rBmTI-6, a Kunitz-BPTI domain protease inhibitor from the tick Boophilus microplus, its cloning, expression and biochemical characterization

Boophilus microplus is a rich source of trypsin inhibitors, numerous Kunitz-BPTI (bovine pancreatic trypsin inhibitor) inhibitors have been described from larvae and eggs, named BmTIs. Among them, were characterized inhibitors for trypsin, human neutrophil elastase, human plasma kallikrein and plasmin. BmTIs elicited a protective immunological response against B. microplus infestation in cattle. However, only a small amount of purified natural BmTIs can be obtained from larvae and eggs by chromatographic methods, thus if BmTIs are to be used as vaccine antigens (immunogens) the production of recombinant BmTIs (rBmTIs) is essential. In this work we describe the cloning, expression, purification and characterization of rBmTI-6. rBmTI-6 is a three-headed Kunitz-BPTI inhibitor, expressed in the Pichia pastoris system. Although rBmTI-6 was processed by proteases and glycosylated during the expression process, these post-translational modifications did not alter the ability of rBmTI-6 to inhibit protease activity. Purified rBmTI-6 inhibited trypsin and plasmin.     

Organic trace minerals on productive performance,egg quality and immune response in Bovans White laying hens

This study was performed in 360 laying Bovans White hens and aimed to evaluate the effect of Carbo‐Amino‐Phosphate‐Chelates (CAFQ) as a mineral premix and its 30% reduction by compared to inorganic trace minerals (ITM) concerning its performance, egg quality, tibia breaking strength and immunity. Sixty‐week‐old hens were assigned into three treatments with 10 replicates of 12 birds each. Treatments were as follows: (a) (100% ITM), (b) (100% CAFQ) and (c) (70% CAFQ). Based on the results obtained during 12 weeks, under the current test conditions, improved yolk colour (p < 0.05); shell breaking strength (p < 0.05); and storage time (p < 0.05) were observed in hens fed with a diet reduced by 70% in CAFQ inclusion showed similar performance behaviour and better egg breaking strength regarding with ITM, suggesting the possibility of minimizing the inclusion of trace minerals in laying hen diets improving environmental impact, in contrast to the use of inorganic sources of minerals.     

Seasonality in Saint Croix male lamb reproductive development in northern Mexico

The aim of this study was to determine the influence of the season of birth on the body weight, testicular size, testosterone concentrations, semen output and sexual behaviour in Saint Croix male lambs. Thirty‐three lambs were born in spring (n = 10), summer (Sum; n = 12) or autumn (n = 11). Body weight and the scrotal circumference were weekly recorded; semen was also weekly collected by electroejaculation; and sexual behaviour towards oestrous ewes was weekly evaluated. Data were recorded from 15 to 33 weeks of age. The reproductive development of Saint Croix male lambs differed according to the period in which they were born. More lambs born in spring than in summer and autumn achieved puberty during this study and were also more precocious in semen production and displayed sexual behaviour earlier. Lambs born in summer delayed their semen production; however, season‐related negative effects were less pronounced at the end of the study than in autumn born lambs born. In conclusion, despite the apparently low seasonality of the breed, Saint Croix male lambs showed in this study a seasonal pattern in their reproductive development at least until they achieve the puberty.     

Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

Recreational sandboxes for children and dogs can be a source of epidemic ribotypes of Clostridium difficile

Different studies have suggested that the sand of public playgrounds could have a role in the transmission of infections, particularly in children. Furthermore, free access of pets and other animals to the playgrounds might increase such a risk. We studied the presence of Clostridium difficile in 20 pairs of sandboxes for children and dogs located in different playgrounds within the Madrid region (Spain). Clostridium difficile isolation was performed by enrichment and selective culture procedures. The genetic (ribotype and amplified fragment length polymorphism [AFLP]) diversity and antibiotic susceptibility of isolates was also studied. Overall, 52.5% (21/40) of samples were positive for the presence of C. difficile. Eight of the 20 available isolates belonged to the toxigenic ribotypes 014 (n = 5) and 106 (n = 2), both regarded as epidemic, and CD047 (n = 1). The other 12 isolates were non‐toxigenic, and belonged to ribotypes 009 (n = 5), 039 (n = 4), and 067, 151 and CD048 (one isolate each). Nevertheless, all isolates (even those of a same ribotype) were classified into different AFLP genotypes indicating non‐relatedness. In conclusion, our results revealed the presence of epidemic ribotypes of C. difficile in children's and dog's sandboxes located nearby, which constitutes a major health risk.     

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (> or =80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick-pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick-pathogen interactions.     

Feulgen-DNA densitometry of embryo sacs permits discrimination between sexual and apomictic plants in Paspalum simplex

The chromosomes of sexual diploid plants of Paspalum simplex were colchicine-doubled and the plant obtained were crossed with their aposporous natural tetraploid counterparts to generate a F1 population segregating for apomixis. Analysis of the DNA content during megagametogenesis indicated that although the nuclei of nucellus and developing embryo sacs were in both the G1 and G2 phases, polar nuclei and egg cells of mature embryo sacs tended to remain in the G1 phase. Because both meiotic and aposporous mature embryo sacs are of the 8-nucleated-type in P. simplex and are barely distinguishable, nuclear DNA content of polar nuclei was used to distinguish apomictic and sexual phenotypes and confirmation obtained by progeny testing. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of urban refuse compost and different tractors tyres on soil physical properties, soil erosion and maize yield

Application of urban refuse compost to agricultural soil could help to solve municipalities' problems related to the increasing production of waste only if soil property improvement and environmental conservation can be demonstrated. The use of low-pressure tractor tyres is another proposal in modern agriculture for reducing soil compaction. This study thus aimed to detect the effects of both compost and low-pressure tractor tyres on soil loss, runoff, aggregate stability, bulk density, penetrometer resistance and maize (Zea mays L.) yield. A 3-year field experiment was carried out on a hilly (15% slope) clay loam soil in central Italy. Twelve plots (200 m² each) were monitored with tipping-pot devices for runoff and soil erosion measurement. Treatments were: compost addition (64 Mg ha⁻¹), mineral fertilisation, use of low-pressure tyres, use of traditional tyres, with three replicates, in a fully randomised block design. Compost was applied once at the beginning of the experiment. Runoff reduction due to compost ranged between 7 and 399 m³ ha⁻¹ during seasons, while soil erosion was reduced between 0.2 and 2.4 Mg ha⁻¹. Mean weight diameter (MWD) of stable aggregates, measured on wheel tracks, increased by 2.19 mm, then progressively decreased. Compost significantly increased bulk density by 0.08 Mg m⁻³ due to its inert fraction content. This effect was less evident in the second and third year, probably due to harrowing. Maize yields were slightly, but significantly, reduced in composted plots by 1.72 Mg ha⁻¹ in the third year. Low-pressure tyres significantly reduced soil loss in the third year by 1 Mg ha⁻¹. Furthermore, they did not significantly influence runoff volumes and soil structural stability. Low-pressure tyres or compost addition were singly able to prevent an increase in penetrometer resistance due to agricultural machinery traffic. Low-pressure tyres increased the maize yield during the 3 years and the difference (0.4 Mg ha⁻¹) became significant in the third year. In conclusion, results show the positive lasting effect of compost in ameliorating soil physical properties and reducing runoff and soil erosion. Low-pressure tyres appear justifiable both for the observed increase of grain production and reduction of soil compaction. This latter effect is, nevertheless, masked by compost addition which is also able to reduce penetrometer resistance. Further research is required to explain the causes of the slight inhibition of grain yield observed when compost was compared with mineral fertilisation.     

A century of fishery data documenting the collapse of smooth‐hounds (Mustelus spp.) in the Mediterranean Sea

相似文献   

High immune diversity in farmed Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

As with any other intensively farmed animal species, the Atlantic salmon has been selectively bred and cross-bred to maximize desirable traits. Selection tends to diminish genetic diversity in target populations, which among other negative effects is hypothesized to decrease their capacity to confront a broad variety of pathogens. We have analyzed mitochondrial (mtDNA) as well as major histocompatibility complex (MHC) DNA sequences from individuals collected from a single aquaculture cage in southern Chile. Interestingly, only two mtDNA haplotypes were obtained; however, several different MH alleles were detected, with divergence values that were compared with those of natural populations of salmonids. Thus, it seems evolutionary processes responsible of keeping MH diversity in the wild managed to retain MH variability in farmed Atlantic salmon, maintaining high immune diversity despite the generally lower levels of observed neutral genetic diversity.