Pro-Inflammatory and Cytotoxic Effects of Polystyrene Microplastics on Human and Murine Intestinal Cell Lines.

Plastic is a polymer extremely resistant to degradation that can remain for up to hundreds or thousands of years, leading to the accumulation of massive amounts of plastic waste throughout the planet's ecosystems. Due to exposure to various environmental factors, plastic breaks down into smaller particles named microplastics (1-5000 µm) and nanoplastics (<1 µm). Microplastics (MPs) are ubiquitous pollutants but, still, little is known about their effects on human and animal health. Herein, our aim is to investigate cytotoxicity, oxidative stress, inflammation and correlated gene modulation following exposure to polystyrene microplastics (PS-MPs) in HRT-18 and CMT-93 epithelial cell lines. After 6, 24 and 48 h PS-MPs treatment, cell viability (MTT) and oxidative stress (SOD) assays were performed; subsequently, expression changes and cytokines release were investigated by Real-Time PCR and Magnetic-beads panel Multiplex Assay, respectively. For each exposure time, a significantly increased cytotoxicity was observed in both cell lines, whereas SOD activity increased only in CMT-93 cells. Furthermore, Magnetic-beads Multiplex Assay revealed an increased release of IL-8 in HRT-18 cells' medium, also confirmed by gene expression analysis. Results obtained suggest the presence of a pro-inflammatory pattern induced by PS-MPs treatment that could be related to the observed increase in cytotoxicity.

Cadmium and wild boar: Environmental exposure and immunological impact on macrophages.

Cadmium (Cd2+) is regarded as one of the most toxic heavy metals, which can enter the food chain through environmental contamination and be bioaccumulated. Its exposure in Ligurian wild boars was monitored between 2016-2020 and revealed high level of this heavy metal in different provinces. In one of these polluted area, 21 wild boars were additionally sampled and the relationship between hepatic and renal Cd2+ concentration suggested that majority of these animals presented chronic intoxication. Cd2+ exposure of wild boar might lead to an immunosuppression status, thus in vitro experiments on wild boar monocyte-derived macrophages (moMФ) were carried out. Effects of Cd2+ scalar doses were evaluated through viability and adsorption assays, ELISA, qPCR. Moderate doses of this environmental pollutant (20 µM) were absorbed by moMФ, with subsequent reduction of their viability. This heavy metal did not trigger release of either IFN- ß, anti-inflammatory or pro-inflammatory cytokines by moMФ, instead 24 h treatment with 20 µM of Cd2+ resulted in down-regulated expression of TNF-α, IL-12p40, several TLRs, CD14, MD2, BD2, MyD88, p65, and NOS2. The results of our monitoring activity suggested that wild boar can be useful to monitor environmental exposure of this heavy metal and can help in understanding the type of contamination. In addition, in vitro experiments on wild boar moMФ revealed that Cd2+ exposure negatively affected the immune function of these cells, likely leading to increased susceptibility to infection.

Analysis of the Gastro-Intestinal Tract of Marine Mammals: A Multidisciplinary Approach with a New Multi-Sieves Tool.

Organs and content of the gastro-intestinal tract (GIT) of marine mammals are relevant for a variety of investigations and provide data to researchers from different fields. Currently used protocols applied to the GIT for specific analysis limit the possibility to execute other investigations and important information could be lost. To ensure a proper sample collection and a multidisciplinary investigation of the GIT of marine mammals, a new multi-sieves tool and a specific protocol have been developed. This new device and approach allowed the simultaneous sampling of the GIT and its content for the main investigations concerned. The samples collected during these preliminary trials were suitable to perform all the different research procedures considered in this work. The obtained results show that with a few and easy procedural adjustments, a multidisciplinary sampling and evaluation of the GIT of marine mammals is possible. This will reduce the risk of losing important data aimed at understanding the cause of death of the animal, but also biology and ecology of marine mammals, and other important data for their conservation and habitats management.

Retrospective immunohistochemical investigation on dolphin morbillivirus infection by comparing the performance of heterologous monoclonal and polyclonal antibodies - Short communication.

Dolphin morbillivirus (DMV) is a pathogen of great concern in free-ranging cetaceans. Confirmation and staging of morbillivirus infections rely on histology and immunohistochemistry (IHC), following molecular detection. As at the present time no specific antibodies (Abs) against DMV are available, two heterologous Abs have been used worldwide for the examinations of morbillivirus infections of cetaceans. One is a monoclonal Ab (MoAb) prepared against the N protein of canine distemper virus (CDV), whereas the other is a polyclonal Ab raised in rabbits against rinderpest virus (RPV). Both Abs are known to show cross-reactivity with DMV. In this study we compared the labelling quality and the neuroanatomical distribution of staining with these two Abs by means of IHC analysis. To this end, serial sections of the target organs from ten free-ranging stranded cetaceans, previously diagnosed as being infected with DMV by PCR and/or serology, were subjected to IHC. The brain, lungs and lymph nodes of one animal were found to be positive with both Abs. From two other animals, the brain and the spleen, respectively, tested positive only with the polyclonal Ab. In the positive brain tissues, multifocal immunostaining was observed, with similar staining location and extent, with the two antibodies tested. Our results suggest that the polyclonal anti-RPV Ab might have a stronger binding activity to DMV than the anti-CDV MoAb. Nevertheless, the elaboration and use of specific anti-DMV Abs might be essential to guarantee conclusive results in diagnostic and pathogenetic investigations.

Prevalence and Antimicrobial Resistances of Salmonella spp. Isolated from Wild Boars in Liguria Region, Italy.

Salmonella spp. is an important zoonotic agent. Wild boars might host this pathogen in the intestinal tract and might represent a risk for Salmonella spp. transmission to humans. Wild boars are widely spread in Liguria, due to the environmental characteristics of the region. The aim of the study was the isolation, typing, and investigation of antimicrobial susceptibility of the isolated strains of Salmonella spp. During the 2013-2017 hunting seasons, 4335 livers of wild boars were collected and analyzed for the presence of Salmonella spp. A total of 260 strains of Salmonella spp. were isolated and characterized, with a prevalence of 6%. The isolated strains belonged to all six Salmonella enterica subspecies. Most of them were identified as Salmonella enterica subs. enterica of which 31 different serotypes were identified. The dominating serotype identified was S. Enteritidis. The antimicrobial resistance profiles of the isolated strains were analyzed against sixteen molecules. Of the isolated strains, 94.6% were resistant to at least one of the tested antimicrobials. This study showed the circulation of resistant Salmonella spp. strains in the wild boar population living in this area of Italy, underling the potential risk for these animals to disseminate this pathogen and its antimicrobial resistances.

Evidence for Unknown Sarcocystis-Like Infection in Stranded Striped Dolphins (Stenella coeruleoalba) from the Ligurian Sea, Italy.

Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of Toxoplasma gondii, additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large Sarcocystis-like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude cross-reaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols precluded a neural infection by Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi, and Neospora caninum. T. gondii coinfection was confirmed only in dolphin SD2. Sarcocystis sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of Sarcocystis-like tissue cysts in the brain of stranded cetaceans along with the first description of Sarcocystis sp. infection in muscle tissue of dolphins from the Mediterranean basin.

Evidence of Antimicrobial Resistance and Presence of Pathogenicity Genes in Yersinia enterocolitica Isolate from Wild Boars.

Yersinia enterocolitica (Ye) is a very important zoonosis andwild boars play a pivotal role in its transmission. In the last decade, the wild boar population has undergone a strong increase that haspushed them towards urbanized areas, facilitating the human-wildlife interface and the spread of infectious diseases from wildlife to domestic animals and humans. Therefore, it is important to know the serotype, antimicrobial resistance and presence of pathogenicity genes of Yersinia enterocolitica (Ye) isolated in species. From 2013 to 2018, we analyzed the liver of 4890 wild boars hunted in Liguria region; we isolated and serotyped 126 Ye positive samples. A decisive role in the pathogenicity is given by the presence of virulence genes; in Ye isolated we found ystB (~70%), ymoA (45.2%), ail (43.6%) and ystA (~20%). Moreover, we evaluated the susceptibility at various antimicrobic agents (Ampicillin, Chloramphenicol, Enrofloxacin, Gentamicin, Kanamycin, Trimethoprim-Sulfamethoxazole, Sulfisoxazole, Ceftiofur and Tetracycline). The antibiotic resistance was analyzed, and we found a time-dependent increase. It is important to shed light on the role of the wild boars as a reserve of potentially dangerous diseases for humans, and also on the antibiotic resistance that represents a public health problem.

Molecular detection of Leptospira spp. in wild boar (Sus scrofa) hunted in Liguria region (Italy).

Leptospirosis is a re-emerging and widespread zoonosis, worldwide distributed, due to a wide variety of wild and domestic animal species able to act as natural or accidental hosts. During last years, in Europe, as in Italy, wild boar (Sus scrofa) population is increased. This animal represents a reservoir for different etiological agents, such as Leptospira. The aim of this investigation was to evaluate the prevalence of Leptospira spp. in wild boar hunted in Liguria region (Italy) during two-year hunting seasons. From 611 hunted wild boar, kidneys were collected. DNA was extracted from each organ and different targets were used to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene) and saprophytic (23S rRNA gene) Leptospira by Taqman-based RealTime-PCR assays. Overall, kidneys were sampled from 282 adults, 155 sub-adults and 174 young wild boar (in total 314 males and 298 females). By RealTime PCR 77 kidneys were positive and, among these, 74 resulted positive for pathogenic (96.10%) and 3 (3.90%) for intermediate Leptospira. No significant differences in pathogenic Leptospira infection ratio were detected between male (11.50%) and female (12.75%). Only 13 sub-adult animals (8.39%) resulted infected by pathogenic Leptospira; 23 young animals (13.22%) and 38 adult animals (13.47%) were positive. The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis, which is able to infect other animal species (domestic and wild) including humans. Rarely, intermediate Leptospira could be able to infect wild boar with a renal localization that can contribute to their shedding and circulation.

First report of the invasive mosquito Aedes koreicus (Diptera: Culicidae) and of its establishment in Liguria, northwest Italy.

BACKGROUND: Invasive mosquito species (IMS) of the genus Aedes are a cause of increasing concern in Europe owing to their ability to vector important human viral diseases. Entomological surveillance to early detect alien mosquito and flavivirus circulation in Liguria, northwest Italy, has been carried out since 2011. RESULTS: The invasive species Aedes koreicus was first detected in Genoa in September 2015, when a male specimen was caught near the international airport; species identity was confirmed by genetic analysis. Over the next three years, 86 more adult specimens were trapped at sites throughout the city, accounting for 0.50% of all mosquitoes and 1.04% of Aedes sp. mosquitoes trapped in Genova in the four-year period 2015-2018. So far, no other monitored sites in Liguria have revealed the presence of this species. Ovitraps at two sites became positive for the species in July-August 2017. All female Ae. koreicus pools analysed were negative in biomolecular assays for Flavivirus. CONCLUSIONS: Our findings of Ae. koreicus in Genoa constitute, to the best of our knowledge, the first report of the species in northwest Italy and in a Mediterranean port city. The species appears to be established; trapping and climatic data support survival of Ae. koreicus in the area through three consecutive winters. Monitoring of adult mosquitoes detected the species two years before its discovery with ovitraps; trapping for adult specimens appears to be a more effective tool for the early detection of IMS. The airport (located near the commercial port area) and the flower market are the most probable sites of introduction; however, the exact time and place of arrival of this IMS in Liguria remain unknown. Based on morphological and genetic data, a common origin for most of the Ae. koreicus populations established in Europe is suspected. So far, no control measures have been adopted in Genoa and the species will probably colonize an even wider area in the next few years.

A survey on zoo mortality over a 12-year period in Italy.

BACKGROUND: The zoo is a unique environment in which to study animals. Zoos have a long history of research into aspects of animal biology, even if this was not the primary purpose for which they were established. The data collected from zoo animals can have a great biological relevance and it can tell us more about what these animals are like outside the captive environment. In order to ensure the health of all captive animals, it is important to perform a post-mortem examination on all the animals that die in captivity. METHODS: The causes of mortality of two hundred and eighty two mammals which died between 2004 and 2015 in three different Italian zoos (a Biopark, a Safari Park and a private conservation center) have been investigated. RESULTS: Post mortem findings have been evaluated reporting the cause of death, zoo type, year and animal category. The animals frequently died from infectious diseases, in particular the causes of death in ruminants were mostly related to gastro-intestinal pathologies. pulmonary diseases were also very common in each of the zoos in the study. Moreover, death was sometimes attributable to traumas, as a result of fighting between conspecifics or during mating. Cases of genetic diseases and malformations have also been registered. DISCUSSION: This research was a confirmation of how conservation, histology and pathology are all connected through individual animals. These areas of expertise are extremely important to ensure the survival of rare and endangered species and to learn more about their morphological and physiological conditions. They are also useful to control pathologies, parasites and illnesses that can have a great impact on the species in captivity. Finally, this study underlines the importance of a close collaboration between veterinarians, zoo biologists and pathologists. Necropsy findings can help conservationists to determine how to support wild animal populations.

Gene expression profile associated with thymus regeneration in dexamethasone-treated beef cattle.

Glucocorticoids (GCs) are illegally used as growth promoters in cattle, and the analytical methods officially applied most likely underestimate the precise frequency of the abuse. As a side effect, the administration of GCs causes fat infiltration, apoptosis, and atrophy of the thymus. However, gross and histological observations carried out previously showed that the thymus preserves an intrinsic ability to regenerate. The aim of this work was to study the transcriptional effects of GCs on genes likely involved in regeneration of the epithelial cell network in the cervical and thoracic thymus of beef cattle treated with dexamethasone (DEX) or prednisolone (PRD) in comparison with a control group. Moreover, the ratio of bax/bcl2 genes was examined to verify a possible antiapoptotic activity occurring at the same time. In the cervical thymus, DEX administration increased the gene expression of c-myc (P < 0.01), tcf3 (P < 0.05), tp63 (P < 0.01), and keratin 5 (krt5; P < 0.01). In the thoracic thymus of DEX-treated cattle, the gene expression of tcf3 (P < 0.01), tp63 (P < 0.01), and krt5 (P < 0.05) was increased. These results suggested that thymic regeneration is underway in the DEX-treated animals. However, the bax/bcl2 ratio was decreased in both cervical and thoracic thymus of DEX-treated cattle (P < 0.01 and P < 0.05, respectively), showing an antiapoptotic effect through the mitochondrial pathway. Conversely, PRD administration caused no change in the expression of all considered genes. These results sustain the hypothesis that regeneration occurs in the thymus parenchyma 6 d after the DEX treatment was discontinued. This hypothesis is also supported by the absence of alterations in the thymus of PRD-treated beef cattle. Indeed, previous studies showed the inability of PRD to induce macroscopic and microscopic lesions in the thymus. Therefore, in this context, it is not surprising that PRD induced no alteration of genes involved in the regeneration pathway.

Reference Gene Selection and Prednisolone Target Gene Expression in Adipose Tissues of Friesian Cattle.

Corticosteroids are frequently used in livestock production, and their use is permitted by the European Union for therapeutic purposes only. However, small doses of corticosteroids are often administered in meat-producing animals to improve zootechnical performance. Prednisolone is one of the most commonly used corticosteroids with a growth-promoting purpose in animal husbandry. This study proposes to identify a gene whose expression is significantly regulated by prednisolone in visceral and subcutaneous adipose tissues. The analysis was conducted on Friesian cattle treated with prednisolone (30 mg day-1). The reference gene expression stability and optimal number for gene expression normalization were calculated. Family with sequence similarity 107 member A (FAM107A) and pyruvate dehydrogenase kinase 4 are the prednisolone target genes identified in adipose tissue. FAM107A was downregulated by ∼2.9-fold by prednisolone in subcutaneous adipose tissue. This result suggests that FAM107A could be a possible indirect biomarker of prednisolone treatment in cattle and encourages a deeper investigation in this direction.

17ß-estradiol upregulates oxytocin and the oxytocin receptor in C2C12 myotubes.

BACKGROUND: The endocrinology of skeletal muscle is highly complex and many issues about hormone action in skeletal muscle are still unresolved. Aim of the work is to improve our knowledge on the relationship between skeletal muscle and 17ß-estradiol. METHODS: The skeletal muscle cell line C2C12 was treated with 17ß-estradiol, the oxytocin peptide and a combination of the two hormones. The mRNA levels of myogenic regulatory factors, myosin heavy chain, oxytocin, oxytocin receptor and adipogenic factors were analysed in C2C12 myotubes. RESULTS: It was demonstrated that C2C12 myoblasts and myotubes express oxytocin and its receptor, in particular the receptor levels physiologically increase in differentiated myotubes. Myotubes treated with 17ß-estradiol overexpressed oxytocin and oxytocin receptor genes by approximately 3- and 29-fold, respectively. A decrease in the expression of fatty acid binding protein 4 (0.62-fold), a fat metabolism-associated gene, was observed in oxytocin-treated myotubes. On the contrary, fatty acid binding protein 4 was upregulated (2.66-fold) after the administration of the combination of 17ß-estradiol and oxytocin. 17ß-estradiol regulates oxytocin and its receptor in skeletal muscle cells and they act in a synergic way on fatty acid metabolism. DISCUSSION: Oxytocin and its receptor are physiologically regulated along differentiation. 17ß-estradiol regulates oxytocin and its receptor in skeletal muscle cells. 17ß-estradiol and oxytocin act in a synergic way on fatty acid metabolism. A better understanding of the regulation of skeletal muscle homeostasis by estrogens and oxytocin peptide could contribute to increase our knowledge of muscle and its metabolism.

Effects and detection of Nandrosol and ractopamine administration in veal calves.

The present study describes different effects of the selective androgen receptor modulator (SARM) nandrolone phenylpropionate (Nandrosol) and the ß-agonist ractopamine administration in veal calves, and it investigates different strategies applied to trace these molecules. Morphological changes of gonads and accessory glands attributed to androgen effects, such as testicular atrophy, seminiferous tubule diameter reduction and hyperplasia of prostate epithelium, were detected, although SARMs are not described to cause these lesions. The gene expression analysis showed an anabolic activity of Nandrosol in Longissimus dorsi muscle, where myosin heavy chain (MYH) was significantly up-regulated. An IGF1 increase was weakly significant only in Vastus lateralis muscle. In conclusion, the anatomo-histopathological observations and the MYH mRNA up-regulation in Longissimus dorsi muscle confirm the androgenic treatment in experimental animals. The biosensor assay was not enough sensitive to detect residues in urines and only the direct chemical analysis of urine samples confirmed both ß-agonist and SARM treatment.

Diagnosis of canine gastric adenocarcinoma using squash preparation cytology.

Adenocarcinoma is the most common gastric tumour in dogs. Clinical signs and laboratory results are often non-specific, with histopathological examination of gastric biopsies being required to reach a definitive diagnosis. Use of cytology would potentially shorten the time to diagnosis and allow early interventional measures to be implemented. However, there are relatively few studies of the cytological features of gastric samples. The present study was designed to investigate whether cytology might be useful for diagnosis of canine gastric adenocarcinomas and to evaluate the performance of squash preparation cytology for this purpose. Squash preparations of gastric biopsies from 94 dogs were reviewed to determine the presence or absence of specific cytological features associated with adenocarcinomas and to compare findings with the results of histopathological examination of gastric biopsies. The presence of signet ring cells, microvacuolation, cellular pleomorphism and single cell distribution of epithelial cells were positively associated with a diagnosis of gastric adenocarcinoma. Combined evaluation (parallel testing) for the presence of signet ring cells and microvacuolation demonstrated excellent results for recognition of adenocarcinomas. Cytological examination of squash preparations from gastric biopsies and identification of signet ring cells and cytoplasmic vacuolation can allow rapid and reliable diagnosis of canine gastric adenocarcinomas.