Low-density colloid centrifugation removes bacteria from boar semen doses after spiking with selected species.

Single-layer centrifugation (SLC) with a low-density colloid is an efficient method for removing contaminating microorganisms from boar semen while recovering most spermatozoa from the original sample. This study tested the performance of this technique, using 50-ml tubes, by spiking commercial semen doses prepared without antibiotics with selected bacterial species followed by storage at 17 °C. The doses were spiked up to 102/ml CFU (colony forming units) of the bacteria Burkholderia ambifaria, Pseudomonas aeruginosa, and Staphylococcus simulans. The semen was processed by SLC (15 ml of sample and 15 ml of colloid) with the colloid Porcicoll at 20% (P20) and 30% (P30), with a spiked control (CTL) and an unspiked control (CTL0), analyzing microbiology and sperm quality on days 0, 3 and 7. SLC completely removed B. ambifaria and S. simulans, considerably reducing P. aeruginosa and overall contamination (especially P30, ∼104 CFU/ml of total contamination on day 7, median). Sperm viability was lower in P20 and P30 samples at day 0, with higher cytoplasmic ROS. Still, results were similar in all groups on day 3 and reversed on day 7, indicating a protective effect of SLC (possibly directly by removal of damaged sperm and indirectly because of lower bacterial contamination). Sperm chromatin was affected by the treatment (lower DNA fragmentation and chromatin decondensation) and storage (higher overall condensation on day 7 as per chromomycin A3 and monobromobimane staining). In conclusion, SLC with low-density colloids can remove most bacteria in a controlled contamination design while potentially improving sperm quality and long-term storage at practical temperatures.

Spatial distribution and pollution evaluation in dry riverbeds affected by mine tailings.

The objective of this study was to evaluate the level of pollution, sources and potential risk of heavy metals (Zn, Cu, Mn, Cd, Cr, Ni, Fe and Pb) and arsenic (As) in four dry riverbeds affected by mine tailing, which drain into one of the biggest coastal lagoon of Europe (Mar Menor). El Beal, La Carrasquilla, Las Matildes and Ponce dry riverbeds sediments were sampled along its course (20, 18, 13, 19 samples were collected, respectively), and total/soluble metal(loid)s, water soluble ions, nitrogen, and organic/inorganic carbon contents were analyzed. Spatial distribution, principal component analysis (PCA), hierarchical cluster analysis (HCA), contamination factor (Cf), pollution load index (PLI) and potential ecological risk index (RI) were used to identify the possible sources of metal(loid)s and to assess the sediment pollution status. The results showed that the mean total concentrations of As, Cu, Cd, Mn, Zn and Pb exceeded the natural background levels of the study area, with the highest values located close to the mining areas. Correlation and cluster analysis identified that Cd and Zn were associated mainly with anthropogenic activities for all riverbeds, while Cr and Ni come from parent. PLI graded the four riverbeds as contaminated by heavy metals, while RI manifested that 100% of samples located in El Beal, La Carrasquilla and Las Matildes had a significantly high ecological risk. Therefore, this study suggests that mine wastes are the main source of metal(loids) contamination in the dry riverbeds, which results can be used to design actions and measures to reduce the environmental impact of metal(loid)s in the Mar Menor coastal lagoon.

Are the soils and vegetation of a forest close to tailings ponds affected by metals and arsenic?

This study aimed to determine the transfer of metals (Cd, Pb and Zn) and As to a Mediterranean forest close to five tailings ponds in Cartagena-La Union mining district (SE Spain). In addition, the effect of the rhizosphere of two native plant species, Olea europaea (OE) and Pistacia lentiscus (PL), on soil properties and chemical speciation of metal(oid)s was evaluated. Results showed there was no influence of the rhizosphere in the total concentration of metal(loid)s in soil, decreasing as Pb > Zn > As > Cd. Chemical partitioning revealed that only Cd and As can be considered hazardous, with a high percentage of these elements in the soil-labile fractions (20-40%). The accumulation in vegetal tissues was only high for Pb in PL roots, which makes it a suitable species for phytostabilization. Additionally, translocation factors showed transfer of Pb and Zn in OE, and Zn in PL to aerial parts, although no toxicity evidences for plants or animals were found. Finally, soil properties affected metal(loid)s accumulation in plants. The OE species was related to soil-labile metal(loid) fractions and pH, total N, organic carbon and silt content. The PL species were associated with immobilized metal(loid) fractions, sand content, electrical conductivity and total concentrations of As, Cd and Pb. Hence, mining activity has affected native adjacent soils, with accumulation of metals in plant species, although translocation was low, likely due to physiological strategies of the studied species to protect themselves against hazardous elements, and to the high soil pH, which limits metals' mobility.

Low density Porcicoll separates spermatozoa from bacteria and retains sperm quality.

Antibiotics are added to semen extenders to control the growth of bacteria contaminating semen during collection but may contribute to the development of antibiotic resistance. An alternative would be physical separation of spermatozoa from bacteria. The objective of the present study was to evaluate two low densities of Porcicoll for removal of bacteria, and for their effect on sperm recovery and sperm quality. Semen was collected from boars at a commercial station. Aliquots of 8 extended ejaculates were subjected to colloid centrifugation through 20% Porcicoll (P20) and 30% Porcicoll (P30) in 500 mL tubes and then stored at 17 °C. Microbiological examination and sperm quality evaluation (computer assisted sperm analysis and flow cytometry) were carried out on controls and all colloid-selected samples immediately after preparation and again after storage for 3 and 7 days. The microorganisms found were mainly bacteria from the environment, gut or skin. There was a considerable reduction or complete removal of some bacteria by both colloids. Recovery rates were 86% for P20 and 81% for P30. Sperm quality was not adversely affected by colloid centrifugation on day 0, and thereafter showed a more gradual deterioration in colloid centrifuged samples than in controls, possibly due to lower bacterial contamination. There were no differences in sperm quality between the two colloid treatments. Thus, these results show that contaminating bacteria in semen can be controlled by centrifugation through low density colloids.

Distribution of metal(loid)s in particle size fraction in urban soil and street dust: influence of population density.

Assessment of street dust is an invaluable approach for monitoring atmospheric pollution. Little information is available on the size distribution of contaminants in street dusts and urban soils, and it is not known how the population density would influence them. This research was carried out to assess the size distribution of trace metal(loid)s in street dust and urban soil, and to understand how population density might influence the size-resolved concentration of metal(loid)s. Three urban areas with a high, medium and low population density and a natural area were selected and urban soil and street dust sampled. They were fractionated into 8 size fractions: 2000-850, 850-180, 180-106, 106-50, 50-20, 20-10, 10-2, and < 2 µm. The concentration of Pb, Zn, Cu, Cd, Cr, Ni, As, and Fe was determined, and enrichment factor and grain size fraction loadings were computed. The results indicated that the concentration of Pb, Zn, Cu, Cd, and Cr was highly size dependent, particularly for particles < 100 µm, especially for street dust. Low concentrations of Ni and As in street dust and urban soil were size and population density independent. Higher size dependency of the metals concentration and the higher degree of elemental enrichment in the street dust fractions than the urban soils indicate higher contribution of human-induced pollution to the dust. Findings also confirm the inevitability of size fractionation when soils or dusts are environmentally assessed, particularly in moderately to highly polluted areas. Otherwise, higher concentrations of certain pollutants in fine-sized particles might be overlooked leading to inappropriate decisions for environmental remediation.

Removal of bacteria from boar semen using a low-density colloid.

Antibiotics are added to semen extenders when preparing commercial semen doses for artificial insemination according to national and international guidelines. However, this addition of antibiotics represents non-therapeutic usage and could be contributing to the development of antibiotic resistance. Colloid centrifugation was shown to reduce the load of bacteria present in boar semen and was capable of removing all bacteria if performed directly after semen collection, albeit with some loss of spermatozoa. The present experiment was conducted with a low density colloid to investigate whether it was possible to separate all of the spermatozoa from seminal plasma i.e. without selection for robust spermatozoa, or whether this would have a detrimental effect on sperm quality. Ejaculates from nine boars were extended in Beltsville Thawing Solution without antibiotics and were transported to the laboratory for Single Layer Centrifugation (SLC) on modified Porcicoll i.e. at a low density (S). A further modification was that a sterile inner tube was included inside some of the 50 mL centrifuge tubes to facilitate harvesting of the sperm pellet (M). Aliquots of all samples (control, S and M) were cultured for bacterial quantification and identification using standard microbiological methods. Sperm quality was evaluated daily. Three of the C and M samples and five of the S samples did not contain any bacteria. Mean bacterial counts for the remaining samples (colony forming units/mL) were as follows: C 259 ±â€¯216; S 30 ±â€¯22; M 33 ±â€¯15 (P < 0.01). Citrobacter spp., Staphylococcus simulans, Klebsiella variicola, Escherichia coli, Myroides odoratimimus, Proteus spp. and Enterococcus faecalis were identified in the control samples. There were marginal differences in sperm quality among treatments, with sperm velocity and linearity being higher in S and M samples than in C at all time points. However, sperm viability, capacitation and acrosome status were marginally better in controls than in S or M on day 0, but these differences disappeared during storage. Conclusions: centrifugation through a low density colloid can remove or reduce bacterial contamination in boar ejaculates without using antibiotics. Furthermore, it is possible to collect boar ejaculates without bacterial contamination by paying strict attention to hygiene.

Antimicrobial resistance of Pasteurella multocida isolates recovered from swine pneumonia in Spain throughout 2017 and 2018.

A total of 32 Pasteurella multocida isolates were obtained from 60 cases of swine pneumonic lungs collected in "Castilla y León" (northwestern Spain) between November 2017 and April 2018. Capsular type A isolates were isolated from 96.9% cases and capsular type D from the remaining 3.1%. All isolates were characterized for their susceptibilities to eight antimicrobial agents and the presence of eight resistance genes. The frequency of susceptibility was lower than 60% in four of the drugs, 84.4% of the isolates showed resistance to at least two compounds, and 46.9% to a combination of three drugs. The resistance patterns suggested that enrofloxacin, chloramphenicol, tetracycline and cefotaxime were the compounds most likely active to P. multocida. The usage of PCR revealed that ermC, bla ROB1, tetB and msrE genes occurred in more than 37.0% isolates, that suggested its putative accountability in the resistance of the strains harbor them. However, most were detected in susceptible strains and only a genetic explanation for the resistance could be linked to erythromycin. Therefore, the resistances to clyndamicin, cotrimoxazol, ß-lactams and tetracyclin observed by phenotypic testing remains genetically unexplained and further investigations are required.

Phytoremediation of mine tailings with Atriplex halimus and organic/inorganic amendments: A five-year field case study.

Mine tailings have adverse chemical and physical conditions, including high concentrations of metals and salts, low organic matter content, and unbalanced rates of nutrients which limit the development of vegetation. A large scale field experiment was conducted to reclaim a tailing pond by triggering the growth of native species by spontaneous colonization by tilling (TL) the tailing pond surface and using marble waste (CaCO3; MW), pig slurry (PS) and their combination (MW + PS) as soil amendments. Soil physicochemical properties and water and DTPA extractable metal concentrations of bulk and rhizosphere soils were analyzed after five year from the application of the treatments. In addition, plants of Atriplex halimus from each treatment were collected and metals in roots, leaves and stems analyzed. Before amendments application, the studied pond showed a neutral pH, high salinity and a moderate organic carbon content. After five years, the pH value was significantly increased only in MW plot. The results showed significant increases of DTPA-extractable Zn in MW and MW + PS plots, Pb in all treatments except MW plot, Cd only in PS plot, and Cu only in MW + PS plot. A. halimus was the most dominant species, growing spontaneously in all plots, with lower vegetation cover in CT and MW plots, 6% and 2% respectively. Application of MW increased leaf Pb accumulation by 2.5-fold and Cd by 55%, when compared to the CT. The high initial salinity and probable substitution of metals by Ca2+ on exchangeable surfaces of soil particles may be the reasons for higher uptake of metals in MW plot when compared to the other plots. Although this plant is widely utilized in contaminated sites for phytostabilization purposes, it may absorb and translocate high concentrations of metals to the aboveground tissues in saline contaminated sites.

Change in metals and arsenic distribution in soil and their bioavailability beside old tailing ponds.

The objectives of this study were to determine the metals and arsenic transfer from mining ponds to agricultural and forest soils, and identify the dynamic of metal(loid)s in the soil-plant system for a native plant species (Ballota hirsuta) in two old mining districts: La Unión and Mazarrón (Spain). Soils and plants from mining ponds and natural and agricultural areas were collected and analyzed for soil properties, and chemical partitioning of Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn and As. Results showed that mine, forest and agricultural soils were contaminated by As, Cd, Cu, Pb, and Zn. Chemical partitioning revealed higher mobility of metals in mining ponds than natural and agricultural soils except for Fe and As which were mostly bound to soil matrix due to the mineralogical compositions of soils. The accumulation of metal(loid)s in B. hirsuta in La Unión decreased as Fe > As > Cr > Ni > Cu > Zn > Cd > Mn > Co > Pb while in Mazarrón was As > Fe > Cr > Pb > Cu > Ni > Co > Mn > Zn > Cd, showing that B. hirsuta has high ability to bio-accumulate Fe, As, Cr, Cu and Ni; and Pb (in Mazarrón), transferring a significant concentration of theses metal(loid)s, except Pb, to edible parts without exceeding the toxicity limits for animals. Therefore, B. hirsuta could be useful as phytoextractor species for Cr, Cu, As and Ni, while it can be used as phytostabilizer species for Zn, Co, Pb and Cd.

Altered indirect hemagglutination method for easy serotyping of Haemophilus parasuis / Método de Hemaglutinação Indireta alterado para facilitar a sorotipificação de Haemophilus parasuis

Glässer's disease is an emergent bacterial disease that affects swine husbandries worldwide causing important economic losses. The aetiological agent, Haemophilus parasuis, is currently divided in fifteen serovars but an increasing number of non-typeable serovars have been reported. Indirect hemagglutination (IHA) is indicated as a serotyping method for H. parasuis. In the present study, we describe an additional step that aims to work around a possible obstacle in the original protocol that may compromise the outcome of this assay. We observed that the choice of anticoagulant for blood collection influences and/or impairs spontaneous adsorption of H. parasuis antigens on sheep red blood cells (SRBCs). However, regardless of the anticoagulant used, chemical treatment of SRBCs with tannic acid induces a stable antigen adsorption (sensitization step). The addition of 1% BSA to SRBCs washing buffer and to antisera dilution augments IHA specificity. Tannic acid treated SRBCs combined with thermo-resistant H. parasuis antigens increases the assay resolution. Thus, our results demonstrate an improvement in the technique of H. parasuis serotyping that will prove valuable to understand Glässer's disease epidemiology and to better characterize serovars involved in outbreaks.(AU)

A Doença de Glässer é uma doença bacteriana emergente que afeta a produção de suínos em todo o mundo e causa importantes perdas econômicas. O agente etiológico, Haemophilus parasuis, é atualmente dividido em quinze sorovares; no entanto, um número crescente de cepas não tipificáveis tem sido relatado. O teste de hemaglutinação indireta (IHA) tem sido utilizado para a sorotipificação de H. parasuis. Neste estudo, descrevemos uma alteração no protocolo original de IHA e que supera uma limitação específica que pode comprometer o uso geral deste ensaio. Descobrimos que o tipo de anticoagulante utilizado para coletar os eritrócitos ovinos (SRBCs) pode comprometer a adsorção espontânea dos antígenos do H. parasuis. Por outro lado, o tratamento químico dos SRBCs com ácido tânico promove uma adsorção antigênica estável (passo de sensibilização) e independente do anticoagulante utilizado. O uso de 1% de BSA durante as lavagens dos SRBCs e na diluição dos antissoros incrementa a especificidade da IHA e, a combinação dos SRBCs tratados quimicamente com antígenos de H. parasuis termo-resistentes aumentam a resolução da IHA. Nossos resultados destacam uma melhoria na principal técnica de sorotipificação de H. parasuis, que auxiliará diretamente no entendimento da epidemiologia da Doença de Glässer e na caracterização dos sorovares envolvidos em surtos da doença.(AU)

Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis.

The molecular analysis of pigs vaccinated with a mutant transferrin-binding protein B (Y167A) from Haemophilus parasuis was compared with that performed for unvaccinated challenged (UNCH) and unvaccinated unchallenged (UNUN) pigs. Microarray analysis revealed that UNCH group showed the most distinct expression profile for immune response genes, mainly for those genes involved in inflammation or immune cell trafficking. This fact was confirmed by real-time PCR, in which the greatest level of differential expression from this group were CD14, CD163, IL-8 and IL-12. In Y167A group, overexpressed genes included MAP3K8, CD14, IL-12 and CD163. Proteomics revealed that collagen α-1 and peroxiredoxins 2 and 6 were overexpressed in Y167A pigs. Our study reveals new data on genes and proteins involved in H. parasuis infection and several candidates of resistance to infection that are induced by Y167A vaccine. The expression of proinflammatory molecules from Y176A pigs is similar to their expression in UNUN pigs.

Influence of population density on the concentration and speciation of metals in the soil and street dust from urban areas.

Street dust and soil from high, medium and low populated cities and natural area were analysed for selected physical-chemical properties, total and chemical speciation of Zn, Pb, Cu, Cr, Cd, Co, Ni to understand the influence of human activities on metal accumulation and mobility in the environment. The pH, salinity, carbonates and organic carbon contents were similar between soil and dust from the same city. Population density increases dust/soil salinity but has no influence on metals concentrations in soils. Increases in metal concentrations with population density were observed in dusts. Cu, Zn, Pb, Cr can be mobilized more easily from dust compared to the soil. In addition, population density increase the percentage of Pb and Zn associated to reducible and carbonate phase in the dust. The behaviour of metals except Cd in soil is mainly affected by physico-chemical properties, while total metal influenced the speciation except Cr and Ni in dusts.

Syrian bean-caper (Zygophyllum fabago L.) improves organic matter and other properties of mine wastes deposits.

The omni-presence of Zygophyllum fabago L. (Syrian bean-caper) natural colonies in post mining areas prompted us to investigate its contributions to reclamation of mine wastes deposits in southeast Spain. Select plant-related (edaphic) characteristics and bio- and water soluble-Cd, Cu, Pb and Zn in rhizosphere of Z. fabago were compared to deposits one year since application of pig slurry and marble waste. Total N in rhizosphere increased up to a factor of 20X (339 vs 17 mg N kg(-1)) in El Gorguel and 27X (85 vs 3.1 mg N kg(-1)) in El Lirio sites. Organic matter accumulation in rhizosphere from litter and roots of Z. fabago increased organic C from 6.6 to 19.5 g kg(-1) in El Gorguel and from 2.1 to 5.7 g kg(-1) in El Lirio in one year. Dissolution of inorganic C takes place due to organic acids from root exudates of Z. fabago. Reduction in bio-available Cd, Cu, Pb, and Zn in rhizosphere of Z. fabago at El Lirio is attributed to increase in pH from 5.3 to 7.7 through marble waste addition, although increased cation exchange capacity may also have played a role. Addition of marble waste to encourage colonization by Z. fabago in acidic mine wastes deposits was recommended.

Marble wastes and pig slurry improve the environmental and plant-relevant properties of mine tailings.

Poor soil fertility is often the biggest challenge to the establishment of vegetation in mine wastes deposits. We conducted field trials in the El Gorguel and El Lirio sites in SE Spain, two representative tailing ponds of similar properties except for pH, to understand the environmental and plant-relevant benefits of marble waste (MW) and pig slurry (PS) applications to mine tailings. Low pH (5.4) tailings (El Lirio) exhibit reduction of up to fourfold in bio-availability of metals as shown by the DTPA-Zn, Pb, water-soluble Zn, Pb and up to 3× for water-soluble Cd. Tailings in El Gorguel have high pH (7.4) and did not exhibit significant trends in the reductions of water-extractable Zn, Pb, Cd and Cu. Improvements to the edaphic (plant-relevant) properties of tailings after the amendments are not as sensitive to pH compared to the environmental characteristics. The two sites had increases in aggregate stability, organic matter (total N and organic C) although total N is higher in the El Gorguel (up to 212 µg N kg(-1)) than the El Lirio (up to 26 µg N kg(-1)). However, cation exchange capacities are similar in both sites at 15.2 cmol(+) kg(-1). We conclude that the characteristics, especially pH, of tailing materials significantly influence the fate of metals but not improvements to plant-relevant properties such as cation exchange capacity and aggregate stability 1 year after the application of MW and PS amendments.

Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin prevent the expression of proinflammatory chemokines and cytokines in pigs.

The expression of chemokines (CCL-2 and CXCL-8) and cytokines (IL-1 α , IL-1 ß , IL-6, TNF- α , and IL-10) was evaluated by RT-qPCR in colostrum-deprived pigs vaccinated and challenged with Haemophilus parasuis serovar 5. Two vaccines containing native proteins with affinity to porcine transferrin (NPAPTim and NPAPTit) were tested, along with two control groups: one inoculated with PBS instead of antigen (challenge group (CHG)), and another one nonimmunized and noninfected (blank group). The use of NPAPTim and NPAPTit resulted in complete protection against H. parasuis (no clinical signs and/or lesions), and both vaccines were capable of avoiding the expression of the proinflammatory molecules to levels similar to physiological values in blank group. However, overexpression of all proinflammatory molecules was observed in CHG group, mainly in the target infection tissues (brain, lungs, and spleen). High expression of CCL-2, CXCL-8, IL-1 α , IL-1 ß , and IL-6 can be considered one of the characteristics of H. parasuis infection by serovar 5.

New insights in cellular immune response in colostrum-deprived pigs after immunization with subunit and commercial vaccines against Glässer's disease.

Four groups of colostrum-deprived pigs were immunized with Porcilis Glässer® (PG) or with subunit vaccines developed by us (rTbpA, NPAPT(M) or NPAPT(Cp)) against Glässer's disease, and they were challenged with 3×10(8)CFU of Haemophilus parasuis. A strong reduction in CD3(+)γδTCR(+) cells was seen in non-immunized control and scarcely protected (rTbpA) groups, suggesting that these cells could represent a target of H. parasuis infection. A significant increase in CD172α(+)CD163(+) cells was detected in all groups but PG, while a reduction in SLAIIDR(+) molecules expression was observed after challenge in control animals. Significant increases in CD3ε(+)CD8α(+)CD8ß(+) and B cells were detected respectively in control and NPAPT groups, and in scarcely (rTbpA) and well-protected (NPAPT(M) and NPAPT(Cp)) groups. Finally, a greater response in CD4(+)CD8α(-) cells was observed in NPAPT(Cp) compared to NPAPT(M) and PG groups. These results state the potential of NPAPT antigen for developing effective vaccines against Glässer's disease.

Comparison of real-time PCR and culture isolation in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis.

AIMS: A real-time PCR (RT-PCR) based on the detection of the infB gene of Haemophilus parasuis is compared with culture isolation (Frandoloso et al., (2011) Clin Vaccine Immunol 18, 50-58.), evaluating different subunit or commercial vaccines. METHODS AND RESULTS: Samples from different tissues of 24 experimentally infected and challenged colostrum-deprived piglets were tested. The RT-PCR gave globally a 23·3% more of positive results than culture, and all samples being positive by culture were positive by RT-PCR also. H. parasuis could not be cultured from any of the samples of the piglets included in the three vaccinated groups resulting in a strong protection, but it could be detected by RT-PCR in six samples in the group immunized with the commercial vaccine, in three in that vaccinated with native proteins with affinity to porcine transferrin (NPAPT) administered intramuscularly and in only two in that immunized with NPAPT intratracheally. CONCLUSIONS: The RT-PCR was more sensitive than culture for H. parasuis detection in the organs compared. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-PCR evidenced that NPAPT vaccines were those yielding the best protection results in terms of H. parasuis clearance.

Salinity increases mobility of heavy metals in soils.

The effect of salinity induced by CaCl(2), MgCl(2), NaCl and Na(2)SO(4) on the mobility of Cu, Cd, Pb and Zn was studied. An increase of ionic strength by any salts promoted a higher release of Cd than the others metals. When CaCl(2) and NaCl were applied, Cd and Pb showed the highest degree of mobilization. When MgCl(2) was applied, Cd and Cu were mobilized the most. Finally, an increase of Na(2)SO(4) also promoted the strongest mobilization of Cd and Cu. As the total heavy metal content was higher, the percentage of Pb and Cu released upon salinization decreased, indicating that these metals are strongly bound to soil constituents. An increase of carbonates in the soil promoted a higher release of Pb for all used salts and for Zn when MgCl(2) and NaCl were used. This indicates that Pb and Zn are adsorbed on the surface of carbonate crystals. An increase of fine particles promoted a decrease of percentage of released Cd for all salts, indicating that Cd is strongly retained in the fine fractions. The main mechanism regulating Pb and Cd mobility was competition with Ca(2+) for sorption sites followed for metal chloro-complexation, association between the Cd/Pb-sulfates and competition with Mg(2+). The main mechanism regulating Cu mobility was the formation of Cu-sulfate, followed by competition with cations (Mg > Ca) and chloride. For Zn, competition with Ca(2+) for sorption sites was the most important process for its mobility; followed by Zn-sulfate association and, finally, chloride and competition with Mg with the same effect.

Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis.

The cellular immune response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized studying changes in peripheral blood mononuclear cells (PBMC) in colostrum-deprived pigs. Five groups were studied, four of those were previously immunized with different formulations and the fifth was maintained as non-immunized control. All groups were challenged with 5 x 10(9) CFU of H. parasuis serotype 5. The non-commercial bacterin conferred a complete protection, while the OMP-vaccine and the exposure to a subletal dose of 10(5) CFU of H. parasuis protected only partially, and the recombinant Tbp B-vaccine induced no protection. PBMC were analyzed using monoclonal antibodies against porcine CD45(+), CD3(+), CD4(+), CD8alpha(+), CD25(+), CD4(+) naïve, alphaIgM(+) and SWC3(+) cells in single-colour fluorescence, and CD4(+)/CD8alpha(+) and CD8alpha(+)/CD8beta(+) combinations in two-colour fluorescence. The different groups showed no significant changes in PBMC subsets following vaccination, and only minor changes were encountered after challenge, consisting mainly of significant increases (P<0.05) in the relative proportions of monocytes and granulocytes (SWC3(+)) and B cells (alphaIgM(+)), as well as a significant reduction in CD3(+) cells (P<0.05). These changes were similar for the five groups compared, except for the significant increase of CD25(+) cells, which was only observed for the bacterin-vaccinated group. These results suggest an increase of trafficking of inflammatory cells and the onset of the adaptive antibody response against H. parasuis infection; in addition, the blood cellular response developed by the different groups was not relevant to protection.

Selective inhibition of vascular endothelial growth factor-mediated angiogenesis by cyclosporin A: roles of the nuclear factor of activated T cells and cyclooxygenase 2.

Cyclosporin A (CsA) is an immunosuppressive drug that inhibits the activity of transcription factors of the nuclear factor of activated T cells (NFAT) family, interfering with the induction of cytokines and other inducible genes required for the immune response. Here we show that CsA inhibits migration of primary endothelial cells and angiogenesis induced by vascular endothelial growth factor (VEGF); this effect appears to be mediated through the inhibition of cyclooxygenase (Cox)-2, the transcription of which is activated by VEGF in primary endothelial cells. Consistent with this, we show that the induction of Cox-2 gene expression by VEGF requires NFAT activation. Most important, the CsA-mediated inhibition of angiogenesis both in vitro and in vivo was comparable to the Cox-2 inhibitor NS-398, and reversed by prostaglandin E(2). Furthermore, the in vivo corneal angiogenesis induced by VEGF, but not by basic fibroblast growth factor, was selectively inhibited in mice treated with CsA systemically. These findings involve NFAT in the regulation of Cox-2 in endothelial cells, point to a role for this transcription factor in angiogenesis, and may provide a novel mechanism underlying the beneficial effects of CsA in angiogenesis-related diseases such as rheumatoid arthritis and psoriasis.