Nanogel-Mediated antigen delivery: Biocompatibility, immunogenicity, and potential for tailored vaccine design across species.

Nanotechnology has emerged as a promising avenue for enhancing the efficacy of vaccine delivery systems. This study investigates the utilization of nanogels as carriers for the model antigen ovalbumin, with a focus on in vivo assessments in equine and murine models. Nanogels, owing to their biocompatibility and tunable physicochemical properties, offer a versatile platform for efficient antigen encapsulation and controlled release. The encapsulation efficiency and physicochemical characteristics of ovalbumin-loaded nanogels were comprehensively characterized. In vitro biocompatibility was evaluated, finding excellent properties of these nanogels. In vivo evaluations were conducted on both equine and murine subjects, assessing immunogenicity through antibody and splenic cell response. Furthermore, the study propose the potential use of nanogels in tailoring immune responses through the modulation of antigen release kinetics. The results obtained in the in vitro assays showed an increase in the uptake of nanogels by APCs compared to free antigen (OVA). In mice, an absence of inflammatory response in the inoculation site was observed, without systemic damage in the evaluated organs. In addition, non-significant humoral response was found nor cellular proliferation and proinflammatory cytokine production, compared with a traditional adjuvant as aluminum hydroxide, in both animal models. These findings allow further insights into nanogel-based delivery systems and offer valuable insights into their application in various animal models. In conclusion, this research establishes the utility of nanogels as effective carriers for antigens-based vaccines, with interesting biocompatibility properties and highly taken affinity by antigen-presenting cells, without inducing inflammation at the injection site. The study underscores the potential of nanogel technology in revolutionizing vaccine design and highlights the importance of tailored approaches for diverse target species.

Evaluation of oral supplementation of free and nanoencapsulated Minthostachys verticillata essential oil on immunological, biochemical and antioxidants parameters and gut microbiota in weaned piglets.

Early weaning is an important stressor that impairs the piglet´s health, and essential oils appear as promising candidates to improve it instead of antibiotics. The aim of this study was to evaluate the effect of oral supplementation of free and nanoencapsulated Minthostachys verticillata essential oil (EO and NEO, respectively) on immunological, biochemical and antioxidants parameters as well as on gut microbiota in weaned piglets. EO was extracted by hydrodistillation and nanoencapsulation was performed by high-energy method using Tween 80 and Span 60 as surfactants. EO and NEO were chemically analyzed by gas chromatography-mass spectrometry (GC-MS). The cytotoxic effects of both EO and NEO was evaluated on Caco-2 cell line. For in vivo assay, male weaned piglets (age: 28 days, mean initial body weight: 11.63 ± 0.37 kg) were randomly distributed in six groups of six animals each (n = 6) and received orally EO (10.0 mg/kg/day) or NEO (2.5, 5.0 and 10.0 mg/kg/day), named hereinafter as EO-10, NEO-2.5, NEO-5 and NEO-10, for 30 consecutive days. Animals not treated or treated with surfactants mixture were evaluated as control and vehicle control. Subsequently, histological, hematological and biochemical parameters, cytokines production, oxidative markers, CD4+/CD8+ T cells and gut microbiota were evaluated. GC-MS analysis was similar in both EO and NEO. The NEO was more toxic on Caco-2 cells than EO. Oral supplementation of EO-10 or NEO-10 improved growth performance compared to control group NEO-2.5 or NEO-5 (p < 0.05) groups. NEO-2.5, NEO-5 and NEO-10 did not alter the morpho-physiology of digestive organs and decreased malondialdehyde (MDA) levels in liver compared to control (p < 0.05) or EO-10 groups (p < 0.05, p < 0.01). In addition, NEO-10 showed an increase in CD4+/CD8+ T cells ratio (p < 0.001), and induced the highest serum levels of IL-10 (p < 0.01). Serum triglycerides levels were significantly lower in animals treated with EO-10 or NEO-2.5, NEO-5 and NEO-10 compared to control group (p < 0.001). Gut microbiota analysis showed that NEO-10 favor the development of beneficial intestinal microorganisms to improve parameters related to early weaning of piglets. In conclusion, EO and NEO improved parameters altered by early weaning in piglets however, NEO was safer and powerful. Therefore, NEO should be further studied to be applied in swine health.

Development and biological evaluation of pNIPAM-based nanogels as vaccine carriers.

"Smart" nanogels are an attractive tool for the development of new strategies of immunization in veterinary medicine. Here, we reported the synthesis and physicochemical characterization of thermoresponsive nanogels based on poly(N-isopropylacrylamide) (pNIPAM) and theirin vitro, ex vivoand in vivo (mice model) performance. Smart nanogels of ca. 250 nm, with a transition temperature of 32 °C were obtained by precipitation polymerization. Assays to evaluatepNIPAM nanogels cytotoxicity were performed in different cell lines showing high biocompatibility (>70 %). The efficient internalization of the system was studied by confocal microscopy as well as flow cytometry. The ability to protect and deliver antigens was analyzed using the outer membrane lipoprotein A (OmlA), an important virulence factor ofActinobacillus pleuropneumoniae(App)cause of porcine pleuropneumonia. This lipoprotein was synthesized by recombinant technology and its technique was also described. The biodistribution ofpNIPAM nanogels administered intranasally was performedinvivo and ex vivo through Pearl Imaging System, which showed that nanogels were kept mostly in the lungs during the evaluated time. Besides, the efficacy of the proposal nanogel-based vaccine was studiedin vivoby measuring the antibody titers of BALB/c mice inoculated with OmlA encapsulated intopNIPAM nanogels compared to OmlA plus aluminum hydroxide adjuvant. The results proved the ability of nanogels to stimulate a humoral immune response. Therefore, we have demonstrated thatpNIPAM nanogels can be used as an efficient platform for vaccine nanocarriers.

Langmuir-Blodgett monolayers holding a wound healing active compound and its effect in cell culture. A model for the study of surface mediated drug delivery systems.

Langmuir and Langmuir-Blodgett films holding a synthetic bioinspired wound healing active compound were used as drug-delivery platforms. Palmitic acid Langmuir monolayers were able to incorporate 2-methyltriclisine, a synthetic Triclisine derivative that showed wound healing activity. The layers proved to be stable and the nanocomposites were transferred to solid substrates. Normal human lung cells (Medical Research Council cell strain 5, MRC-5) were grown over the monomolecular Langmuir-Blodgett films that acted as a drug reservoir and delivery system. The proliferation and migration of the cells were clearly affected by the presence of 2-methyltriclisine in the amphiphilic layers. The methodology is proposed as a simple and reliable model for the study of the effects of bioactive compounds over cellular cultures.

Characterization and comparison of strains of Pasteurella multocida associated with cases of progressive atrophic rhinitis and porcine pneumonia in Argentina.

BACKGROUND: Pasteurella multocida (Pm) is the causative agent of progressive atrophic rhinitis (PAR) and pneumonic pasteurellosis (PN) in pigs. Pm is a member of the porcine respiratory complex responsible for important economic loss in the pig industry. AIM: This study aimed to characterize the Pm strains recovered from clinical cases of PN and PAR and to elucidate the antibiotic susceptibility profiles of the strains. MATERIALS AND METHODS: Sixty strains were characterized molecularly by polymerase chain reaction to determine species-specific gene, capsular type (A or D), and toxin A production. The agar diffusion method was employed to evaluate antibiotic resistance profiles. RESULTS: We found that 65% of strains belonged to capsular type A or D, and 15% of those were positive to toxA gene. The antibiotic susceptibility profiles found were sensitive in decreasing order to: Enrofloxacin, ceftiofur (CTF), ampicillin, tilmicosin (TIL), florfenicol (FFN), spectinomycin (SPC), gentamicin, oxytetracycline (OTC), and trimethoprim-sulfamethoxazole (TMS). Strains were resistant in decreasing order to: Lincomycin (LIN), tylosin (TYL), erythromycin (ERY), TMS, SPC, OTC, FFN, TIL, and CTF. CONCLUSION: The toxA gene was detected in many Pm isolates from pneumonic lungs. Capsule type A or D was the most frequently found among the collected isolates. LIN, TYL, and ERY are the drugs which showed higher percentages of resistant isolates.

Formation of dendrimer-guest complexes as a strategy to increase the solubility of a phenazine N, N'-dioxide derivative with antitumor activity.

Poly(amidoamine) and Poly(propylenimine) dendrimers with different generations and peripheral groups were studied as solubility enhancers and nanocarriers for 7-bromo-2-hydroxy-phenazine N 5,N 10-dioxide. This compound possesses potential antitumoral and anti-trypanosomal activity, but its low solubility in physiological media precludes its possible application as therapeutic drug. The amino terminated dendrimers association with the active compounds as observed trough NMR studies showed that electrostatic interactions are essential in the solubilization enhancement process. The obtaining of a stable and no cytotoxic formulation makes the drug-carried association a suitable strategy for the generation of a drug delivery system for phenazine derivatives.

Cytotoxicity and bioadhesive properties of poly-N-isopropylacrylamide hydrogel.

Several hydrogel surfaces present properties that simulate the mechanical and physicochemical features of extracellular matrix (ECM), providing a platform that mimic the native cellular milieus. Poly-N-isopropylacrylamide (PNIPAM) hydrogels are receiving attention in biomedical field due to their thermosensibility and soft texture. However, more extensive biocompatibility and cellular interactions studies with cell lines are needed. Therefore, the aim of this study is focus on evaluating the biocompatibility of PNIPAM through cytotoxicity, genotoxicity, and proliferation tests in murine preadipose cells (3T3-L1), human embryonic kidney cells (HEK293) and human carcinoma-derived cells (A549) in presence of hydrogel surfaces. Bioadhesive capacity above PNIPAM surfaces was also analyzed. MTT and neutral red uptake assays shown non-cytotoxic effect of PNIPAM in the studied cell lines. Genotoxicity was evaluated by the single-cell gel electrophoresis assay, where DNA damages were not detected. [3H]-thymidine staining allowed to corroborate that cell proliferation had progressed correctly. Adopted morphologies for each cell line over PNIPAM were similar to cell growing observed on polystyrene, indicating that the surfaces favor the cell attachment during 5 days' culture. The good biocompatibility of PNIPAM surfaces make it an interesting scaffold with clinical potential in tissue regeneration engineering, and a possible adipose and kidney tissue-engineered construct.

Nanocomposites based on pH-sensitive hydrogels and chitosan decorated carbon nanotubes with antibacterial properties.

The present work aimed to study the properties of a novel nanocomposite with promising biomedical applications. Nanocomposites were prepared by the addition of different concentrations of chitosan decorated carbon nanotubes to acrylamide-co-acrylic acid hydrogels. The nanocomposites chemical structure was characterized by Fourier Transform Infrared Spectroscopy (FT-IR). The FT-IR shows the typical bands due to the hydrogel and additionally the peaks at 1750 cm-1 and 1450 cm-1 that correspond to the carbon nanotubes incorporated into the polymer matrix. Mechanical properties and swelling measurements in different buffer solutions were also performed. The nanocomposites showed improved mechanical properties and a stronger pH-response. In order to evaluate antimicrobial activity, the growth and adhesion of Staphylococcus aureus to nanocomposites were studied. Cytocompatibility was also evaluated by MTT assay on MDCK and 3T3 cell lines. The nanocomposites were found to be cytocompatible and showed a reduced bacterial colonization.

Presence and characterization of Escherichia coli virulence genes isolated from diseased pigs in the central region of Argentina.

BACKGROUND: The main pathogen of neonatal and post weaning diarrhea and edema disease (ED) is Escherichia coli and pathotypes involved are enterotoxigenic, enteropathogenic, and shiga toxigenic (ETEC, EPEC, and STEC, respectively). Those diseases cause economic loss in pig production. AIM: The aim of this work was to evaluate the presence of strains expressing virulence markers genes and the antibiotic susceptibility profiles of E. coli from clinical cases of post weaning diarrhea and ED in farms in the central area of Argentina. MATERIALS AND METHODS: Intensive pig farms from the central region of Argentina were sampled. Intestinal mucosa swabs from pigs with diarrhea were taken, seeded on MacConkey agar plates, biochemically typified and tested by polymerase chain reaction (PCR). Antibiograms were made by disk-diffusion method. RESULTS: A total of 54 strains from clinical cases studied showed PCR findings: 88.88% (48/54) expressed at least one gene coding for a virulence factor. Colonization factors found were: 39.58% of strains had F18, 33.33% were F4 and 31.25% adhesin involved in diffuse adherence-I; 29.17%, 25%, and 2.1% expressed LT, STb, and STa, respectively. 25% were STx and 16.67% were eae positive. Only 2.1% were STx2. The most active antibiotics against most strains were gentamicin and ceftiofur, but resistance profiles against many antibiotics were found. CONCLUSION: High circulation of pathogens strains of E. coli among pigs with diarrhea with an extended antibiotic resistance profile.

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.

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.

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.

IgY against enterotoxigenic Escherichia coli administered by hydrogel-carbon nanotubes composites to prevent neonatal diarrhoea in experimentally challenged piglets.

In previous studies, the applicability of polymeric hydrogels for the protection of egg yolk immunoglobulin (IgY) against simulated gastric conditions was established. Thereafter, the performance of the hydrogels was improved with the addition of chitosan wrapped carbon nanotubes and the in vitro toxicity for porcine intestinal cells of these nanocomposites was assessed. The objective of the present study was to evaluate in vivo the protective efficacy of the nanocomoposite matrix for IgY when the immunoglobulin is used against enterotoxigenic Escherichia coli (ETEC) in challenged piglets. Groups of piglets orally challenged with 10(11)CFU/mL of ETEC were treated with non-protected and protected IgY. The clinical response of each group was monitored and evaluated in terms of dehydration, rectal temperature, faecal consistency score and body weight gain. Blood parameters and histological aspects were also studied. The results showed that treatment of infected piglets with protected IgY reduced significantly the severity of diarrhea. Non-protected IgY group show a lower recovery rate. Blood parameters and histological aspects were normal in both groups. Collectively, these results support previous in vitro studies showing that the nanocomposites can be an effective method of IgY protection against gastric inactivation.

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

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.

Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina / Detección molecular de cepas patógenas de Escherichia coli aisladas de terneros neonatos de tambo en la provincia de Córdoba, Argentina

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.

El objetivo de este trabajo fue realizar una caracterización molecular actualizada de cepas patógenas bovinas de Escherichia coli aisladas de un muestreo aleatorio en tambos de una de las principales zonas lecheras de Argentina. Se obtuvieron hisopados rectales de 395 terneros neonatos sanos (63,7 %) y 225 diarreicos (36,3 %) pertenecientes a 45 tambos de la provincia de Córdoba, Argentina. Los genes de virulencia f5, f41, f17, sta, stb, lt, eae y vt se analizaron mediante PCR y se investigó la prevalencia de los perfiles de virulencia en función de la distribución geográfica. La prevalencia de aislamientos de E. coli patogénicos con al menos un gen de virulencia fue del 30,1 %. Once perfiles de virulencia fueron identificados, dependiendo de la combinación de genes presentes. La mayor parte de las muestras presentó un solo gen de virulencia, y no predominó ninguna combinación de genes de fimbrias y toxinas. No hubo asociación entre la frecuencia y la distribución de los genes de virulencia y el estado de salud de los terneros. La mayoría de los perfiles de virulencia fueron compatibles con cepas ECET y se distribuyeron cubriendo toda el área geográfica muestreada. No se reconoció ningún patrón de agrupamiento espacial para dichos perfiles. Este trabajo provee información actualizada sobre la caracterización molecular de E. coli patógena en rodeos lecheros de Córdoba, Argentina. Estos resultados serían importantes para formular programas preventivos y terapias eficaces contra la diarrea bovina causada por E. coli.

Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina / Detección molecular de cepas patógenas de Escherichia coli aisladas de terneros neonatos de tambo en la provincia de Córdoba, Argentina

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.(AU)

El objetivo de este trabajo fue realizar una caracterización molecular actualizada de cepas patógenas bovinas de Escherichia coli aisladas de un muestreo aleatorio en tambos de una de las principales zonas lecheras de Argentina. Se obtuvieron hisopados rectales de 395 terneros neonatos sanos (63,7 %) y 225 diarreicos (36,3 %) pertenecientes a 45 tambos de la provincia de Córdoba, Argentina. Los genes de virulencia f5, f41, f17, sta, stb, lt, eae y vt se analizaron mediante PCR y se investigó la prevalencia de los perfiles de virulencia en función de la distribución geográfica. La prevalencia de aislamientos de E. coli patogénicos con al menos un gen de virulencia fue del 30,1 %. Once perfiles de virulencia fueron identificados, dependiendo de la combinación de genes presentes. La mayor parte de las muestras presentó un solo gen de virulencia, y no predominó ninguna combinación de genes de fimbrias y toxinas. No hubo asociación entre la frecuencia y la distribución de los genes de virulencia y el estado de salud de los terneros. La mayoría de los perfiles de virulencia fueron compatibles con cepas ECET y se distribuyeron cubriendo toda el área geográfica muestreada. No se reconoció ningún patrón de agrupamiento espacial para dichos perfiles. Este trabajo provee información actualizada sobre la caracterización molecular de E. coli patógena en rodeos lecheros de Córdoba, Argentina. Estos resultados serían importantes para formular programas preventivos y terapias eficaces contra la diarrea bovina causada por E. coli.(AU)

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.

Frequency of virulence genes of Escherichia coli among newborn piglets from an intensive pig farm in Argentina.

The enterotoxigenic and porcine enteropathogenic Escherichia coli (EtEc and PEPEc) strains are agents associated with swine neonatal diarrhea, causing economic losses in swine production. The main goal of this study was to identify virulence genes of EtEc, verotoxigenic (VtEc) and PEPEc in intestinal strains responsible for swine diseases, by molecular typing using Pcr in newborn piglets from an intensive farm system. Two hundred and sixty seven rectal swabbings from 7-15 days- old landrace x large White crossbred piglets were taken, and 123 randomly selected samples, biochemically compatible with E. coli, were tested for E. coli virulence genes by Pcr. A frequency (%) compatible with: 68 EtEc, 24 VtEc, and 8 EPEc were found. of all E. coli strains studied, 19.51 % carried at least one virulence gene. These data showed conclusively that, in spite of the application of strict sanitary measures in the intensive farm, genes encoding virulence factors of intestinal pathogens compatible with EtEc are still detected; therefore these strains will probably keep circulating among animals.

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.

Prevalence of Cryptosporidium spp. and Giardia spp., spatial clustering and patterns of shedding in dairy calves from Córdoba, Argentina / Prevalência de Cryptosporidium spp. e Giardia spp., distribuição espacial e padrões de eliminação em bezerros de propriedades leiteiras em 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 percent (95 percent CI: 16.14; 22.54) and 34.50 percent (95 percent 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 percent CI: 2.27; 6.26). Giardia spp. shedding showed a similar age pattern (RR: 1.33, 95 percent 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.

Os objetivos deste estudo foram determinar a prevalência de Cryptosporidium spp. e Giardia spp., a presença de aglomerados, a distribuição espacial de acordo com o tipo de solo e padrões de eliminação de cistos e oocistos em bezerros de propriedades leiteiras em Córdoba, Argentina. Amostras fecais foram colhidas de 620 bezerros com menos de sete semanas de idade, provenientes de 43 propriedades leiteiras e examinadas pelas técnicas de formol-éter e Ziehl-Neelsen modificada. Foram realizadas uma análise univariada e o teste de Kruskal-Wallis e, em seguida, uma análise multivariada com a intensidade de eliminação de cistos e oocistos, como um evento. A presença de aglomerados foi determinada com o método de varredura e a análise espacial foi realizada para explorar a sobreposição de rebanhos com alta prevalência e tipo de solo. A prevalência de Cryptosporidium spp. e Giardia spp. foi de 19,35 por cento (IC 95 por cento: 16,14; 22,54) e 34,50 por cento (IC 95 por cento: 30,69; 38,34), respectivamente. A probabilidade de infecção por Cryptosporidium spp. foi quase quatro vezes maior para bezerros com menos de 2 semanas em comparação com os bezerros mais velhos (RR: 3,78, IC 95 por cento 2,27; 6,26). O mesmo padrão de infecção relacionada à idade foi observado para Giardia spp. (RR: 1,33, IC 95 por cento 1,02; 1,75). Foi encontrado um aglomerado primário com alta prevalência de Cryptosporidium spp., e rebanhos com alta prevalência foram localizados em solos mal drenados.