Fabrisio Alustiza

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals.

Transgenic farm animals are attractive alternative mammalian models to rodents for the study of developmental, genetic, reproductive and disease-related biological questions, as well for the production of recombinant proteins, or the assessment of xenotransplants for human patients. Until recently, the ability to generate transgenic farm animals relied on methods of passive transgenesis. In recent years, significant improvements have been made to introduce and apply active techniques of transgenesis and genetic engineering in these species. These new approaches dramatically enhance the ease and speed with which livestock species can be genetically modified, and allow to performing precise genetic modifications. This paper provides a synopsis of enzyme-mediated genetic engineering in livestock species covering the early attempts employing naturally occurring DNA-modifying proteins to recent approaches working with tailored enzymatic systems.

Prevalence of Cryptosporidium spp. and Giardia spp., spatial clustering and patterns of shedding in dairy calves from Córdoba, Argentina.

The objectives of this study were to estimate calf and herd prevalence of Cryptosporidium spp. and Giardia spp., the herd prevalence clustering, spatial distribution according to soil type and shedding patterns in dairy calves from Cordoba, Argentina. Six hundred twenty calves younger than 7 weeks of age from 43 dairy herds were sampled. Samples were processed with the formol-ether and modified Ziehl-Neelsen techniques. Univariate analysis and Kruskall-Wallis tests were used. Factors associated were subjected to multivariate analysis with calf shedding intensity as the response variable. Clustering of herd prevalence was assessed by a scan method, and spatial analysis was applied to explore the overlapping of high prevalence herds and soil type. Overall calf prevalence for Cryptosporidium spp. oocysts and Giardia spp. cysts were 19.35% (95% CI: 16.14; 22.54) and 34.50% (95% CI: 30.69; 38.34), respectively. Calves younger than two weeks of age were almost four times more likely to be infected with Cryptosporidium, in comparison to older ones (RR: 3.78, 95% CI: 2.27; 6.26). Giardia spp. shedding showed a similar age pattern (RR: 1.33, 95% CI: 1.02; 1.75). A primary cluster of high Cryptosporidium prevalence was found, and high prevalence herds were located in areas with poor drained soil.

Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina

Abstract The aim of this study was to perform a current molecular characterization of bovine pathogenic Escherichia coli strains isolated from random samplings in Argentinean dairy farms. Rectal swabs were obtained from 395 (63.7%) healthy and 225 (36.3%) diarrheic calves, belonging to 45 dairy farms in Cordoba Province, Argentina. E. coli isolates were examined for virulence genes (f5, f41, f17, sta, stb, lt, eae, vt) using PCR and the prevalence of E. coli virulence profiles was spatially described in terms of spatial distribution. A total of 30.1% isolates were found to be positive for at least one of the virulence genes. Depending on the different gene combinations present, 11 virulence profiles were found. Most of the isolates analyzed had a single gene, and no combination of fimbrial and enterotoxin gene was predominant. There was no association between the frequency and distribution of E. coli virulence genes and calf health status. Most of the virulence profiles were compatible with ETEC strains and showed a homogeneous distribution over the sampled area. A clustering pattern for E. coli virulence profiles could not be recognized. This work provides updated information on the molecular characterization of pathogenic E. coli strains from dairy herds in Cordoba, Argentina. These findings would be important to formulate prevention programs and effective therapies for diarrhea in calves caused by E. coli.

Characterization of egg yolk immunoglobulin (IgY) against enterotoxigenic Escherichia coli and evaluation of its effects on bovine intestinal cells

Enterotoxigenic Escherichia coli (ETEC) infection is common in calves. Egg yolk antibodies (IgY) have been used to treat gastrointestinal infectious diseases. This study aimed to characterize IgY against bovine ETEC and to evaluate its effects on bovine intestinal cell culture challenged with a bovine ETEC strain. IgY was isolated from the egg yolks of hens immunized with ETEC. The characteristics of IgY were determined by Bradford, ELISA, gel electrophoresis and immunoblotting. Significant differences in anti-ETEC activity between anti-ETEC IgY and non-specific IgY were found in lyophilized fractions. In the bacterial growth assay, anti-ETEC IgY (40 mg/mL) showed growth inhibition of ETEC after 2 h of incubation (p<0.05). The difference in bacterial growth between anti-ETEC IgY and non-specific IgY groups was 0.51 log CFU/ml after an 8 h incubation (p<0.05). The bacterial adhesion assay indicated that anti-ETEC IgY (40 mg/ml) significantly decreased the adhesion of ETEC to bovine intestinal epithelial cells within 4 h (about 1.36 log units compared with the control group; p<0.05). This study demonstrates that anti-ETEC IgY inhibits the growth and adherence of ETEC to bovine intestinal cells and is a potential alternative to traditional treatments of E. coli infections.

Physicochemical properties of ionic and non-ionic biocompatible hydrogels in water and cell culture conditions: Relation with type of morphologies of bovine fetal fibroblasts in contact with the surfaces

Cationic, anionic and non-ionic hydrogels having acrylamide polymer backbones were synthesized via free radical polymerization with N,N-methylenebisacrylamide (BIS) as crosslinker. The chemical structures of the hydrogels were characterized by Fourier Transform Infrared Spectroscopy (FTIR). Physicochemical properties such as swelling kinetic, maximum swelling capacity, volume phase transition temperature (VPTT) and wettability (static water contact angle) of hydrogels swollen in aqueous and cell culture medium, at room and cell culture temperatures were studied. In order to correlate the surface properties of the hydrogels and cellular adhesivity of bovine fetal fibroblasts (BFFs), cellular behaviour was analyzed by inverted fluorescence optical microscopy and atomic force microscopy (AFM). MTT assay demonstrated that the number of viable cells in contact with hydrogels does not significantly change in comparison to a control surface. Flattened and spindle-shaped cells and cell spheroids were the adopted morphologies during first days of culture on different hydrogels. Cell spheroids were easily obtained during the first 5days of culture in contact with PNIPAM-co-20%HMA (poly (N-isopropylacrylamide-co-20%N-acryloyl-tris-(hydroxymethyl)aminomethane)) hydrogel surface. After 15days of culture all hydrogels showed high adhesion and visual proliferation. According to obtained results, non-ionic and hydrophilic surfaces with moderated wettability induce the formation of BFFs cell spheroids. These hydrogel surfaces could be used in clinical and biochemical treatments at laboratory level to cell growth and will allow generating the base for future biotechnologic platform.

Immunohistochemical distribution of early pregnancy factor in ovary, oviduct and placenta of pregnant gilts

Abstract Early pregnancy factor (EPF) is an immunosuppressant that promotes maternal immune system tolerance of the allogenic fetus. Little is known about localization of this factor in different tissues and nothing has been reported about localization in swine reproductive and placental tissues. We determined the concentration of EPF in serum of gilts and porcine placenta conditioned medium (PPCM). We also analyzed the expression of EPF in different reproductive tissues of pregnant gilts at 10, 30, 60 and 90 days of pregnancy. EPF concentration in serum and PPCM was determined by western blot and densitometry. EPF expression in reproductive tissue was assessed by immunohistochemistry. The highest concentration of EPF was observed at 30 days in serum and PPCM; the concentration was higher in PPCM than in serum at the stages we evaluated. All reproductive tissues from the gestational stages analyzed showed specific labeling of EPF, but this labeling did not appear in non-pregnant gilts. At 30 days pregnancy, the EPF expression in the ovary was predominantly in follicular lutein cells, probably owing to its function as a luteotrophic factor. In the oviduct, EPF was expressed in unciliated secretory epithelial cells and in the cilia of ciliated cells. In the placenta, EPF was expressed in the fetal portion (mesoderm chorioallantois and epithelium of endoderm). EPF acts as an autocrine and paracrine growth factor for the trophoblast during the peri-implantation period.

Formation and characterization of Langmuir and Langmuir-Blodgett films of Newkome-type dendrons in presence and absence of a therapeutic compound, for the development of surface mediated drug delivery systems

Organic macromolecules with dendrimeric architectures are polymeric materials potentially useful as nanocarriers for therapeutic drugs. In this work, we evaluate a series of Newkome-type dendrons in Langmuir and Langmuir-Blodgett films as platforms capable of interacting with a potential antitumoral agent. The nanocomposite is proposed as model for the development of surface mediated drug delivery systems. We were successful in the formation and characterization of pure (dendrons) and composite (drug-dendron) stable and reproducible monolayers, and their transfer to solid substrates. A detailed study of topographic characteristics of the generated surfaces by atomic force microscopy was conducted. Furthermore, we probed dendron monolayer films as anchorage surfaces for mammalian cells. Normal cell attachment and proliferation on the surfaces were observed. No evident cytotoxic effects were detected, demonstrating the adequate biocompatibility of the surfaces.

Gut-Associated Lymphoid Tissue: A Key Tissue Inside the Mucosal Immune System of Hens Immunized with Escherichia coli F4

Immunoglobulin Y (IgY) is the predominant antibody found in hen’s (Gallus domesticus) egg yolk. This antibody, developed against several microorganisms in hen egg yolk, has been successfully used as an alternative to immunoglobulins from mammals for use in immunodiagnostics and immunotherapy. Enteropathogenic Escherichia coli (E.coli) F4 is the main etiological agent associated with swine neonatal diarrhea, and it causes notable economic losses in swine production. The aim of the present study was to evaluate the relationship between humoral immune response and the activation of gut-associated lymphoid tissue (GALT) in laying hens intramuscularly immunized with E. coli F4. Adult laying Shaver hens were immunized with a bacterin based on an inactivated lysate E. coli F4 strain that was originally isolated from neonatal piglet diarrhea, following a recommended schedule. The percentage of B lymphocytes in blood and spleen homogenates was determined by flow cytometry. Villi histomorphometry and the size of germinal centers (GC) activated in GALT and the spleen were measured in histological samples either stained with hematoxylin/eosin or through immunofluorescence. Antibody and isotype-specific antibodies in serum and egg yolk were measured using indirect enzyme-linked immunosorbent assay (ELISA). Secretory and serum immunoglobulin A (IgA) were measured by ELISA tests. Laying hen with intramuscular immunization with E. coli F4 lysate, activated both mucosal and systemic protection. Mucosal protection was provided through B lymphocytes, and most of them were activated on Peyer’s patches and esophageal tonsils, in GALT. Furthermore, increased B lymphocyte number in the lamina propria of the gut, and increased intraepithelial plasmatic cell number, produced high levels of mucosal IgA. Activated B lymphocytes interacted with absorptive cells, immune cells, and microbiota in the gut, producing signals that were translated into a powerful physical defense by producing a greater volume of mucin from an increased number of goblet cells. Systemic protection was provided through B lymphocyte activation of spleen GC, which produced hugely specific IgY serum levels. One week later, this specific IgY was deposited in the yolk. This suggests that GALT is a key immunologic tissue inside the mucosal immune system, acting as the “command center” for humoral reaction.

IL-2 is involved in immune response of prenatally stressed rats exposed to postnatally stimulation

espanolRESUMEN: Las condiciones ambientales influyen en el crecimiento y desarrollo de los mamiferos durante la vida prenatal, posnatal temprana y pueden ejercer efectos a largo plazo en la vida adulta. Altas concentraciones circulantes de glucocorticoides durante el embarazo (estres prenatal) afecta a la actividad del eje hipotalamo-pituitario-adrenal (HPA) de la descendencia y se encuentra en relacion con alteraciones de las respuestas del sistema inmunologico. La estimulacion postnatal temprana de los animales estresados prenatalmente genera efectos beneficiosos a largo plazo en la reactividad del eje HPA y en la funcion del sistema inmunologico. El objetivo del estudio fue investigar el efecto de las manipulaciones posnatales tempranas sobre la respuesta inmune de ratas machos y dilucidar la posible relacion con la actividad del eje HPA. Crias controles y estresadas prenatalmente (PS) por inmovilizacion (IMO) se manipularon durante la primera semana de vida. Los animales controles y PS fueron sometidos a estres agudo por la IMO. Los niveles de corticosterona (COR) plasmatica se midieron por el ensayo de RIA, la proliferacion de linfocitos T por [3H] timidina y los niveles de IL-2 por la tecnica de ELISA directa. El estres prenatal cronico por IMO aumento los niveles plasmaticos basales de COR en la madre, mientras que las crias PS sometidas a una sesion de estres agudo posnatal mostraron disminuida la proliferacion de celulas T y niveles menores de IL-2. En conclusion, las estimulaciones postnatales tempranas revirtieron los efectos negativos provocados por el estres prenatal sobre la proliferacion de los linfocitos T y la liberacion de IL-2. EnglishABSTRACT: Environmental cues influence growth and development of mammals during prenatal and particularly early postnatal life and can exert long-lasting effects in adult life. High circulating concentration of glucocorticoids during pregnancy (prenatal stress) affects the activity of the hypothalamic-pituitary-adrenal axis (HPA) of offspring and has been linked to alter immune system responses. Early postnatal stimulation (handling) of prenatally stressed animals generates long-term beneficial effects on the reactiveness of the HPA axis and immune system function. The aim of this study was to further investigate the effect of handling on immune response in prenatally stressed male rats and to elucidate a possible relationship with the HPA axis activity. Control and prenatally stressed (PS) offspring by immobilization (IMO) were handled during the first week of life. Animals from both treatments were subjected to acute stress by IMO. Corticosterone (COR) plasma concentration was measured by RIA assay, T lymphocyte proliferation by [3H] thymidine assay and IL-2 levels by direct ELISA technique. Chronic IMO prenatal stress caused an increase in mother plasma COR basal levels. Furthermore, prenatally stressed rats subjected to an acute stress session had lower T cell proliferation and decreased IL-2 release. In addition, early postnatal stimulation reversed the negative effects of prenatal stress on proliferation of T lymphocytes and IL-2 release.