is more prone to ingest, following the standardised Marine Strategy Framework Directive protocols. Moreover, we integrated to this protocol the Fourier Transform Infrared Spectroscopy, which allowed us to determine the polymer type of each item. We analysed samples from 226 sea turtles from 2008 to 2017 in two areas of the western Mediterranean sub-region (sensu MSFD). In the Lazio area we found a frequency of occurrence of plastic ingestion of 78.33%, while in Sardinia 41.79%. The analysis of the litter categories, among all individuals, highlights a prevalence of user-sheet (Use-She; 69.13%) and user-fragment plastics (Use-Fra; 20.84%). In addition, the polymer analysis showed a dominance of polyethylene (65.98%) and polypropylene (26.23%). As a result, by looking at other works that have investigated polymer types and items sources, we are able to infer that 77.25% of the objects ingested by the C. caretta individuals are attributable to disposable daily-life objects managed in an improper way. Therefore, C. caretta apart from being an efficient bio-indicator for plastic pollution, highlighting spatial and temporal concentration differences, it could also be used to verify the effectiveness of the Single-use Plastic Directive (EU 2019/904).

Systemic Infection by Angiostrongylus vasorum in a Fennec (Vulpeszerda) in an Italian Zoological Garden.

This paper reported a case of a metastrongyloid nematode Angiostrongylus vasorum infection in a fennec (Vulpes zerda) kept in a zoo in central Italy. The fennec had shown paralysis of the hind limbs, anorexia, weakness and respiratory signs before death. Cardiomegaly and granulomatous pneumonia were the major anatomopathological findings. Inflammatory lesions associated with parasitic larvae were observed in the lungs, brain, liver, heart, spinal cord and kidney of the fennec at histology. A. vasorum diagnosis was confirmed by both morphological and molecular identification of adult worms recovered at necropsy. Fennecs are active predators and maintain their hunting behaviour in captivity. Hence, it is likely that the animal was exposed to infection by preying on parasitised gastropods, intermediate hosts of A. vasorum, entering zoo enclosures from the surrounding environment. This is the first report of A. vasorum systemic infection in a captive fennec (V. zerda) in a zoo in Italy.

The Italian Network of Laboratories for Veterinary Oncology (NILOV) 2.0: Improving Knowledge on Canine Tumours.

Advances in tumour research are crucial, and comparative oncology can improve the knowledge in several ways. Dogs are not only models of specific naturally occurring tumours but can also be sentinels of environmental exposures to carcinogens, as they share the same environment with their owners. The purpose of this work was to describe the data collected by The Italian Network of Laboratories for Veterinary Oncology in the first 9 years of activity (2013-2021) and to evaluate their potential epidemiological significance. Frequencies of tumour topographies and main morphologies in dogs were described, analysed and compared, calculating age-adjusted proportional morbidity ratios and considering several risk factors (breed, sex, period and region of residence). These observations allowed us to highlight differences not only in morphology and topography of some tumours but also to formulate hypotheses on the potential role of some risk factors, e.g., neutering/spaying or geographical location. In our opinion, the results of this case series confirm the importance of initiating and consolidating animal cancer registration initiatives that would facilitate the possibility of conducting multicentric collaborative studies to deepen the knowledge of the epidemiology of tumours in dogs from a comparative perspective.

Potential SARS-CoV-2 Susceptibility of Cetaceans Stranded along the Italian Coastline.

Due to marine mammals' demonstrated susceptibility to SARS-CoV-2, based upon the homology level of their angiotensin-converting enzyme 2 (ACE2) viral receptor with the human one, alongside the global SARS-CoV-2 occurrence and fecal contamination of the river and marine ecosystems, SARS-CoV-2 infection may be plausibly expected to occur also in cetaceans, with special emphasis on inshore species like bottlenose dolphins (Tursiops truncatus). Moreover, based on immune and inflammatory responses to SARS-CoV-2 infection in humans, macrophages could also play an important role in antiviral defense mechanisms. In order to provide a more in-depth insight into SARS-CoV-2 susceptibility in marine mammals, we evaluated the presence of SARS-CoV-2 and the expression of ACE2 and the pan-macrophage marker CD68. Aliquots of tissue samples, belonging to cetaceans stranded along the Italian coastline during 2020-2021, were collected for SARS-CoV-2 analysis by real-time PCR (RT-PCRT) (N = 43) and Immunohistochemistry (IHC) (N = 59); thirty-two aliquots of pulmonary tissue sample (N = 17 Tursiops truncatus, N = 15 Stenella coeruleoalba) available at the Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padua (Legnaro, Padua, Italy) were analyzed to investigate ACE2 expression by IHC. In addition, ACE2 and CD68 were also investigated by Double-Labeling Immunofluorescence (IF) Confocal Laser Microscopy. No SARS-CoV-2 positivity was found in samples analyzed for the survey while ACE2 protein was detected in the lower respiratory tract albeit heterogeneously for age, gender/sex, and species, suggesting that ACE2 expression can vary between different lung regions and among individuals. Finally, double IF analysis showed elevated colocalization of ACE2 and CD68 in macrophages only when an evident inflammatory reaction was present, such as in human SARS-CoV-2 infection.

Morbilliviral encephalitis in a striped dolphin Stenella coeruleoalba calf from Italy.

A free-living, striped dolphin Stenella coeruleoalba calf was stranded on the Latium coast of Italy in November 2009. Significant neuropathological findings included non-suppurative meningoencephalitis, microgliosis, neuronal degeneration, astrocytosis, and occasional multinucleate syncytia. Immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR) for Morbillivirus were positive exclusively from the brain, with morbilliviral antigen and nucleic acid being detected in neurons and, to a lesser extent, in astrocytes. A low neutralizing antibody titer (1:10) against Morbillivirus (Canine distemper virus, CDV) was also found in blood serum, with no simultaneous presence of serum antibodies to Brucella spp. or Toxoplasma gondii. Furthermore, no pathogenic bacteria were isolated from any tissue or biological sample. This is the second report of morbilliviral encephalitis in a striped dolphin stranded along the Italian coastline in a 16 yr period (1993 to 2009). The neurohistopathological, IHC, and biomolecular features of this case are of additional interest, as antigenic and genomic positivity were exclusively confined to the brain of this dolphin, which may have acquired morbilliviral infection either postnatally or transplacentally.

Plasmodium matutinum Causing Avian Malaria in Lovebirds (Agapornis roseicollis) Hosted in an Italian Zoo.

Avian malaria is a worldwide distributed, vector-born disease of birds caused by parasites of the order Haemosporida. There is a lack of knowledge about the presence and pathogenetic role of Haemosporida in Psittacidae. Here we report a case of avian malaria infection in lovebirds (Agapornis roseicollis), with the genetic characterization of the Plasmodium species involved. The birds were hosted in a zoo located in Italy, where avian malaria cases in African penguins (Spheniscus demersus) were already reported. Animals (n = 11) were submitted for necropsy after sudden death and were subjected to further analyses including histopathology, bacteriology, and PCR for the research of haemosporidians. Clinical history, gross lesions and histopathological observation of schizonts, together with positive PCR results for Plasmodium spp., demonstrated that avian malaria was the cause of death for one animal and the possible cause of death for the other nine. The sequences obtained were compared using BLAST and analyzed for similarity to sequences available at the MalAvi database. Genetic analyses demonstrated a 100% nucleotide identity to Plasmodium matutinum LINN1 for all the obtained sequences. To our knowledge, this is the first report describing avian malaria in lovebirds.

Plasmodium matutinum Transmitted by Culex pipiens as a Cause of Avian Malaria in Captive African Penguins (Spheniscus demersus) in Italy.

Avian malaria is a parasitic disease of birds caused by protozoa belonging to the genus Plasmodium, within the order Haemosporida. Penguins are considered particularly susceptible, and outbreaks in captive populations can lead to high mortality. We used a multidisciplinary approach to investigate the death due to avian malaria, occurred between 2015 and 2019, in eight African penguins (Spheniscus demersus) kept in two Italian zoos located in central Italy, and situated about 30 km apart. We also provided information about the presence and circulation of Plasmodium spp. in mosquitoes in central Italy by sampling mosquitoes in both zoos where penguin mortalities occurred. In the eight dead penguins, gross and histopathological lesions were consistent with those previously observed by other authors in avian malaria outbreaks. Organs from dead penguins and mosquitoes collected in both zoos were tested for avian malaria parasites by using a PCR assay targeting the partial mitochondrial conserved region of the cytochrome b gene. Identification at species level was performed by sequencing analysis. Plasmodium matutinum was detected in both dead penguins and in mosquitoes (Culex pipiens), while Plasmodium vaughani in Culex pipiens only. Parasites were not found in any of the PCR tested Aedes albopictus samples. Based on our phylogenetic analysis, we detected three previously characterized lineages: Plasmodium matutinum LINN1 and AFTRU5, P. vaughani SYAT05. In Culex pipiens we also identified two novel lineages, CXPIP32 (inferred morphospecies Plasmodium matutinum) and CXPIP33 (inferred morphospecies P. vaughani). Significantly, LINN1 and AFTRU5 were found to be associated to penguin deaths, although only LINN1 was detected both in penguins (along the years of the study) and in Culex pipiens, while AFTRU5 was detected in a single penguin dead in 2017. In conclusion, in our study Plasmodium matutinum was found to cause avian malaria in captive penguins kept in Europe, with Culex pipiens being its most probable vector. Our results are in agreement with previous studies suggesting that Culex pipiens is one of the main vectors of Plasmodium spp. in Europe and the Northern Hemisphere. Zoos maintaining captive penguins in temperate areas where Culex pipiens is abundant should be well aware of the risks of avian malaria, and should put every effort to prevent outbreaks, in particular during the periods when the number of vectors is higher.

Evaluation of intrinsic and extrinsic risk factors for dog visceral hemangiosarcoma: A retrospective case-control study register-based in Lazio region, Italy.

The genetic and breed susceptibility of visceral hemangiosarcoma in dogs has been studied, but there is no evidence of environmental risk factors as reported in human medicine. We conducted a case-control study in which the sampling population was the list of canine oncology cases of the Animal Tumour Registry of Lazio region, Italy (2009-2017). We defined cases as dogs with visceral hemangiosarcoma and controls as dogs affected by another neoplasm. The ratio between controls and cases was 3:1. Analysed variables were: age, weight, sex, reproductive status, size, breed, nutrition habit, living environment and location of the house. We performed a preliminary univariate analysis to select potential risk factors (p-value < 0.2) then entered in a forward stepwise logistic regression model. Ninety-three cases enrolled in the study were compared with 279 controls. The multivariable logistic regression identified age, reproductive status and breed as significant risk factors. Results showed an increasing risk with increasing age for age classes 6-10 and > 10 years old (OR = 9.69, 95 % CI: 1.21-77.62; OR = 14.01, 95 % CI: 1.65-119.03). Neutered animals (male and female) were at greater risk compared to intact ones. The breeds at greatest risk were German shepherd (OR = 4.17, 95 % CI: 1.25-13.86) and mixed breed (OR = 3.50, 95 % CI: 1.44-8.51). The last finding could be explained by the genetic origin of the animals, which may include German shepherd or another possible breed at risk. No other individual or environmental variables were identified as risk factors. The findings of this work indicate that genetic predisposition is the key element in visceral hemangiosarcoma development.