Silage review: Factors affecting dry matter and quality losses in silages.

An overview was made of dry matter (DM) and quality losses that occur during the ensiling process from the field through the feeding phase. The aim was to review the relevant published literature of the last 15 yr focusing on developments achieved after the publication of the book Silage Science and Technology. This review discusses the factors affecting DM and quality losses in terms of field and pre-ensiling conditions, respiration and temperature at ensiling, fermentation patterns, methods of covering and weighting the silage cover, and management of aerobic deterioration. The possibility of reducing DM and quality losses during the ensiling process requires knowledge of how to measure losses on farm and establish the status of the silage during the feed-out phase, implementing the most effective management practices to avoid air exposure during conservation and reduce silage aerobic deterioration during feeding. The paper concludes with future perspectives and recommended management practices to reduce losses and increase efficiency over the whole ensiling process in view of increasing sustainability of the livestock production chain.

Extracting DNA from 'jaws': high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material.

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield.

Age and growth of the tiger shark Galeocerdo cuvier off the east coast of Australia.

Total lengths (L(T)) at age and growth rates for south-west Pacific Galeocerdo cuvier were estimated from vertebral growth-band counts of 202 sagitally sectioned centra from 112 females (71-430 cm L(T)), 79 males (72-351 cm L(T)) and 11 of unknown sex. Captive growth data were also examined to complement vertebral age estimations. The sexes combined modelled growth coefficient (k = 0.08) was smaller than previously reported for G. cuvier populations elsewhere. Split-band and narrow banding patterns were identified as potential sources of age underestimation in this species.

Evidence-informed healthcare through integration of health research.

A foundational element of modern healthcare is an evidence-based practice. However, the conduct of research to generate evidence and the subsequent application of research findings are viewed by many Canadian healthcare organizations as separate from healthcare delivery. This mindset impedes effective translation of knowledge into practice. In this article, underlying issues that enable this disintegrated model to persist are described, and strategies to help healthcare organizations achieve integration of research are summarized.

A review of the application of molecular genetics for fisheries management and conservation of sharks and rays.

Since the first investigation 25 years ago, the application of genetic tools to address ecological and evolutionary questions in elasmobranch studies has greatly expanded. Major developments in genetic theory as well as in the availability, cost effectiveness and resolution of genetic markers were instrumental for particularly rapid progress over the last 10 years. Genetic studies of elasmobranchs are of direct importance and have application to fisheries management and conservation issues such as the definition of management units and identification of species from fins. In the future, increased application of the most recent and emerging technologies will enable accelerated genetic data production and the development of new markers at reduced costs, paving the way for a paradigm shift from gene to genome-scale research, and more focus on adaptive rather than just neutral variation. Current literature is reviewed in six fields of elasmobranch molecular genetics relevant to fisheries and conservation management (species identification, phylogeography, philopatry, genetic effective population size, molecular evolutionary rate and emerging methods). Where possible, examples from the Indo-Pacific region, which has been underrepresented in previous reviews, are emphasized within a global perspective.

Nonatopic wheezy children have reduced interferon-gamma.

Viruses cause asthmatic exacerbations in schoolchildren. We tested the hypothesis that children who wheezed with viral respiratory tract infections secrete higher levels of the type 1 cytokine interferon-gamma (IFN-gamma) in the peripheral circulation than children who had never wheezed. Blood was taken from 13 children (eight atopic) with episodic wheeze and 11 controls. CD4 and CD8 cells were separated from peripheral blood mononuclear cells and stimulated with phorbol 12-myrisate 13-acetate (PMA) and ionomycin for 24 h. IFN-gamma, IL-4, and IL-5 were measured in the supernatant by ELISA. IFN-gamma production by CD4 and CD8 cells was lower in children with a history of wheeze (CD4, P = 0.046; CD8, P = 0.037). These children were then analysed according to atopic status. CD4 and CD8 IFN-gamma production in nonatopic wheezy children was reduced (CD4, P=0.009; CD8, P=0.003). IFN-gamma production by atopic wheezy children was lower than by controls, but the differences were not significant (CD4, P = 0.2831; CD8, P = 0.1372). CD8 IL-5 was lower in children who wheezed (P=0.012). Release of IL-4 and IL-5 by CD4 cells did not differ between the three groups. We propose that defective IFN-gamma secretion by CD4 and CD8 cells may contribute to viral-induced wheeze in nonatopic children.

Ovalbumin-specific, MHC class I-restricted, alpha beta-positive, Tc1 and Tc0 CD8+ T cell clones mediate the in vivo inhibition of rat IgE.

In the following study, we demonstrate that medium responder PVG rats immunized i.p. with OVA complexed to the adjuvant aluminum hydroxide exhibit a moderate IgE response (400-1000 ng/ml). In these rats, we demonstrate that underlying the MHC class II-restricted CD4+ T cell response, there is an MHC class I-restricted CD8+ T cell component that plays an important role in restricting the magnitude and duration of the IgE response. We show that in vivo depletion of CD8+ T cells effects a massive increase in IgE (20-fold), and that they are MHC class I-restricted, OVA-specific, cytolytic cells that universally produce IFN-gamma (25-69 ng/ml) and IL-2 (7.6-22 U/ml), and occasionally secrete IL-4 (68-81 U/ml IL-4), and when adoptively transferred into CD8-depleted recipients, can effect a significant reduction in IgE (3- to 50-fold). We also demonstrate that this in vivo inhibition of IgE is dependent on the Ag-specific activation of the CD8+ T cells, and that the activated CD8+ T cells will suppress total/bystander IgE in an Ag-nonspecific manner. These data are consistent with a growing literature demonstrating sensitization of MHC class I-restricted CD8+ T cells by exogenous protein Ags delivered to mucosal sites, and may represent a mechanism whereby a selective pressure can be applied on the functional outcome of an immunoglobulin response to environmental allergens.

Antigen-specific CD8+ T cells inhibit IgE responses and interleukin-4 production by CD4+ T cells.

There is a growing body of evidence which suggests that CD8+ T cells play an important part in regulating the IgE response to non-replicating antigens. In this study we have systematically investigated their role in the regulation of IgE and of CD4+ T cell responses to ovalbumin (OVA) by CD8+ T cell depletion in vivo. Following intraperitoneal immunization with alum-precipitated OVA, OVA-specific T cell responses were detected in the spleen and depletion of CD8+ T cells in vitro significantly enhanced the proliferative response to OVA. Depletion of CD8+ T cells in vivo 7 days after immunization failed to enhance IgE production, while depletion of CD8+ T cells on days 12-18 greatly enhanced the IgE response, which rose to 26 micrograms/ml following a second injection of anti-CD8 on day 35 and remained in excess of 1 microgram/ml over 300 days afterwards. Reconstitution on day 21 of rats CD8-depleted on day 12 with purified CD8+ T cells from animals immunized on day 12 completely inhibited the IgE response. This effect was antigen specific; CD8+ T cells from OVA-primed animals had little effect on the IgE response of bovine serum albumin immunized rats. In vivo, CD8+ T cell depletion decreased interferon (IFN)-gamma production but enhanced interleukin (IL)-4 production by OVA-stimulated splenic CD4+ T cells. Furthermore, CD8+ T cell depletion and addition of anti-IFN-gamma antibody enhanced IgE production in vitro in an IL-4-supplemented mixed lymphocyte reaction. These data clearly show that antigen-specific CD8+ T cells inhibit IgE in the immune response to non-replicating antigens. The data indicate two possible mechanisms: first, CD8+ T cells have direct inhibitory effects on switching to IgE in B cells and second, they inhibit OVA-specific IL-4 production but enhance IFN-gamma production by CD4+ T cells.

Depletion of CD8+ T cells following primary immunization with ovalbumin results in a high and persistent IgE response.

The role of CD8+ T cells in the regulation of IgE was investigated by in vivo CD8+ T-cell depletion. Following intraperitoneal immunization with ovalbumin (OVA), OVA-specific T cell responses were first detected in the draining lymph nodes (LN) and subsequently in the spleen. In vitro depletion of LN CD8+ cells reduced the OVA-specific LN CD4+ T cell response; while depletion of splenic CD8+ cells enhanced the OVA-specific splenic CD4+ T cell response, suggesting an early helper and later suppressor function. CD8+ cell depletion in vivo up to 7 days after immunization failed to enhance IgE production, but depletion of CD8+ T cells between day 12 and day 18 increased IgE levels to over 10 microg/ml. Adoptive transfer of 1 x 10(7) purified CD8+ T cells (> 95% CD8+ > 98% CD3+ < 1% NK, < 1% CD4+) from parallel immunized rats or of 1 x 106 cells of an OVA-specific, MHC class I restricted CD8+ T cell line suppressed the IgE, but not IgG, response by > 95%. These results provide further evidence that CD8+ T cells inhibit IgE production, possibly by preventing the generation of adequate amounts of appropriate CD4+ T cell help.

Generation of rat MHC class-I-restricted ovalbumin-specific IgE inhibitory CD8+ T cell clones.

Allergic disease presents an increasing health problem in industrialised countries. Despite improved diagnosis and drug therapy, the number of fatalities from asthma and anaphylaxis continues to rise. A key element in the aetiology of the allergic response to inert protein antigens is an inherent imbalance in the atopic allergen specific CD4+ T cells, resulting in a dominance of the pathological, IL-4 producing, T helper 2 phenotype which favours B cell IgE production. Previous work from our laboratory, and elsewhere, has established that CD8+ T cells play an important role in regulating the magnitude and duration of IgE responses and may be vital for the restoration of IgE immunologic homoeostasis in 'normal' non-atopic humans and low/medium responder rodents. We have found that CD8+ T cell depletion during a critical period after immunization produces a high and persistent IgE response in otherwise IgE hyporesponsive animals. We and others have demonstrated that small, but important, quantities of soluble antigen can enter the MHC class I pathway and, using ovalbumin (OVA)-transfected antigen-presenting cells, have succeeded in cloning OVA-specific, MHC class I-restricted rat alpha/beta T cell receptor (TcR) positive CD8+ T cells. These cells universally secrete IFN-gamma (5-69 ng/ml) and IL-2 (7.6-37.0 U/ml) and occasionally IL-4 (16.0-8/1.0 IU). When adoptively transferred, 1 x 10(6) of these cloned cells inhibit IgE production in vivo by as much as 50-fold and this effect can be titrated down to 1 x 10(3) cells. The IgE regulatory activity of the clones appears unrelated to their cytolytic potential or to their capacity to make IFN-gamma.

The contrasting effects of CD8+ T cells on primary, established and Nippostrongylus brasiliensis-induced IgE responses.

Recent data have indicated that CD8+ T cells suppress rodent IgE responses. In this study we investigated the effect of CD8+ T cells on primary and established IgE responses in euthymic and athymic nude rats. Euthymic PVG rats were depleted of CD8+ T cells by intraperitoneal injection of a CD8-specific monoclonal antibody (OX8), which resulted in an apparent loss of 92% of splenic and 98% of peripheral blood CD8+ T cells. The CD8+ T-cell depleted animals failed to mount a significant IgE response compared with control animals given an irrelevant monoclonal antibody (OX21). Furthermore, PVG nude rats reconstituted with purified CD4+ thoracic duct lymphocytes (TDL) alone failed to mount a significant IgE response, while animals given unfractionated TDL (containing CD4+ and CD8+ T cells) did. Depletion of CD8+ T cells 7 days prior to immunization and subsequent reconstitution at the time of immunization restored the IgE response. In contrast, removal of CD8+ T cells 1 month after induction of IgE by immunization with ovalbumin (OVA) and ricin prolonged the IgE response. In all cases IgG antibody responses were unaffected by the presence or absence of CD8+ T cells. This study shows that some CD8+ T cells are required for IgE, but not IgG, production to soluble antigen in a primary immune response. However, later in the immune response CD8+ T cells were shown to inhibit IgE production. These effects were apparently restricted to the immune response to soluble antigen, as Hooded Lister rats infected with 9000 larvae of the nematode Nippostrongylus brasiliensis produced high sustained levels of circulating IgE, in excess of 10 micrograms/ml, regardless of whether CD8+ T cells were depleted before or 1 month after infection.

The role of CD8+ T cells in immunoglobulin E regulation.

The link between immunoglobulin E (IgE) antibodies and environmental allergens, allergic sensitization and atopic diseases such as asthma, rhinitis and eczema is well established, yet treatment of these diseases is largely symptomatic and fails to correct the underlying immune disorder. B-cell production of IgE depends on interleukin 4 (IL-4) and is inhibited by interferon (IFN-gamma). These cytokines are produced by T-helper 2 (Th2) and T-helper 1 (Th1) CD4+ T cells respectively. In atopic dermatitis skin and asthmatic lung, Th2 cells predominate but antigen-specific CD4+ T-cell clones derived from peripheral blood are a mixture of Th1, Th2, and Th0. Circulating allergen-specific CD4+ T cells differentiate into Th1- and Th2-like clones in the presence of IL-12 and IL-4 respectively. In rats, CD8+ T cells are potent regulators of IgE responses in vivo, although the mechanism for this is still unclear. CD8+ T-cell depletion in immunized animals prolonged the IgE response but prior depletion did not. Indeed, prior depletion of CD8+ T cells actually inhibited IgE production, suggesting that there may be more than one type of IgE regulatory CD8+ T cell. This was supported by the observation that CD8+ T cells inhibited the differentiation of both Th1 and Th2 CD4+ T cells in vitro as well as IL-4- but not IL-2-induced Th2 CD4+ T-cell growth. The existence of distinct CD8+ T-cell subsets is supported by the observation that IL-4 and IFN-gamma regulate the growth and differentiation of CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune regulation: a new role for the CD8+ T cell.

During an immune response, peripheral T cells develop into functionally distinct subpopulations that effect cell-mediated immunity and regulate humoral immune responses through the secretion of specific cytokines. Recent data suggest that CD8+ T cells, which have long been regarded simply as cytotoxic cells, play a more active role in the regulation of the immune response. In this article, Mike Kemeny and colleagues suggest that there are functionally distinct subsets of CD8+ T cells that produce different combinations of cytokines and appear to play an important part in determining the pattern of cytokines produced by CD4+ T cells and the isotype of immunoglobulins expressed by B cells.

Moxalactam therapy of difficult infections in patients with serious underlying conditions.

Moxalactam was the single therapeutic agent used to treat a variety of infections in sixty-three patients, most of whom had serious concomitant illnesses. Fifty-three patient case reports qualified for evaluation, including those with pneumonia (8), urinary tract infections (18), superficial infections (6), orthopaedic infections (7), osteomyelitis (8), septicaemia (4), pansinusitis (1), and meningitis (1). Preliminary in vitro studies had indicated that most organisms, including those resistant to other antibacterial agents, would respond to moxalactam. Infecting bacteria from the fifty-three evaluable patients included a wide variety of Gram-positive and Gram-negative organisms. Doses of moxalactam ranged from 1 to 16 g/day administered intravenously or intramuscularly for 5 to 41 days. With few explainable exceptions, clinical and bacteriologic responses were adequate and satisfactory. Adverse effects were inconsequential. Allergic reactions were not observed, even in patients with a past history of reactions to penicillin.

Antigenic relationships of common rhinovirus types from disabling upper respiratory illnesses.

The frequency of the common cold has been though to be due to the existence of more than 89 different serologic types of rhinoviruses. However, in the civilian and military population studied in this area from 1962 through 1970, types 1A, 1B, 2, 23, 29, 30 and 31 accounted for 81 percent of 487 rhinoviruses isolated from individuals with respiratory illnesses. Antigenic relationships between these types and others have been demonstrated by neutralization in WI-38 cell cultures and by immunodiffusion. Types 1A and 1b, 2 and 49, 23 and 30, 29 and 44 and others are related. With specific animal antisera, heterotypic antibody titers are minimal. However, following natural infection in man with one of these related types, antibody responses to the other was almost equal to the homotype and could predictably provide cross-protection. Even with inactive T13 rhinovirus vaccine in man, protective levels of neutralizing antibody to T41 were produced. It is clear that the rates of rhinovirus colds would be greatly increased if more than 89 immunologic distinct types actually existed. An effective vaccine could be easily prepared by selection of a few appropriate types, such as 1A, 2, 23, 29 and 31.