Effect of 1-24ACTH administration on sheep blood granulocyte functions

The objective of the present study was to determine the efficiency of blood neutrophils (PMN) taken from sheep during acute stress. Ten healthy Charolle sheep were sampled before treatment (T0) and 1 (T1), 2 (T2), 24 (T24) and 48 (T48) hours after 1-24ACTH administration. Ten sheep serving as the controls were sampled at the same time intervals, using saline solution instead of 1-24ACTH. At each time sampling, rectal temperature, heart rate, cortisol, glucose, non-esterified fatty acids (NEFA), total and differential leukocyte counts were evaluated. PMN were isolated after centrifugation of whole blood and hypotonic lysis of RBC. Chemotaxis was evaluated on a modified Boyden chamber using a nitrate cellulose filter and both Zymosan activated serum (ZAS) and interleukin-8 (IL-8) as chemoattractants. Phagocytosis was measured using both non-opsonized latex beads and fluoresceinated yeasts opsonized with homologous serum. Superoxide (O(-)2) production was evaluated by measuring superoxide dismutase-inhibitable reduction of ferricytochrome C, and adherence by a colorimetric assay of acid phosphatase activity of adherent cells. The administration of 1-24ACTH induced an acute stress reaction, indicated by the presence of clinical, biochemical and hematological changes. Adherence significantly increased from T0 to T2 in treated sheep. This might be responsible for the depression of non-specific immunity in stressed animals. Studies using stressors other than 1-24 ACTH are needed to verify the influence of other components of the stress reaction on PMN functions.     

Characteristics of the response of ovine granulocytes (PMNs) to zymosan-activated serum (ZAS) and to recombinant human interleukin-8 (IL-8)

The chemotactic activity of zymosan-activated serum (ZAS) and of two concentrations of recombinant human IL-8 (IL-8(25), 25 ng/ml; IL-8(50), 50 ng/ml) for ovine polymorphonuclear granulocytes (PMNs) was tested in a modified Boyden chamber. Thick cellulose acetate filters and the leading front method were used to quantify the movements of the cells. Both ZAS and IL-8(25) exerted a chemotactic effect on ovine PMNs (P < 0.01): IL-8(50) induced a more homogeneous response (P < 0.001). To verify the characteristics of the responsiveness to the chemokines after short-term (st) or long-term (lt) repeated samplings, chemotaxis was investigated 1 (T1st), 2 (T2st), 24 (T3st) and 48 h (T4st) after the basal sampling (T0st) and 15 days (T1lt) after the basal sampling (T0lt). No differences in chemotaxis were found in long-term repeated samplings. In contrast an increase in the responsiveness to IL-8(25) and to IL-8(50) (P < 0.05) was detected at T2st in comparison with T0st. Furthermore, the significance of the distance run by activated PMNs compared with the controls, increased from T0st to T2st, as a sign of a more homogeneous response to the chemokines. In the absence of evident changes in circulating leucocyte numbers and in serum cortisol concentrations, these findings could be interpreted as a consequence of a different expression of chemoattractant receptors on the membrane of PMNs collected at different times.     

Microbiological Quality Assessment of Watershed Associated with Animal-Based Agriculture in Santa Catarina, Brazil

Environmental problems many times could evolve when manure-containing pathogens are distributed into an open environment with no effort made to reduce the content of pathogens or limit their movement in the environment. Wind, surface flow, and subsurface flow can all carry enough pathogens to receiving waters to exceed water quality standards. This study was conducted to assess the microbiological quality of water associated with animal-based agriculture in the sub-basin of Pinhal River located in the rural area of Concordia, Santa Catarina, Brazil. Six sampling points representing different agricultural land uses (LU1—dairy cattle; LU2—without animals; LU3—dairy + pigs + poultry + crops; LU4—pigs + poultry + crops; LU5—dairy + pigs + poultry + crops + human; and LU6—dairy+pigs+crops) along the Pinhal River sub-basin (north to south) were sampled biweekly from August 2006 to December 2008. Concentrations of fecal coliforms and Escherichia coli varied significantly (p?≤?0.05) with land use (LU), but there was no interaction effect of LU, season, and time. Water samples from the catchment area of LU1 had the highest concentration of fecal coliforms (4,479?±?597 CFU ml^?1) when compared with other catchment areas. Catchment area associated with LU2 (no animal) had the lowest concentrations of fecal coliforms (39.2?±?5.2 CFU ml^?1). With the exception of LU2 (control site), all the maximum concentrations of E. coli exceeded the single maximum allowable concentration for E. coli (100 CFU ml^?1). When LU1 was compared with other catchment areas (LU3, 50%; LU4, 67%; LU5, 58%; and LU6, 44%), it had the lowest counts (39%) of Salmonella sp. Our results suggest that spatial pattern of bacterial water quality is evident, which can be linked to the different land uses and associated practices (present or absent of animal activities). Therefore, varying responses associated with the different land uses would be critical in identifying the importance of different sources of bacteria in the catchment area and the mechanisms transferring them.     

alpha(1)-Acid glycoprotein modulates apoptosis in bovine monocytes

alpha(1)-Acid glycoprotein (AGP, orosomucoid) is a normal constituent of bovine blood. AGP is an immunocalin, a binding protein that can also exert several immunomodulatory functions. In this paper we investigated the effect of bovine alpha(1)-acid glycoprotein (boAGP) on spontaneous and staurosporine-induced apoptosis of blood derived monocytes purified using magnetic cell sorting techniques. Bovine AGP was purified from blood following a chromatographic protocol. The homogeneous protein was used to stimulate the cells as well to raise a polyclonal antibody, that was used throughout all the experiments. When monocytes were incubated with high concentrations of boAGP (0.9 mg/ml), similar to those found in bovine plasma during systemic reaction to inflammation, their spontaneous apoptosis rate was suppressed, as determined by caspase-3/7 enzymatic activity assay. Similar results were obtained when apoptosis was induced by adding staurosporine, a potent protein kinase inhibitor. The apoptosis-modulating activity of boAGP was dependent on its concentration, since physiological concentrations of boAGP (0.3 mg/ml) did not exhibit a statistically significative anti-apoptotic activity. We also investigated whether this apoptosis-modulating activity was dependent on the terminal sialic acid residues exposed on the surface of the protein. Enzymatic treatment with neuraminidase, that cleaves terminal sialic acid residues, completely abolished boAGP's anti-apoptotic activity. These results suggest that the protective effect of AGP is likely mediated by its sialic acid terminal groups.     

Mass Production of Juveniles of the Fat Snook Centropomus parallelus in Brazil

A pilot‐scale trial to rear fat snook Centropomus parallelus through larval, weaning and nursery phases was conducted in Florianópolis, Brazil. Eggs (96% fertilization) from captive broodstock, induced to spawn using 50 μg/kg LHRHa were stocked in two 4,000‐L cylindrical fiberglass tanks at a mean density of 19.2 eggs/L. Nannochloropsis oculata was stocked with the eggs and maintained at a mean density of 0.5–1.0 ± 10⁶ cells/mL up to 31 dph (31 dph). Hatching averaged 90%. Larvae were fed rotifers Brachionus rotundiformis enriched with an oil emulsion from 3 dph to 36 dph (30–40 rotifers/mL) and Artermia meta‐nauplii enriched with Selco from 22 dph to 60 dph (mean 2.9 meta‐nauplii/mL). Weaning began at 45 dph with an artificial dry diet NRG (50% protein), supplied together with concentrated and enriched Artemia meta‐nauplii. No critical period of mortality was observed during larval rearing. During the 43 days of weaning and nursery, less than 1% mortality was recorded. Food conversion rate during nursery was 1.17, with a change in the coefficient of variation of mean total length of 1.3%. Specific total growth rate in weight was 13.0 %/d and mean growth in total length and total weight were 0.65 mm/d and 24.0 mg/d over the whole rearing trial respectively. Mean total length and total weight of juveniles were 57.6 ± 0.1 mm and 2.11 ± 0.12 g, respectively, and the length‐weight relationship was W = 8.29931 ± 10–⁵ TL^3.049607 (r= 0.9986). on 88 dph when the trial was terminated. The condition factor on 88 dph was 1.104. On 88 dph a total of 35,000 juveniles were harvested, overall survival was 25.5% with mean final density of 4.4 fishn and biomass of 6.9 kg/m³. The present trial demonstrated the feasibility of mass production of fat snook. Possible improvements necessary for commercial cultivation of fat snook C. parallelus are discussed.     

Linoleic acid increases adhesion,chemotaxis, granule release,intracellular calcium mobilisation,MAPK phosphorylation and gene expression in bovine neutrophils

Neutrophils are critical to the innate immune response; therefore, the proper function of neutrophils is critical to avoid the development of certain diseases. Linoleic acid, a polyunsaturated long-chain fatty acid, is one of the most abundant long-chain fatty acids found in the plasma of cows after giving birth. In this study, we evaluated the effects of linoleic acid treatment on bovine neutrophil adhesion, chemotaxis, metalloproteinase (MMP)-9 release, CD11b expression, intracellular calcium mobilisation, mitogen-activating protein kinase (MAPK) phosphorylation and COX-2 and IL-8 expression. Bovine neutrophils isolated from healthy heifers were incubated with different concentrations of linoleic acid, and then neutrophil responses were evaluated. Our results show that the treatment of neutrophils with 100 μM linoleic acid increased their adhesion to the bovine endothelial cell line CPA47. The results of a transwell migration assay revealed that linoleic acid could also promote the chemotaxis of bovine neutrophils. Furthermore, linoleic acid treatment increased MMP-9 activity and CD11b cell surface expression in neutrophils. Fifty and 100 μM linoleic acid also increased intracellular calcium mobilisation in neutrophils loaded with Fluo-4 AM dye. Linoleic acid also rapidly (2–5 min) stimulated the phosphorylation of ERK1/2 and p38 MAPK as evaluated by immunoblot. Finally, COX-2 and IL-8 mRNA expression increased after 2 h of linoleic acid treatment. In conclusion, linoleic acid stimulates adhesion, chemotaxis, granule release and intracellular responses in bovine neutrophils.     

Advanced glycation end products and sorbitol in blood from differently compensated diabetic dogs

Canine diabetes mellitus (DM) is a common metabolic disorder with long term complications, most of which are caused by glycosylation of structural proteins, decreases in antioxidant concentrations, altered osmotic balance and hypoxia due to impaired oxygen transport. Previous studies have demonstrated that under hyperglycemic conditions canine erythrocytes undergo swelling, probably due to activation of the polyol pathway. The present work aimed to assess the plasma concentration of advanced glycation end (AGE) products, stable Amadori-products generated by non-enzymatic glycosylation of proteins and the intracellular concentration of sorbitol, produced by the activation of polyol pathway in 34 blood samples from diabetic dogs and in 14 controls. AGE products were significantly higher (p<0.01) in plasma from diabetic dogs compared with control animals. The sorbitol concentration in erythrocytes was also significantly higher in diabetic dogs and, in particular, in poorly compensated animals and in dogs with ketonuria. In five cases that were analysed before and after clinical improvement, sorbitol concentration was found to correlate with improvement. These results suggest that non-specific glycosylation is increased and that the polyol pathway is activated in diabetic dogs in a manner that is proportionate to the severity of disease. Moreover, the concentration of AGE products and sorbitol may be useful for monitoring the onset of diabetic complications and assessing the most appropriate therapeutic approaches for management of canine DM.