Halobacteriovorax isolated from marine water of the Adriatic sea, Italy, as an effective predator of Vibrio parahaemolyticus, non-O1/O139 V. cholerae, V. vulnificus.

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.

Dietary Lactobacillus acidophilus positively influences growth performance, gut morphology, and gut microbiology in rurally reared chickens.

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.

A comparative study of the in vitro activity of iodopropynyl butylcarbamate and amphotericin B against Prototheca spp. isolates from European dairy herds.

The objective of this study was to assess the in vitro effect of iodopropynyl butylcarbamate (IPBC) and amphotericin B (AMB) on Prototheca zopfii genotype 2 and Prototheca blaschkeae isolates recovered from dairy herds of Belgium, France, Italy, Germany, and Poland. The combination of IPBC with AMB on Prototheca isolates and toxicity of IPBC to the bovine mammary epithelial cells were also evaluated. The in vitro activity of IPBC and AMB against 96 isolates of P. zopfii genotype 2 and 42 isolates of P. blaschkeae was performed. Minimum inhibitory concentrations (MIC) and minimum algicidal concentrations (MAC) of IPBC and AMB were determined. To determine any synergistic, additive, or antagonistic effect of the combination of IPBC and AMB, 2-dimensional checkerboard combination tests were also performed to calculate fractional inhibitory concentrations. Cytotoxicity analysis of IPBC to the bovine mammary epithelial cell line was performed using a 3-(4,5-dimethyl-2-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MIC for 50 and 90% of isolates (MIC50 and MIC90, respectively) for IPBC were 4 and 8 mg/L versus 0.5 and 1 mg/L for AMB, respectively. The MIC profiles differed between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to both compounds. The MIC50 and MIC90 of IPBC were 4 and 8 mg/L for P. zopfii genotype 2 and 1 and 2 mg/L for P. blaschkeae, respectively. The MIC50 and MIC90 of AMB were both 1 mg/L for P. zopfii genotype 2 and 0.25 and 1 mg/L for P. blaschkeae, respectively. Both IPBC and AMB exhibited the ability to kill Prototheca spp. The MAC for 90% of isolates of IPBC was twice the MIC90, whereas an 8-fold increase of the MIC90 was algicidal in the case of AMB. Overall, the combined use of IPBC and AMB exhibited an increased algicidal effect, albeit the fractional inhibitory concentration index showed synergistic activity only against 3 P. zopfii genotype 2 isolates. For all the remaining isolates (87.5%), this combination produced only an additive effect. The MTT assay results showed both IPBC and AMB, at the concentrations employed in the study, to be nontoxic to the epithelial mammary gland cells (cell viability >90%). Notably, only IPBC at the highest concentration (i.e., 8 mg/L) exerted a slight cytotoxic effect on the cell line tested (mean cell viability: 88.54 ± 3.88 and 90.66 ± 3.0, after 2 and 4 h of MTT treatment, respectively). The anti-Prototheca activity of IPBC was here demonstrated for the first time. In addition, the combined use of IPBC with AMB enhanced each other's effect, creating an additive rather than synergistic interaction. Both agents, used at concentrations corresponding to MIC values against Prototheca spp., showed no toxic effect for the mammary epithelial cells. In conclusion, IPBC, used either alone or in combination with AMB, can be considered a promising option in the treatment armamentarium for protothecal mastitis in dairy cows.

Study on cortisol, cortisone and prednisolone presence in urine of Chianina cattle breed.

The Chianina, one of the oldest and most important cattle breeds of Italy, is now reared all over the world. The Chianina has been known and appreciated since ancient times because, from a nutritional point of view, its meat has no proper rivals. To date, studies have been performed to evaluate the genetic profile of the breed, but knowledge about the chemical profile is generally lacking. Due to the increased interest from farmers regarding breeding of the Chianina, this study proposes a preliminary evaluation of main endogenous urinary corticosteroids (cortisol and cortisone) and most commonly used synthetic one (dexamethasone). Moreover, after recent findings regarding the presence of endogenous prednisolone in the urine of more popular breeds, particular attention was given to analysis of the presence of prednisolone and prednisone, as well. For this aim, the urine samples of 12 young cows and 30 young bulls was collected at the farms and analysed using a fit-for-purpose LC-MS/MS method. The preliminary results of this study show that prednisolone was found only in Chianina females (3 out of 12). Cortisol and cortisone were found at concentrations that showed a high inter-individual variability, and that were higher in female urine compared to that of males.

Effects of two different probiotics on microflora, morphology, and morphometry of gut in organic laying hens.

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 < 0.001). Clostridium spp. decreased (P < 0.001) in both L and B subjects. The probiotic supplementation appeared to affect the intestinal microbial population, promoting the presence of beneficial bacteria such as Lactobacillus spp. and Bifidobacterium spp. and reducing potential harmful bacteria such as E. coli, clostridia and staphylococci. Morphological and morphometric analyses did not reveal substantial differences among groups. At T3, the plasma cell infiltrate in the villi of the CTR hens was more severe than that observed in the L and B groups (P = 0.009).

Immunosuppression in Dogs During Mammary Cancer Development.

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.

Plasmodium spp. In a captive raptor collection of a Safaripark in northwest Italy.

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.

Atypical Yersinia pseudotuberculosis serotype O:3 isolated from hunted wild boars in Italy.

Atypical Yersinia pseudotuberculosis serotype O:3 was isolated from rectal contents of two wild boars hunted in Italy within a regional wildlife management program. No outbreak of yersiniosis was reported in this area in the same period and no lesions were found by the veterinarian at post-mortem inspection. Nevertheless, after histological examination, granulomatous lesions were detected in submandibular lymph nodes of one of the two wild boars. Microbiological and bio molecular characterization of the isolates revealed a melibiose-negative, biotype 2, wbyK+O:3 genotype, carrying inv, yop (yopH and yopB), virF, and R-HPI. Strains showing the same profile, matching to the criteria of genetic group 5, have been recently reported in fatal cases of yersiniosis in cynomolgus macaques and in farmed deer and atypical O:3 serotype has been suggested as a pathogenic subtype of O:3. This is the third report of an atypical O:3 Y. pseudotuberculosis strain, the first outside the American continent and the first one not associated to fatal yersiniosis. Wild boars could be a possible reservoir of this emerging pathogen.

Canine pancreatic clear acinar cell carcinoma showing an unusual mucinous differentiation.

A small solitary pancreatic nodule was identified in a 10-year-old mixed breed male dog. Microscopically, the lesion consisted of neoplastic clear acinar cells with diffuse, marked mucin production. Immunohistochemical and ultrastructural investigations confirmed the exocrine acinar origin of the tumour. The tumour was classified as a pancreatic clear acinar cell carcinoma (ACC) showing an unusual mucinous differentiation.

An outbreak of bovine besnoitiosis in beef cattle born in central Italy.

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 µ 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.

Cancer antigen 15/3: possible diagnostic use in veterinary clinical oncology. Preliminary study.

The cancer antigen 15/3 is a mucin that is associated with the cell membrane, encoded by the MUC1 gene, and recognized by the monoclonal-clone DF3 antibody. The latter antigen was discovered to be specific for both the identification of human mammary neoplasia and during patient follow-up evaluations. The aim of this study is to report and compare the results of the application of direct chemiluminescence in canine blood sera and the kit utilized in human medicine for the determination of Ca 15/3 to verify the diagnostic efficiency of the kit in cases presenting mammary tumors. Specifically, CA 15/3 has proven to be measurable in all samples assayed to distinguish clinically healthy subjects from those with mammary neoplasia.

Dientamoeba fragilis in swine population: a preliminary investigation.

Dientamoeba fragilis, a protozoan with worldwide distribution is considered to be responsible for enteric disease in humans. A wide spectrum of clinical symptoms including; diarrhoea (acute or prolonged), flatulence, abdominal pains and other unspecific bowel symptoms have been ascribed to this parasite. Asymptomatic infection has also been reported. Dientamoeba fragilis is as its name indicates an extremely delicate protozoon and only the trophozoite has ever been demonstrated in stool samples. The definitive diagnosis of this infection is based on demonstration in permanently stained stool samples. In Italy examination of ova and parasite (O&P) samples are not currently performed. This protozoan is extremely difficult to cultivate but molecular techniques such as the Polymerase Chain Reaction offer promise as a means of diagnosing infection. The epidemiology of Dientamoebiasis is not clear. This paper will present preliminary results from a study looking for the parasite's presence in swine faeces. The possible role of pigs as a reservoir of infection was studied; 121 faecal samples from breeding and fattening pigs were examined using a Giemsa permanent stain. Dientamoeba fragilis was found in 53 (43.8%) of the stool samples examined.

Reversibility of hepatocyte nuclear modifications in mice fed on genetically modified soybean.

In the literature, the reports on the effects of a genetically modified (GM) diet are scanty and heterogeneous; in particular, no direct evidence has so far been reported that GM food may affect human or animal health. Hepatocytes represent a suitable model for monitoring the effects of a GM diet, the liver potentially being a primary target. In a previous study, we demonstrated that some modifications occur in hepatocyte nuclei of mice fed on GM soybean. In order to elucidate whether such modifications can be reversed, in the present study, 3 months old mice fed on GM soybean since their weaning were submitted to a diet containing wild type soybean, for one month. In parallel, to investigate the influence of GM soybean on adult individuals, mice fed on wild type soybean were changed to a GM diet, for the same time. Using immunoelectron microscopy, we demonstrated that a one-month diet reversion can influence some nuclear features in adult mice, restoring typical characteristics of controls in GM-fed animals, and inducing in control mice modifications similar to those observed in animals fed on GM soybean from weaning. This suggests that the modifications related to GM soybean are potentially reversible, but also that some modifications are inducible in adult organisms in a short time.

Unusual finding in a nipple discharge of a female dog: dirofilariasis of the breast.

We report a case of dirofilariasis of the breast occurring in a 6-yr-old Bernese female dog living in central Italy presenting with a breast lump, diagnosed on cytological examination of nipple secretion fluid smears. The direct smears of nipple discharge revealed clusters of glandular cells characterized by mild pleomorphic appearance and the presence of some nematodes of about 350 microm in length in the background. The smears also contained several inflammatory cells, including eosinophils. Both the Knott and the antigen tests were negative for Dirofilaria immitis. The morphological features of this round worm is compatible with that of Dirofilaria repens. The case describes an unusual finding of dirofilariasis presenting as a breast lump, simulating an inflammatory breast carcinoma.

The effect of Mycoplasma mycoides ssp. mycoides LC of bovine origin on in vitro fertilizing ability of bull spermatozoa and embryo development.

Several Mycoplasma species may adversely affect bovine spermatozoa viability and embryo development. Mycoplasma mycoides ssp. mycoides large-colony (LC) has been isolated from naturally aborted bovine fetuses and from bull semen. The objective of this study was to evaluate whether M. mycoides ssp. mycoides LC contaminated bovine ejaculates could (i) impair in vitro fertilizing ability of bull spermatozoa, (ii) impair embryo development, and (iii) evaluate potential spread by reproductive technologies. In the present study, spermatozoa of 10 fertile bulls were contaminated with M. mycoides ssp. mycoides LC, at a final concentration of 1.5 million CFU/ml and incubated for 60 min before evaluating spermatozoa motility and acrosome reaction inducibility with calcium ionophore. In addition, in vitro contaminated semen of a bull previously shown to have a good in vitro fertilizing ability, was used in an IVF procedure. Embryo development stage on Day-7 of culture was evaluated. Spermatozoa and embryos at morula and blastocyst stages were routinely processed for transmission electron microscopy observation. Both mean total and progressive motility decreased (P < 0.01 ) upon spermatozoa incubation with Mycoplasma. One-hour incubation with calcium ionophore increased the percentage of acrosome-reacted spermatozoa, although Mycoplasma contamination reduced calcium ionophore treatment efficacy (P < 0.05). Ultrastructurally, Mycoplasma microorganisms appeared as moderately electron-dense sphere-shaped particles, adhering to cell membranes. Sperm mid-piece sections showed numeric aberrations of the central singlets such as nine + zero or nine + one of the axonemal complex. Further morphological abnormalities included partial or total absence of dinein arms and radial fibers, with lack of the bridge and the central ring in 35.00 +/- 4.20% of contaminated cells, whereas these abnormalities were not observed in uninfected ones. The IVF trials showed that two-four cell blocks were higher (P < 0.05) in the infected group. Ultrastructure of Day-7 contaminated embryos showed Mycoplasma particles adhering and infiltrating the outer layer of the zona pellucida. Our investigations suggest that M. mycoides ssp. mycoides LC contaminating the bovine ejaculate induced adverse effects on in vitro spermatozoa-fertilizing ability and embryonic development. Some satisfactory quality transferable embryos could be produced in contaminated IVF systems. This could imply a potential transmission of this microorganism through reproductive technologies.

Detection of Neospora caninum in an aborted goat foetus.

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.

Fine structural analyses of pancreatic acinar cell nuclei from mice fed on genetically modified soybean.

We carried out ultrastructural morphometrical and immunocytochemical analyses on pancreatic acinar cell nuclei from mice fed on genetically modified (GM) soybean, in order to investigate possible structural and molecular modifications of nucleoplasmic and nucleolar constituents. We found a significant lowering of nucleoplasmic and nucleolar splicing factors as well as a perichromatin granule accumulation in GM-fed mice, suggestive of reduced post-transcriptional hnRNA processing and/or nuclear export. This is in accordance to already described zymogen synthesis and processing modifications in the same animals.