Luciana Rossi

Effects of retinoids on proliferation and plasminogen activator expression in a bovine mammary epithelial cell line

Effects of two natural (retinol and retinoic acid, RA) and one synthetic N-(4-hydroxyphenyl) retinamide (4-HPR) retinoids on proliferation and expression of urokinase-plasminogen activator (u-PA) by bovine mammary epithelial cells were examined. The BME-UV1 established bovine mammary epithelial cell line was used as a model system. All retinoids tested (retinol, RA and 4-HPR) were effective inhibitors of cell proliferation. When cells were cultured in the absence of fetal bovine calf serum (FBCS), inhibition occurred at concentrations as low as 1 nM for all retinoids tested. The effect of retinoids on cell proliferation was not dose-related when cells were cultured in the absence of FBCS. All retinoids (retinol, RA, 4-HPR), when used in the range 1 nM-10 microM (noncytotoxic concentrations), were equally effective and had identical inhibition patterns. Inhibition of cell proliferation by RA was apparent by 6 h and was higher after 24 h in culture. In contrast, when cells were cultured in the presence of FBCS, the effect of RA and retinol on cell proliferation was dose-related. RA and retinol inhibited cell proliferation (P<0.01) when added to the culture medium in concentrations as low as 10 nM and 100 nM, respectively. 4-HPR was inhibitory (P<0.01) in concentrations as low as 1 nM. Higher concentrations of 4-HPR in the range 1 nM-1 microM had no further effect on cell proliferation. None of the retinoids tested, when added to cultures in the presence or absence of FBCS, could completely arrest cell proliferation at noncytotoxic concentrations. RA at 1 microM inhibited (P<0.05) insulin or IGF-I-induced cell proliferation but had no effect (P>0.05) on u-PA mRNA levels or u-PA activity. Furthermore, RA inhibited cell proliferation in the presence of FBCS but had no effect (P>0.05) on u-PA mRNA levels. Thus, retinoids are effective inhibitors of bovine mammary epithelial cell proliferation and this growth inhibition does not seem to correlate with any changes in u-PA mRNA or u-PA activity.

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

Evaluation of the Fourier Frequency Spectrum Peaks of Milk Electrical Conductivity Signals as Indexes to Monitor the Dairy Goats’ Health Status by On-Line Sensors

The aim of this study is a further characterization of the electrical conductivity (EC) signal of goat milk, acquired on-line by EC sensors, to identify new indexes representative of the EC variations that can be observed during milking, when considering not healthy (NH) glands. Two foremilk gland samples from 42 Saanen goats, were collected for three consecutive weeks and for three different lactation stages (LS: 0–60 Days In Milking (DIM); 61–120 DIM; 121–180 DIM), for a total amount of 1512 samples. Bacteriological analyses and somatic cells counts (SCC) were used to define the health status of the glands. With negative bacteriological analyses and SCC < 1,000,000 cells/mL, glands were classified as healthy. When bacteriological analyses were positive or showed a SCC > 1,000,000 cells/mL, glands were classified as NH. For each milk EC signal, acquired on-line and for each gland considered, the Fourier frequency spectrum of the signal was calculated and three representative frequency peaks were identified. To evaluate data acquired a MIXED procedure was used considering the HS, LS and LS × HS as explanatory variables in the statistical model.Results showed that the studied frequency peaks had a significant relationship with the gland’s health status. Results also explained how the milk EC signals’ pattern change in case of NH glands. In fact, it is characterized by slower fluctuations (due to the lower frequencies of the peaks) and by an irregular trend (due to the higher amplitudes of all the main frequency peaks). Therefore, these frequency peaks could be used as new indexes to improve the performances of algorithms based on multivariate models which evaluate the health status of dairy goats through the use of gland milk EC sensors.

On-Line Monitoring of Milk Electrical Conductivity by Fuzzy Logic Technology to Characterise Health Status in Dairy Goats

Intramammary infection affects the quality and quantity of dairy goat milk. Health status (HS) and milk quality can be monitored by electrical conductivity (EC). The aim of the study was to determine the detection potential of EC when measured on-line on a daily basis and compared with readings from previous milkings. Milk yields (MYs) were investigated with the same approach. To evaluate these relative traits, a multivariate model based on fuzzy logic technology – which provided interesting results in cows – was used. Two foremilk samples from 8 healthy Saanen goats were measured daily over the course of six months. Bacteriological tests and somatic cells counts were used to define the HS. On-line EC measurements for each gland and MYs were also considered. Predicted deviations of EC and MY were calculated using a moving-average model and entered in the fuzzy logic model. The reported accuracy has a sensitivity of 81% and a specificity of 69%. Conclusions show that fuzzy logic is an interesting approach for dairy goats, since it offered better accuracy than other methods previously published. Nevertheless, specificity was lower than in dairy cows, probably due to the lack of a significant decrease of MY in diseased glands. Still, results show that the detection of the HS characteristics with EC is improved, when measured on-line, daily and compared with the readings from previous milkings.

Antigenic recombinant proteins expressed in tobacco seeds as a model for edible vaccines against swine oedema

L. Rossi1, A. Baldi1*, V. Dell’Orto1 and C. Fogher2 1Department of Veterinary Sciences and T echnology for Food Safety, Faculty of Veterinary Medicine, University of Milan, V ia Celoria 10, 20133, Milano; 2Botanic and Genetic Institute, U.C.S.C., 29100, Piacenza, Italy *Correspondence: Dipartimento di Scienze e T ecnologie Veterinarie per la Sicurezza Alimentare, Facoltà di Medicina Veterinaria, V ia T rentacoste 2, 20133 Milano, Italy E-mail: antonella.baldi@unimi.it

Evaluation of the damage induced by ochratoxin A and the protective role of α-tocopherol in cultured bovine mammary epithelial cells

Evaluation of the damage induced by ochratoxin A and the protective role of α-tocopherol in cultured bovine mammary epithelial cells E. Fusi & R. Rebucci & C. Pecorini & L. Rossi & F. D’Ambrosio & A. Baldi Published online: 7 August 2008 # Springer Science + Business Media B.V. 2008

Signal spectral analysis to characterize gland milk electrical conductivity in dairy goats

Intramammary infection affects quality and quantity of milk. Having as final target the improving of animal health’ monitoring, this research studied the gland milk electrical conductivity (EC) signal in order to identify a possible parameter more representative of the EC variations that can be observed, during a milking, when not healthy (NH) glands are considered. Two foremilk gland samples, from 40 Saanen goats, were acquired for three weeks and lactation stages (LS: 0-60 Days In Milking; 61-120 DIM; =>120 DIM), for a total amount of 1440 samples. Bacteriological analyses and somatic cells counts (SCC) were used to define glands health status. In case of negative bacteriological analyses and SCC <1,000,000 cells/mL, glands were classified as healthy; alternatively, when bacteriological analyses were positive or SCC higher than 1,000,000 cells/mL, for two or more consecutive days, glands were classified as NH. A spectral analysis, to calculate the frequency spectrum and the bandwidth length of the milk EC signal, was performed. To validate data acquired, A MIXED procedure was used considering the HS, LS and the LS x HS as explanatory variables of the statistical model. Results showed that spectral analysis allows characterizing the milk EC variations thorough the bandwidth length parameter. This parameter has a significant relationship with the gland health status and it provides more accurate information than other traits, like the statistical variance of the signal. Therefore, it could be useful to improve the performances of multivariate models/algorithms that detect dairy goat health status.

Plant bioreactors for the antigenic hook-associated flgK protein expression

Plants engineered with genes encoding for the antigenic proteins of various microorganisms have shown to correctly express the proteins that elicit the production of antibodies in mammalian hosts. In livestock, plant-based vaccines could represent an innovative strategy for oral vaccination, especially to prevent infection by enteric pathogens. The aim of this study was to evaluate tobacco plants as a seed-specific expression system for the production of the flgK flagellar hook-associated protein from a wild type Salmonella typhimurium strain, as a model of an edible vaccine. The flgK gene is the principal component of bacterial flagella and is recognised as virulence factor by the innate immune system. It was isolated from the Salmonella typhimurium strain by PCR. The encoding sequence of flgK was transferred into a pBI binary vector, under control of soybean basic 7S globulin promoter for the seed-specific. Plant transformation was carried out using recombinant EHA 105 Agrobacterium tumefaciens. A transgenic population was obtained made up of independently kanamycin-resistant transgenic plants, which had a similar morphological appearance to the wild-type plants. Molecular analyses of seeds confirmed the integration of the gene and the average expression level of flgK was estimated to be about 0.6 mg per gram of seeds, corresponding to 0.33% of the total amount of soluble protein in tobacco seeds. This study showed that the foreign flgK gene could be stably incorporated into the tobacco plant genome by transcription through the nuclear apparatus of the plant, and that these genes are inherited by the next generation.

Milk Choline, α-Tocopherol and Neutrophil Chemotaxis in the Periparturient Dairy Cow

L. Pinotti*, R. Rebucci, E. Fusi, L. Rossi and A. Baldi Department of Veterinary Sciences and T echnology for Food Safety, Faculty of Veterinary Medicine, University of Milan, Milan, Italy *Correspondence: Dipartimento di Scienze e T ecnologie Veterinarie per la Sicurezza Alimentare, Facolta di Medicina Veterinaria, Universita degli Studi di Milano, V ia T rentacoste 2, 20134 Milano, Italy E-mail: luciano.pinotti@unimi.it

Evaluation the of PL-PG-PA system in relation to quality of bovine milk.

Evaluation the of PL-PG-PA system in relation to quality of bovine milk L. Rossi & F. Galante & E. Fusi & M. Luini & V. Dell’Orto & A. Baldi Published online: 17 July 2009 # Springer Science + Business Media B.V. 2009