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In the last decade in Europe, the number of autochthonous cases of hepatitis E has significantly increased. Most of the cases arise from foodborne infections caused by the zoonotic hepatitis E virus (HEV) genotypes HEV-3 and HEV-4. Several human cases have been linked to consumption of raw or undercooked animal products of both pork (liver sausages) and wild boar meat. In this study, the occurrence of HEV infection was investigated in 611 livers and 88 paired lungs from wild boars collected during the hunting seasons of 2016-2020 in the Umbria-Marche Apennines (Central Italy). Using real-time reverse transcription polymerase chain reaction, 15 liver samples (2.45%) and one lung sample were found to be positive for HEV RNA. The phylogenetic tree built on the partial ORF2 gene revealed that the detected HEV strains belonged to HEV-3f (n = 5), HEV-3e (n = 1) and HEV-3c (n = 1) subtypes. Interestingly, 8 strains were genetically placed in a different cluster, further away from all other subtypes. To corroborate this finding, four complete genomes were obtained by next generation sequencing. The full genome of the HEV strains clustered together with another wild boar strain previously detected in Southern Italy in 2015 but the strains were divergent from all the HEV-3 strains classified in any subtype defined so far. Thus, these strains represent a novel subtype that might have originated in Italy, which we have tentatively named HEV-3n.

Objectives Naturally occurring tumours in domestic cats are less common than in dogs and represent the leading cause of death among older animals. The main objective of this study was to analyse a large data set of histologically diagnosed tumours to highlight the most common World Health Organization (WHO) tumour histotypes, the effect of age and sex, and the International Classification of Diseases for Oncology (ICD-O) topographical site predilections of feline breed-specific tumours. Methods A total of 680 feline tumours diagnosed in European Shorthair cats by three veterinary diagnostic laboratories located in central Italy from 2013 to 2019 were collected. Data on age, sex and topography of lesions were recorded. Samples were morphologically and topographically coded using the WHO and the ICD-O-3 classification system. Results Skin and soft tissue neoplasms comprised 55.9% of all tumours, followed by mammary gland (11%), alimentary tract (7.9%), oral cavity and tongue (7.3%), nasal cavity and middle ear (6%), lymph node (3.1%), bone (1.8%) and liver/intrahepatic bile duct (1.3%) tumours. Squamous cell carcinoma (SCC), sarcoma, lymphoma and basal cell tumours were the most diagnosed neoplasms. Malignant tumours were 82.9% of the total and the topographical sites mainly involved were skin (C44), connective/subcutaneous/other soft tissues (C49), mammary gland (C50), small intestine (C17), nasal cavity and middle ear (C30), and gum (C03). Conclusions and relevance This study aimed to provide an in-depth evaluation of spontaneous feline tumours in the European Shorthair cat breed. Results identify SCC as the most commonly represented skin neoplasm. It is likely that the analysed feline population, living in southern latitudes, was more subject to prolonged exposure to ultraviolet light, explaining the discrepancy with previous studies in which SCC was less represented.

Background Comamonas kerstersii is rarely associated with infections in humans and has never been reported in animals until now. Case presentation Herein, we describe a case of urinary tract infection caused by C. kerstersii in a young goat. A seven-month-old male goat showed lethargy, generalised weakness and anorexia and in the last hours before its death, severe depression, slight abdominal distention, ruminal stasis, and sternal recumbency. Grossly, multifocal haemorrhages in different organs and tissues, subcutaneous oedema and hydrocele, serous fluid with scattered fibrin deposition on the serosa of the abdominal organs and severe pyelonephritis with multifocal renal infarction were detected. Histopathological examination confirmed severe chronic active pyelonephritis with renal infarcts, multi-organ vasculitis and thrombosis suggestive of an infectious diseases of bacterial origin. The bacterium was identified using routine methods, matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), and sequencing of the gyrB gene. Conclusions To the best of our knowledge, this is the first report of C. kerstersii infection in animals (goat). Our findings support the possibility of C. kerstersii isolation from extraintestinal sites and suggest this organism as a possible cause of urinary tract infection.

Tuberculosis is a worldwide zoonosis involving a wide range of hosts among domestic and wild animals. We describe tuberculosis caused by Mycobacterium bovis in a wild crested porcupine (Hystrix cristata) found dead in the district of Macerata, Marche Region, Italy in 2019.

Surveillance of illegal use of growth promoters such as β2-agonists in food producing animals rely on the detection of drug residues by LC-MS/MS. Screening strategies focusing on indirect physiological responses following administration of active compounds are promising approaches to strengthen existing targeted methods and ensure food safety. A metabolomics analysis based on LC-HRMS was carried out on liver extracts from bulls experimentally treated with clenbuterol combined with dexamethasone (n = 8) to mimic a potential anabolic practice, and control animals (n = 8). Nicotinic acid and 5′-deoxy-5′-methylthioadenosine were identified as biomarkers of treatment. Ratio values of such markers to others of the same metabolic pathways (nicotinamide or methionine) were used to develop a classification model to assign animals as treated with clenbuterol or non-treated. The classification model was tested on an external validation set comprising 74 animals either treated with different anabolic compounds (β2-agonists, sexual steroids, corticosteroid), or non-treated, showing 100% sensitivity and specificity.

A 40 years old male Nile crocodile (Crocodylus niloticus) was diagnosed with pulmonary mycobacteriosis caused by a member of Mycobacterium chelonae/abscessus group. Post-mortem examination showed a severe systemic visceral granulomatous involvement, with lesions in lungs, heart, liver, spleen and kidneys. Histopathological examination of lung, spleen, heart and liver revealed multifocal to coalescing granulomas showing eterophils in central zone and outer rim of epithelioid histiocytes, multinucleated giant cells and lymphocytes. The Ziehl–Neelsen histological staining revealed rare vacuoles containing numerous alcohol-acid resistant bacteria. Mycobacterial infection was confirmed by culture and PCR targeting rRNA 16S gene. Sequence analysis of the DNA amplicon revealed a 100% homology with the M. chelonae/ abscessus group. Even if the classification of the memebrr of this group is still on updating, to the best of our knowledge, this is the first report of M. chelonae/abscessus member infection in a Nile crocodile species.

Canine testicular tumors account for about 90 % of tumors affecting the male genitalia. Seminomas (SEM), Sertoli cell tumors (SCT), and interstitial cell tumors (ICT) are the most common histological diagnoses, but their incidence shows high variability among studies. Our aim is to report the results on the analysis of testicular tumors recorded by the population-based Umbria Canine Cancer Registry (CCR) for a 5-year time period and to assess the value of tumor major diameter, measured during trimming, in discriminating neoplastic from non-neoplastic lesions. The study was conducted on 388 testicular tumors (on 1969 total male tumors) diagnosed in 355 dogs from 2014 to 2018. The median incidence was 35 cases/100,000 dogs, with a proportion equal to 19,7 % of all tumors. The incidence on neutered male dogs was 352/100,000. Most tumors were ICTs (50 %), with fewer SEMs and SCTs (29 % and 17 %, respectively). Only 3 % of tumors were mixed germ cell-sex cord-stromal tumors (MGC-SCST). Ten percent of cases had multiple tumors in the same testicle, with SEM-ICT being prevalent (69.2 %). Tumors in cryptorchid testes were 5.9 % of the total, mostly SCT (60.9 %). Mean age at diagnosis was 10.7 ± 2.7, with similar values for different tumor types. Significant incidence ratios (IRR) were found in Golden retriever (IRR 7.18, CI95 % 4.72-10.92) and in English cocker spaniel (IRR 2.30, CI95 % 1.28-4.13) when compared with mixed breed dogs. A value of 0.3 cm (major diameter) of lesions at the moment of trimming was helpful in discriminating a final tumor histological diagnosis from a non-tumor lesion. Since the number of tumors included in this evaluation was limited, further studies to confirm the diagnostic value of this measure are recommended. Our results provided information on the incidence of canine testicular tumors in Umbria region that can be compared with future results and incidence from other geographical areas if provided with reliable data on the total population, can offer solid information on the incidence and proportion of different tumor types in specific territories, contributing also to the supervision of its inhabitants' health. Moreover, pathological data such as the major diameter of tumors can be obtained and contribute to diagnostic routine and standardization.

A multiclass screening method to detect fifty-three forbidden substances by liquid-chromatography coupled to hybrid high-resolution mass spectrometry (LC-Q-Orbitrap) was developed and validated in bovine bile and urine. Eight classes of compounds were included in the method's scope (β-agonists, corticosteroids, nitroimidazoles, progestins, resorcylic acid lactones (RALs), sedatives, steroids and stilbenes) plus chloramphenicol and dapsone. After hydrolysis, the sample was divided in two aliquots, which followed two parallel purification steps. The reunified extracts were injected and two chromatographic runs performed in positive and negative ionization mode, respectively. The validation data (60 different samples per matrix) proved that the method was fit for purpose with detection capabilities lower than 1 μg L−1 in both matrices. The combined application of accurate mass acquisition and two-stage mass spectrometry (parallel reaction monitoring) was crucial to achieve suitable selectivity, which is the most critical parameter mainly for urines. Finally, the long-standing problem of the high rate of false positive results for RALs, due to the natural ingestion of mycotoxin, zearalenone, was taken on including all their labelled standards. That allowed a very satisfactory management of this screening test.

In poultry production, probiotics have shown promise to limit campylobacteriosis at the farm level, the most commonly reported zoonosis in Europe. The aim of this trial was to evaluate the effects of Saccharomyces supplementation in Campylobacter jejuni challenged chickens on performance and intestinal ecosystem. A total of 156 day old male Ross 308 chicks were assigned to a basal control diet (C) or to a Saccharomyces cerevisiae boulardii CNCM I-1079 supplemented diet (S). All the birds were orally challenged with C. jejuni on day (d) 21. Live weight and growth performance were evaluated on days 1, 21, 28 and 40. The histology of intestinal mucosa was analyzed and the gut microbiota composition was assessed by 16S rRNA. Performance throughout the trial as well as villi length and crypt depth were positively influenced by yeast supplementation. A higher abundance of operational taxonomic units (OTUs) annotated as Lactobacillus reuteri and Faecalibacterium prausnitzii and a lower abundance of Campylobacter in fecal samples from S compared to the C group were reported. Supplementation with Saccharomyces cerevisiae boulardii can effectively modulate the intestinal ecosystem, leading to a higher abundance of beneficial microorganisms and modifying the intestinal mucosa architecture, with a subsequent improvement of the broilers&rsquo, growth performance.

In poultry production, probiotics have shown promise to limit campylobacteriosis at the farm level, the most commonly reported zoonosis in Europe. The aim of this trial was to evaluate the effects of Saccharomyces supplementation in Campylobacter jejuni challenged chickens on performance and intestinal ecosystem. A total of 156 day old male Ross 308 chicks were assigned to a basal control diet (C) or to a Saccharomyces cerevisiae boulardii CNCM I-1079 supplemented diet (S). All the birds were orally challenged with C. jejuni on day (d) 21. Live weight and growth performance were evaluated on days 1, 21, 28 and 40. The histology of intestinal mucosa was analyzed and the gut microbiota composition was assessed by 16S rRNA. Performance throughout the trial as well as villi length and crypt depth were positively influenced by yeast supplementation. A higher abundance of operational taxonomic units (OTUs) annotated as Lactobacillus reuteri and Faecalibacterium prausnitzii and a lower abundance of Campylobacter in fecal samples from S compared to the C group were reported. Supplementation with Saccharomyces cerevisiae boulardii can effectively modulate the intestinal ecosystem, leading to a higher abundance of beneficial microorganisms and modifying the intestinal mucosa architecture, with a subsequent improvement of the broilers’ growth performance.

Dexamethasone (DXM) is a synthetic adrenal corticosteroid with anti-inflammatory properties used for therapeutic purposes in a wide range of pathologies and of the most common corticosteroids used for anabolic purposes in beef cattle. It is proven that DXM induces histological changes, traceable as increasing fatty infiltration of the thymus associated with a concurrent decrease of the cortex-medulla ratio, so the histological examination of the thymus gland has been established as an indirect morphological biomarker. The aim of the present study is to compare thymus histology and DXM concentrations in biological fluids collected at slaughterhouse after 1 month of DXM treatment. Our findings demonstrate that a low dosage of DXM administered to 12 months-old-Chianina beef cattle induces severe thymic atrophy with concurrent reduction of the cortex/medulla ratio, demonstrable even when DXM residues are not found in serum and urine samples. It is worth to note that, at the slaughterhouse, DXM residues are detectable in bile samples, indicating the ability of this biological fluid to bio-concentrate the administered drug if compared to serum and urine. Therefore, bile could be candidates as new liquid matrix for the screening programs planned to contrast the illegal use of anabolic substances.

The Umbria Canine Cancer Registry (CCR) is a web-based platform for cancer registration set up in order to estimate the incidence of spontaneous tumors. It is an integral part of the regional canine demographic registry in which veterinary practitioners and pathologists interact. Veterinary pathologists perform double-blind comparisons and classify neoplasms in an automated classification process using the WHO criteria for canine neoplasms and the ICD-O tumor topographical and morphological keys. Here we describe the organization, on-line procedures and the methods used to assess canine demography, a pre-requisite for accurately estimating the incidence of cancer. In its first 4 years the CCR recruited 4857 cases of suspected tumors, as diagnosed by practitioners, clinics and a veterinary hospital. After the first year the number of enrolled cases increased by 63%, suggesting growing interest from the regional veterinary community.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 μmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

The current study investigated the effects of Lactobacillus acidophilus and Bacillus subtilis, used as probiotics, on the microflora, morphology, and morphometry of the gut in organic laying hens. The birds (180 Hy-Line laying hens) were divided into 3 homogenous groups and received a pre-deposition diet from 16 to 20 wk of age and a deposition diet for the remaining 7 months of the experiment. The control group ( CTR: ) was fed a corn-soybean cake-based diet, the second group ( L: ) received the same diet supplemented with 0.1% of L. acidophilus while in the third group ( B: ) the basal diet was supplemented with 0.05% of B. subtilis At 18 wk of age ( T1: ) and at 5 ( T2: ) and 7 months ( T3: ) from the beginning of deposition, 9 subjects per group were humanely killed for microbiological, morphological and morphometric analyses of the intestinal tract. The 2 probiotic-supplemented diets increased Lactobacillus spp. and Bifidobacterium spp. counts compared with the CTR diet. The lowest viable counts of E. coli, coliforms and staphylococci were observed in the L group (P

In livestock production corticosteroids are licensed only for therapy; nevertheless, they are often illegally used as growth promoters. The aim of this study was to identify morphological or biomolecular alterations induced by prednisolone (PDN) in experimentally treated beef cattle, because PDN and its metabolites are no longer detectable by LC-MS/MS methods in biological fluids. Moreover, PDN does not induce any histological alterations in the thymus, different from dexamethasone treatments. Therefore, a marker of illicit treatment for this growth promoter could be useful. Eight male Italian Friesian beef cattle were administered prednisolone acetate 30 mg day–1 per os for 35 days, and seven beef cattle represented the control group. Six days after drug withdrawal, the animals were slaughtered. Morphological and morphometric modifications were evaluated in the epididymis and testis, whereas transcriptomic changes induced by PDN administration were investigated in peripheral blood mononuclear cells (PBMC...

Hepatic lipidosis is the most diffused hepatic disease in the lactating cow. A new methodology to estimate the degree of fatty infiltration of the liver in lactating cows by means of texture analysis of B-mode ultrasound images is proposed. B-mode ultrasonography of the liver was performed in 48 Holstein Friesian cows using standardized ultrasound parameters. Liver biopsies to determine the triacylglycerol content of the liver (TAGqa) were obtained from each animal. A large number of texture parameters were calculated on the ultrasound images by means of a free software. Based on the TAGqa content of the liver, 29 samples were classified as mild (TAGqa50mg/g), 6 as moderate (50mg/gTAGqa100mg/g) and 13 as severe (TAG100mg/g) in steatosis. Stepwise linear regression analysis was performed to predict the TAGqa content of the liver (TAGpred) from the texture parameters calculated on the ultrasound images. A five-variable model was used to predict the TAG content from the ultrasound images. The regression model explained 83.4% of the variance. An area under the curve (AUC) of 0.949 was calculated for50mg/g vs50mg/g of TAGqa; using an optimal cut-off value of 72mg/g TAGpred had a sensitivity of 86.2% and a specificity of 84.2%. An AUC of 0.978 for100mg/g vs100mg/g of TAGqa was calculated; using an optimal cut-off value of 89mg/g, TAGpred sensitivity was 92.3% and specificity was 88.6%. Texture analysis of B-mode ultrasound images may therefore be used to accurately predict the TAG content of the liver in lactating cows.

Cancer immunosuppression that facilitates tumor progression and metastasis evolves by development of an immunosuppressive network. The aim of this study was to assess this network in dogs with benign or malignant tumors with or without confirmed metastasis. The authors showed that the number of various T cell subpopulations was constant during tumor development; however the number of regulatory T cells (Tregs) was significantly higher in tumor-bearing dogs than in healthy individuals. The number of myeloid-derived suppressor cells (MDSCs) and their p-STAT3 expression (which is a negative regulator of hematopoiesis and regulates VEGF expression) were higher in cancer patients than in control dogs, however their number increased significantly in late-stage cancer patients. Canine mammary carcinomas with confirmed metastases to either lymph nodes or internal organs had greater MDSCs and Treg infiltration than benign mammary tumors or malignant mammary tumors for which metastases had not been detected. Similarly, expression of p-STAT3 and VEGF-C was the highest in tumors with confirmed metastases. This research shows changes occurring in the blood (n = 30 patients) and tumor tissue of patients (n = 100) during canine mammary tumor development. The findings should be considered preliminary because of the small number of samples. Nonetheless, the findings suggest that a high level of Tregs and MDSCs as well as high expression of p-STAT3 and VEGF-C may significantly contribute to mammary tumor progression and metastasis in dogs.

We report cystic echinococcosis in a free-living wild boar (Sus scrofa) in Europe. Parasites were identified by histopathology and molecular techniques, revealing Echinococcus granulosus of the G3 genotype.

The reproductive cycle of an invasive alien Italian grey squirrel population was studied to understand its adaptation and limit its spread, in order to conserve the autochthonous red squirrel. Female and male genital traits were evaluated throughout the reproductive cycle, including the ovary, uterus, testicle, epididymis, seminiferous tubule morphometry, and germinative epithelium histology. Moreover, individual female fecundity was determined by counting uterine scars. Ovary width and uterus weight, length, and width reached their highest values in the luteal and pregnancy phases. On conducting a histological evaluation of the testicular germinal epithelium, four morphotypes related to the different reproductive phases of the male squirrels were identified: immature, pubertal, spermatogenesis, and regressive. Testicle and epididymis weights and seminiferous tubule diameters reached their largest values during spermatogenesis. Uterine scar analysis showed that 69% of the females had given birth to one or two litters, while 31% had no uterine scars. Litters were larger in the first breeding period than in the second, annual fecundity was 4.52 &plusmn, 1.88 uterine scars/female. Umbrian grey squirrels have adapted to their non-native range, showing two annual mating periods at times similar to those in their native range, and high reproductive success.

The aim of the present study was to evaluate the potential for diagnosis of fatty liver by means of ultrasonographic measurement of liver and perivisceral adipose tissue as an alternative to blood indicators of lipomobilization and liver biopsy in periparturient high-yielding dairy cows. Thirty cows were enrolled and divided into two groups. The evaluation of body condition score (BCS), non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), liver and perivisceral adipose tissue ultrasonographic measurement and histological liver lipid content (GdL) was performed at 15 ± 5 d prepartum (T0), 10 ± 2 d postpartum (T1), 30 ± 2 d postpartum (T2) and 50 ± 2 d postpartum (T3). Mesenteric fat thickness (the thickness of the perivascular adipose tissue) measured on ultrasound was shown to be an independent determinant of fatty liver. The cut-off of the ultrasonographic evaluation of the liver may be useful as a first and practical approach in identifying fatty liver. In conclusion, a non-invasive and reliable diagnostic method for predicting the risk of fatty liver in high yielding dairy cows has been demonstrated.

In a market undergoing constant evolution, the production of chicken meat that consumers would perceive as “natural” and “animal friendly” is crucial. The use of probiotics in rurally reared chickens could represent a major opportunity to achieve mutual benefit for both the industry and consumers. A total of 264 male Kabir chicks were randomly distributed to one of 2 dietary treatments: the L group received a commercial feed supplemented with 2.0 g/100 kg of Lactobacillus acidophilus D2/CSL, while the C group received the same basal diet without the additive. To assess the effects of probiotic supplementation in the chickens’ diet, productive performance was evaluated at d 21 and 42, whereas microbiological analyses of the intestinal content and intestinal histology and morphometry were performed at the end of the trial (d 42). At d 21 and 42, L birds showed better (P < 0.001) performance in terms of body weight, average daily gain, and feed conversion ratio. Enterococci, staphylococci, and Escherichia coli populations were not influenced by dietary treatment. On the contrary, Lactobacillus population increased (P = 0.032) in the L group. Furthermore, a tendency (P = 0.069) was observed for the coliforms to be influenced by diet, with lower values in the L group in comparison to the C group. Histological techniques revealed that the number of goblet cell containing neutral mucins was lower in the C group. Morphometric evaluations demonstrated that the probiotic supplementation increased the height of the mucosal layer by improving (P = 0.040) villus height, while crypt depth was unaffected. In conclusion, the results obtained in this study demonstrate that it is possible to use Lactobacillus acidophilus D2/CSL (CECT 4529) in rurally reared chicken breeds with positive effects on performance and gut health.

During the transition period, high-yielding dairy cows suffer from negative energy balance, intense lipomobilization and impaired lipid metabolism; this metabolic condition can lead to overburdened triglycerides accumulation in the liver, known as liver lipidosis, which has been associated to impaired fertility in dairy cows. The mechanisms of this impairment can be in principle correlated with the presence and the extent of ectopic fat depots. However, current methods for evaluating fat accumulation in liver and in the reproductive tract suffer from low resolution, sensitivity, and specificity. Confocal microscopes are equipped with Gallium arsenide phosphide detectors, thus enabling the acquisition of intense signals from tissue biopsies. This method could differentiate whether fat deposition occurred without requiring sample sectioning. Here, we examined with this technique liver, uterine and ovarian samples of heifers and regularly slaughtered repeat breeder and overconditioned dairy cows, to quantify lipid droplets and depots at a submicrometer scale with high specificity. With the aid of this technique, we found lipid depots in uterine and ovarian specimens. Moreover, we found that the size and number of depots increased with the degree of liver lipidosis. Further studies are needed to elucidate the relationship between the severity and extent of these deposits and the fertility of lactating dairy cows. Since tissues other than liver display different characteristic lipid droplet distributions, this technique can be potentially employed to shed new light on the pathogenesis of lipidosis and to assess new risk factors for infertility.

Aim To detect marine Bdellovibrio and like organisms (BALOs) which are able to infect Vibrio parahaemolyticus from seawater of the Adriatic, Italy. To test, prey specificity and predation efficiency of our Halobacteriovorax isolate, named HBXCO1, towards 17 Vibrio and 7 non-Vibrio strains linked to the Adriatic sea, Italy. Methods and results Double layer agar plating technique was used to enumerate BALOs and to evaluate their prey specificity and predation efficiency. Transmission electron microscopy and 16S rRNA analysis were used to identify them. Means of BALOs counts ranged from 5·0 PFU per ml (March 2017) to 98·6 PFU per ml (August 2016). HBXCO1 had the ability to attack all tested prey strains of V. parahaemolyticus, Vibrio cholerae non-O1/O139 and Vibrio vulnificus, but it did not prey on non-Vibrio strains and V. alginolyticus under the tested conditions. Conclusions Bdellovibrio and like organisms capable of infecting pathogenic vibrios are naturally present in seawater of the Adriatic, Italy. Isolate HBXCO1 shows prey specificity preferentially for the Vibrio genus and high predatory efficiency towards a wide range of pathogenic strains. Significance and impact of the study The public impact of V. parahaemolyticus, non-O1/O139 V. cholerae and V. vulnificus in bivalves is relevant and current decontamination processes are not always effective. We believe that the predator HBXCO1 represents a potential candidate for the development of strategies of biocontrol of pathogenic vibrios in bivalves from harvesting to trade.

The aim of this study was to assess the severity and distribution of hepatic lipidosis in high-yielding dairy cows during the transition period by the evaluation of body condition score (BCS), serum levels of non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHB) and histological liver lipid content (GdL). Twenty-seven dairy cows, with a milk production of ~10 000 kg per year were enrolled in an experiment. Clinical examination was performed to evaluate the health status or the presence of periparturient diseases after calving. Animals were divided into two groups: healthy (HG; n = 11) and sick (SG; n = 16). The evaluation of BCS, NEFA, BHB and liver biopsies were performed at 15 ± 5 days prepartum (T0), 10 ± 2 days postpartum (T1), 30 ± 2 days postpartum (T2) and 50 ± 2 days postpartum (T3). Two-way repeated-measure ANOVA was applied to assess statistical significance of sampling time and between groups for all variables. Pearson’s correlation coefficient was used to investigate the relationship between all variables. Results showed BCS loss from T0 to T3 in SG and a significant increase of NEFA and BHB at T1. The GdL began to be mild at T0 increasing and becoming moderate to severe at T1, fairly regressing, but not disappearing, at T2 and T3, in both groups. This study showed that high lipomobilisation with a mild to moderate fat infiltration does not imply that ketosis or other periparturient diseases might be present. Our results suggest that hepatic lipidosis is associated with long-term histological and metabolic changes in dairy cows.

Red blood cells (RBCs) loaded with iron oxide nanoparticles have been proposed as biomimetic constructs with long half-life (ca. 20 days) in the blood compartment and potentially interesting properties (such as relaxivity) as intravascular contrast agents for magnetic resonance imaging. However, the encapsulation of nanoparticles into RBCs might affect their magnetic properties and relaxivity, which may be significantly different from the native suspension. Here, we present a relaxometric study of P904, a novel ultra small iron oxide nanoparticle developed by Guerbet, enclosed in human RBCs. We measured longitudinal (r1) and transverse (r2) relaxivity over a wide range of Larmor frequencies (0.01–300 MHz) in samples of P904-loaded RBCs, and in control samples with P904 nanoparticles dispersed in blood. Internalization of P904 into RBCs resulted in smaller r1, and in a very high r2/r1 ratio (232) at the highest field. Moreover, a shift of the Curie peak to high fields was observed in P904-loaded RBCs, possibly the result of nanoparticle size selection caused by the internalization process. High r2 relaxivity together with a high r2/r1 ratio and a very long blood half-life make P904-loaded RBCs a promising blood-pool negative contrast agent for MR diagnostic applications. Copyright © 2014 John Wiley & Sons, Ltd.

A widespread deadly outbreak occurred in captive birds belonging to the family Turdidae in Italy. The present study was performed on 46 dead birds coming from 3 small decoy-bird breeders in central Italy. Only Turdus pilaris, Turdus iliacus, Turdus philomelos and Turdus merula were affected. No other species of bird held by these breeders died. A change of diet before the hunting season was reported from all breeders. Full necropsy of the animals and histological investigations of representative tissue samples were performed. Microscopical examination showed marked iron deposits in liver samples. Bacteriological investigations and molecular analysis to exclude bacterial and viral diseases were carried out. Contamination of food pellet samples by mycotoxins and analysis to detect heavy metal contaminants in food pellet samples were considered. An interesting result was the high iron content found in food pellets. It was higher than that considered suitable for birds, especially for species susceptible to development iron storage disease (ISD). Taken together, the results suggested an outbreak of ISD caused by the high iron content of food given to the birds before the hunting season. The high mortality recorded only in species belonging to the family Turdidae suggests a genetic predisposition in the affected birds.

Illegal use of growth promoter compounds in food production exposes consumers to health risk. Surveillance of such practices is based on direct detection of drugs or related metabolites by HPLC-MS/MS. Screening strategies focusing on indirect biological responses are considered promising tools to improve surveillance. In this study, an untargeted shotgun proteomics approach based on tandem mass tags (TMTs) is carried out to identify proteins altered in bovine liver after different anabolic treatments. Three controlled pharmacological treatments with dexamethasone, a combination of dexamethasone and clenbuterol, or a combination of sexual steroids (trenbolone and estradiol) are analyzed. Untargeted TMT analysis of liver digests by high resolution MS allowed for the relative quantification of proteins. Thanks to partial least squarediscriminant analysis, a set of proteins capable to classify animals treated with dexamethasone alone (11 proteins), or in combination with clenbuterol (13 proteins) are identified. No significant difference is found upon administration of sexual steroids. After relative quantification of candidate markers by parallel reaction monitoring (PRM), two predictive models are trained to validate protein markers. Finally, an independent animal set of control bulls and bulls treated with dexamethasone is analyzed by PRM to further validate a predictive model giving an accuracy of 100%.

Made available in DSpace on 2018-11-26T15:31:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-11-01 Italian Ministry of Health (Ricerca Corrente) DG SANTE of the European Commission The flagellated protozoan Dientamoeba fragilis is often detected in humans with gastrointestinal symptoms, but it is also commonly found in healthy subjects. As for other intestinal protozoa, the hypothesis that genetically dissimilar parasite isolates differ in their ability to cause symptoms has also been raised for D. fragilis. To date, only two D. fragilis genotypes (1 and 2) have been described, of which genotype 1 largely predominates worldwide. However, very few markers are available for genotyping studies and therefore the extent of genetic variation among isolates remains largely unknown. Here, we performed metagenomics experiments on two D. fragilis-positive stool samples, and identified a number of candidate markers based on sequence similarity to the phylogenetically related species Trichomonas vaginalis. Markers corresponding to structural genes and to genes encoding for proteases were selected for this study, and PCR experiments confirmed their belonging to the D. fragilis genome; two previously described markers (small subunit ribosomal DNA and large subunit of RNA polymerase II) were also included. Using this panel of markers, 111 isolates of human origin were genotyped, all of which, except one, belonged to genotype 1. These isolates had been collected at different times from symptomatic and asymptomatic persons of different age groups in Italy, Denmark, Brazil and Australia. By sequencing approximately 160 kb from 500 PCR products, a very low level of polymorphism was observed across all the investigated loci, suggesting the existence of a major clone of D. fragilis with a widespread geographical distribution. (C) 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved. Ist Super Sanita, Rome, Italy IRCCS San Matteo Hosp Fdn, Lab Parasitol Microbiol & Virol, Pavia, Italy Statens Serum Inst, Dept Microbiol & Infect Control, Copenhagen, Denmark Sao Paulo State Univ, Inst Biosci, Dept Parasitol, Sao Paulo, Brazil Ist Zooprofilatt Sperimentale Umbria & Marche, Parasitol Lab, Perugia, Italy Western Diagnost Pathol, Myaree, WA, Australia Sao Paulo State Univ, Inst Biosci, Dept Parasitol, Sao Paulo, Brazil Italian Ministry of Health (Ricerca Corrente): RC04/12

Blood parasites infect all vertebrates (Clayton and Moore 1997). Avian malaria parasites (Plasmodium spp., Plasmodiidae) are cosmopolitan in their distribution and are responsible for severe diseases in domestic and wild birds.In September 2009, nine raptorial birds that either arrived recently or were maintained as permanent residents at the Safaripark Pombia (northwest Italy) showed loss of stamina, developing listlessness, anorexia and regurgitation. Within one month three animals died and were necropsied.Following the diagnosis of Plasmodium infection all other raptorial birds were treated: clinical improvement was observed in all birds, and blood smears made after one month resulted negative for parasites.

Aim: To develop a multilevel approach that includes different toxicity tests and gene-expression studies for toxicity evaluation of engineered nanomaterials developed for biomedical applications. Materials & methods: K-562, MCF-7 and U-937 human-derived cell lines were used as models for in vitro toxicity tests. These tests included viability assays (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium [MTS] assay); evaluation of apoptosis/necrosis by propidium iodide staining and DNA laddering assay; evaluation of mitochondrial toxicity (5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethyl-benzimidazolcarbocyanine iodide [JC-1] assay); transmission electron microscopy analysis and gene expression analysis by DNA microarray. For in vivo toxicity evaluation, Swiss mice were used for monitoring acute or chronic effects. Two superparamagnetic contrast agents approved for human use (Resovist® and Primovist®) and two new lanthanide-based luminescent nanoparticles were tested. Results & discussion: The nanomaterials approved for human use did not show significant toxicities in our assays. Toxicity studies performed on lanthanide-based nanoparticles (EDTA120 and EDTA120D) complexed with the chelating agent EDTA revealed that these nanomaterials induced necrosis in U-937 and K-562 cells while no toxicity was observed in MCF-7 cells. Moreover, no in vivo effects have been observed. The comparative analysis of the nanomaterials and their separated components showed that the toxicity in U-937 and K-562 cells was mainly due to the presence of EDTA. Conclusion: The multilevel approach proved to be useful for nanomaterial toxicity characterization. In particular, for the lanthanide-based nanoparticles tested in this work, the EDTA was identified as the main cause of the toxicity in vitro, suggesting a possible applicability of these nanoparticle suspensions for in vivo optical imaging.

Summary An outbreak of bovine besnoitiosis in three female, 15–18 months old beef cattle in central Italy is here described. All the animals were born in central Italy without any recent contact with imported animals. The animals were in poor body conditions and showed symptoms and clinical signs consistent with chronic besnoitiosis. The diagnosis was confirmed by histopathologic examinations of skin biopsies and whole body at necropsy, showing typical 50–100 l cysts engulfing superficial dermis in skin and lamina propria in mucosae; lesions were confined to skin and respiratory mucosae, and cysts were not seen in any other tissue. Bovine besnoitiosis is rapidly spreading among European countries and in our case the affected animals were born in the farm and not recent admission was referred, so it is likely to consider this as an autoctone outbreak of the disease in Italy. This case, taken together with other recently reported ones, suggest to consider Italy among potentially endemic areas for bovine besnoitiosis.

This study shows that a diet including insect-resistant Bt176 maize, fed to 53 ewes and their progeny for 3 years, did not have adverse effects on their health or performance and that no horizontal gene transfer to ruminal microorganisms or animal tissues was detected. No differences were observed regarding performance, reproductive traits, haematological parameters, antioxidant defences, lymphocyte proliferative capacity, phagocytosis and intracellular killing of macrophages, and ruminal microbial population characteristics between control and genetically modified (GM) maize-fed animals. Immune response to Salmonella abortus ovis vaccination was more efficient in GM maize fed sheep. No modifications of histological features of tissues were found; however, cytochemical analyses of ruminal epithelium by Ki67 staining provided evidence of proliferative activation of basal cells in all GM maize-fed ewes. Preliminary electron microscopy analyses of the liver and pancreas revealed smaller cell nuclei containing increased amounts of heterochromatin and perichromatin granules in GM maize-fed lambs. Meat protein content and water loss by cooking were slightly affected by the dietary treatment. No transgenic DNA was detected in tissues, blood, and ruminal fluid or ruminal bacteria. Longitudinal studies should be included in evaluation of food safety whenever possible and sheep may be a useful animal model for toxicological assessment.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important cause of acute food- borne zoonoses worldwide, typically carried by pigs. It is well known that Salmonella has evolved a wide array of strategies enabling it to invade the host, but little information is available on the specific host responses to Salmonella infections. In the present study, we used an in vivo approach (involving piglets infected with a virulent or an attenuated S. Typhimurium strain) coupled to histological and proteomic analysis of the cecum mucosa, to highlight the host pathways activated during S. Typhimurium infection. We confirm the complex host-pathogen interaction. Our data showed that the metabolic and the cytoskeleton organization functions were the most significantly altered. In particular, the modifications of energy metabolic pathway could suggest a "nutriprive" mechanism, in which the host reduce its metabolic and energetic status to limit Salmonella infection. This study could represent a preliminary approach, providing information useful to better understand the host-Salmonella interaction.

Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inflamed intestinal environment exploiting inflammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modifications are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inflammation and a reduction of specific protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inflammatory response, was unable to successfully sustain competition with the microbiota.

In Italy Neospora caninum has been reported in cattle, in buffaloes and in dogs. No data are available about the infection in sheep and goats. In this paper, the authors report the detection of protozoan cysts, identified as N. caninum by PCR, in the brain of an aborted goat foetus.