Characterization of humus-protease complexes extracted from soil

Pyrophosphate (140 mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: A_I with nominal molecular weight (nmw) > 100 kD, A_II with nmw between 10 kD and 100 kD and R with nmw < 10 kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-l-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-l-phenylalanyl l-leucine (ZPL), specific for carboxypeptidases; and casein, essentially a non-specific substrate. The derivative fractions were also analysed for their amino acid N and humic (HA) and fulvic (FA) acid contents. The organic matter of extracts and derivative fractions obtained from the pasture soil was analysed by isoelectric focusing (IEF) and that of fractions analysed by pyrolysis gas chromatography (Py-GC). Activities of the extract were monitored for their thermal stability and those of the extract and derivative fractions for their optimal pH.Due to the mechanical disintegrating action of sodium pyrophosphate over the humic substances during the fractionation process the amount of total organic C and FA in the fractions was ranked as R > A_II > A_I. The lowest amino acid N/organic C was found in the R fraction, whereas A_II fraction was rich in humic acids, carbohydrates and amino acid N and A_I fraction showed the lowest carbohydrate content. At least 70% of the total BAA- and ZPL-hydrolysing activity was associated to particles with nmw higher than 10 kD and at least 30% of these activities were present in particles with nmw higher 100 kD. Casein-hydrolysing activity was quite evenly distributed among the three fractions (A_I, A_II and R). The extracted protease-organic complexes were resistant to thermal denaturation and some of them showed optimal activity at pH values higher than 10 as a result of the polyanionic characteristics of the humic material surrounding enzyme molecules and of the presence of alkaline protease. Comparison of data obtained in Py-GC analyses and in protease activity suggests that BAA-hydrolysing activity was associated to a highly condensed humic matter and ZPL-hydrolysing activity to less resistant humic substances, while at least some of the extracted casein-hydrolysing activity was present as glyco-proteins not associated to humus. BAA-hydrolysing activity was probably inhibited by fresh organic matter of carbohydrate origin whereas lignin derived organic matter probably inhibited ZPL- and casein-hydrolysing activity.     

First attempts towards the restoration of gorgonian populations on the Mediterranean continental shelf

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Dynamics of porcine circovirus type 2 infection in a herd of pigs with postweaning multisystemic wasting syndrome

OBJECTIVE: To determine the pattern of infection for porcine circovirus type 2 (PCV2) in a herd of pigs with postweaning multisystemic wasting syndrome (PMWS). ANIMALS: 29 sows and 250 pigs. PROCEDURE: Blood samples were collected from all 3-, 7-, and 12-week old pigs and 59 pigs at 28 weeks of age. Pigs that died during the study were necropsied. Porcine parvovirus and PCV2 antibodies were assayed. A polymerase chain reaction (PCR) was used to detect PCV2 genome in serum of selected pigs. RESULTS: The PMWS started when pigs were 8 weeks old, with a prevalence of 30% in 8- to 10-week-old pigs. Eighty-three pigs died during the period between 3 and 12 weeks of age. Microscopic lesions consistent with PMWS were observed, and PCV2 nucleic acid was detected (50 of 68 pigs). Antibodies to PCV2 decreased from 3 to 7 weeks of age, increased at 12 weeks of age, and were maintained until 28 weeks of age. One sow had a positive result for PCR of serum. Nine, 37 and 8 pigs had PCV2 genome in serum obtained at 7, 12, and 28 weeks of age, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with PCV2 coincided with severe clinical signs; however, infected 28-week-old pigs did not have evidence of disease. Immunity declined over time in young pigs. A long duration of PCV2 viremia was apparent in a high percentage of infected pigs, which may affect transmission and persistence of the virus in a herd.     

Effects of exercise conditioning on semen characteristics from hyper- and hypotensive lines of turkeys

Twenty-one male Broad Breasted White turkeys which had been selected for high or low blood pressure were put on an exercise regimen to determine its effect on semen quality. Eleven were from the hypertensive line and 10 from the hypotensive line. Eleven (6 hypertensive and 5 hypotensive) turkeys were placed on an exercise regime of walking (herding) about 1 mile daily for 6 weeks before the experiment began, and then for a further 6 weeks during semen collection. Semen was collected from 11 turkeys on the exercise regime and 10 nonexercised control birds which were kept in their experimental pens during the trial. Both hypertensive and exercised groups showed an improved semen colour score at the first collection period (P less than or equal to 0.09). Only the exercised group showed a significantly (P less than or equal to 0.05) better score than the control group during periods 2 and 3. The hypertensive line showed significantly higher spermatozoa count than the hypotensive line during the first collection period. There was no effect of either exercise or selection for high or low blood pressure on semen volume.     

Effect of drought on diameter increment of Quercus ilex, Phillyrea latifolia, and Arbutus unedo in a holm oak forest of NE Spain

The present study was carried out to elucidate the drought growth responses of Quercus ilex L., Phillyrea latifolia L., Arbutus unedo L., and other accompanying woody species of the Mediterranean holm oak forest. We submitted holm oak forest stands in Prades mountains (NE Spain) to a 2-year experimental drought. We reduced soil water availability about 15% by plastic strips and funnels that partially excluded rain throughfall and by ditch interception of water runoff. Mean stem diameter increment showed a great variation depending on the species. A. unedo had larger growth rates than Q. ilex and P. latifolia, but it was also the species that experimented the highest growth reduction in the drought plots (77%), suggesting a higher drought sensitivity than Q. ilex (55%) and P. latifolia (no drought effect). The growth reduction was specially marked in the larger trees. Aboveground stand biomass increment, estimated from stem diameter by allometric relationships, was 1.9 Mg ha⁻¹ per year in the control plots. The 15% reduction in the upper soil moisture produced 42% reduction in this biomass increment. In the drier conditions predicted in this Mediterranean area in the frame of climate change, an important reduction of growth rates can be hence expected, accompanied by a gain of dominance of drought-tolerant species such as P. latifolia in detriment of more mesic species such as Q. ilex.     

Effects of Rapid Cooling Prior to Freezing on the Quality of Canine Cryopreserved Spermatozoa

The aim of this study was to evaluate the effects of rapid cooling prior to freezing on frozen-thawed canine sperm quality. In experiment 1, centrifuged ejaculates from 6 dogs were pooled, split into 4 aliquots and cryopreserved by the Uppsala procedure using different cooling rates (control, cooling speed 18 C/90 min and average cooling rate 0.2 C/min; rapid, cooling speed 18 C/8 min and average cooling rate 2.25 C/min) in combination with 2 glycerol addition protocols (fractionated or unfractionated). In experiment 2, centrifuged ejaculates from 4 dogs were processed individually using the same cooling rates described in experiment 1 in combination with an unfractionated glycerol addition protocol. Each of the experiments was replicated 5 times. Sperm quality was evaluated after 30 and 150 min of post-thawing incubation at 38 C. Total motility (TM), progressive motility (PM) and quality of movement parameters were assessed using a computerized system, and sperm viability (spermatozoa with intact plasma and acrosome membranes) was assessed using flow cytometry (H-42/PI/FITC-PNA). Values for TM, PM, viable spermatozoa and the quality of movement parameters after thawing were not significantly affected by the cooling rate. The interaction between the cooling rate and the added glycerol protocol was not significant. There were significant differences among the males (P<0.01) in the sperm quality parameters evaluated after thawing. The interaction between the males and the cooling rate was not significant. In conclusion, canine spermatozoa can be cryopreserved using the Uppsala method at an average cooling rate of 2.25 C/min prior to freezing together with addition of fractionated or unfractionated glycerol.     

A Large‐scale,Rapid Public Health Response to Rabies in an Organ Recipient and the Previously Undiagnosed Organ Donor

This article describes and contrasts the public health response to two human rabies cases: one organ recipient diagnosed within days of symptom onset and the transplant donor who was diagnosed 18 months post‐symptom onset. In response to an organ‐transplant‐related rabies case diagnosed in 2013, organ donor and recipient investigations were conducted by multiple public health agencies. Persons with potential exposure to infectious patient materials were assessed for rabies virus exposure. An exposure investigation was conducted to determine the source of the organ donor's infection. Over 100 persons from more than 20 agencies spent over 2700 h conducting contact investigations in healthcare, military and community settings. The 564 persons assessed include 417 healthcare workers [5.8% recommended for post‐exposure prophylaxis (PEP)], 96 community contacts (15.6% recommended for PEP), 30 autopsy personnel (50% recommended for PEP), and 21 other persons (4.8% recommended for PEP). Donor contacts represented 188 assessed with 20.2% recommended for PEP, compared with 5.6% of 306 recipient contacts recommended for PEP. Human rabies cases result in substantial use of public health and medical resources, especially when diagnosis is delayed. Although rare, clinicians should consider rabies in cases of encephalitis of unexplained aetiology, particularly for cases that may result in organ donation.     

Digestibility, faeces recovery, and related carbon, nitrogen and phosphorus balances of five feed ingredients evaluated as fishmeal alternatives in Nile tilapia, Oreochromis niloticus L.

This study shows that alternatives for fishmeal in a fish diet affect not only fish growth but also faeces stability and nitrogen (N) and phosphorus (P) waste production. Wheat gluten diet (WGD), soybean meal extract diet (SBE), soybean meal diet (SBM), duckweed diet (DWD) and single‐cell protein diet (SCP) were evaluated as a fishmeal replacement on a 15% weight weight^?1 basis in tilapia diets. Fishmeal replacement affected dry matter (dm), protein, ash and P digestibility significantly. Faeces recovery (6.8–11.2%) was not significantly affected, although the amount of non‐recovered faeces and total faeces showed significant differences. Duckweed diet and SCP resulted in the largest amounts of non‐recovered and total faeces (199–210, 224–225 g dm kg^?1 feed dm). Compared with fishmeal diet (FMD), the WGD and SBE resulted in similar growth, but higher non‐faecal N losses (471–495 vs. 416 g N kg^?1 N). Soybean meal diet, DWD and SCP resulted in lower growth but less non‐faecal loss (409–450 g N kg^?1 N). The DWD and FMD had the highest N retention (480 g N kg^?1 N) compared with the other diets (431–451 g N kg^?1 N). Carbon retention, faecal and non‐faecal losses and P retention were similar for all diets (302–358, 142–176 and 489–523g C kg^?1 C, 606–704 g P kg^?1 P). Phosphorus faecal loss was lower for all diets (329–381 g P kg^?1 P) than for the FMD (401 g P kg^?1 P).     

Leishmania-specific isotype levels and their relationship with specific cell-mediated immunity parameters in canine leishmaniasis

In the current retrospective study, Leishmania infantum-specific IgG, IgA and IgM levels were determined by ELISA in 106 untreated dogs with clinically-patent leishmaniasis (Sx) and in 171 clinically healthy dogs (Asx) from Spain to investigate the relationship between these Ig isotypes and clinical status. In addition, we studied if different Leishmania-specific humoral pattern exists between Asx dogs with and without cellular mediated immunity (CMI). Fifty-six dogs were assessed by means of lymphoproliferation assay (LPA), interferon production (IFN) and leishmanin skin test (LST), 71 dogs by means of both LPA and IFN and 44 only by LST. Both Sx and Asx dogs produced Leishmania-specific IgG, IgA and IgM antibodies, however the levels and proportion of positive dogs for each Ig isotype were significantly higher in Sx than in Asx ones (P<0.001). Analysis of immunological profiles determined for each cellular technique (positive and negative cellular response for each technique combined with positive or negative specific humoral response) showed that Asx dogs constituted a high heterogeneous group. No correlations were observed between CMI tests and specific IgG or IgM levels. However, a significant inverse correlation was demonstrated between specific IgA levels and LPA response (Spearman's r=-0.220; P=0.035). In general, the low correlation detected between CMI tests and isotype levels might indicate that the immune response is not strongly polarized in the majority of Asx dogs. Additionally, this study suggests that dogs developing T-cell response are probably able to avoid the dissemination of the parasite at least to mucosal surfaces and, as a consequence, to produce low or background specific IgA levels. Further studies are needed to investigate the relationship between specific IgA and parasite load, especially at mucosal site.