Pathological consequences of low atmospheric pressure stunning in broiler chickens.

Low atmospheric pressure stunning (LAPS) is a novel approach to pre-slaughter stunning of chickens using progressive hypobaric hypoxia by the application of gradual decompression (280s cycle) according to a set of prescribed pressure curves. Low atmospheric pressure stunning produces a non-recovery state. Concerns have been raised relating to the possible pathological and welfare consequences of expansion of air in the body during LAPS. In a randomised trial, we compared the gross pathology of broilers exposed to LAPS with a control group euthanised by intravenous injection of pentobarbital sodium (60 mixed sex broilers per treatment). The birds were exposed to each treatment in triplets and all birds were subject to necropsy examination to detect and score (1 to 5, minimal to severe) haemorrhagic lesions or congestion for all major organs and cavities (e.g. air sacs, joints, ears and heart) as well as external assessment for product quality (e.g. wing tips). Behavioural data (latency to loss of posture and motionless) and chamber cycle data (temperature, humidity, pressure and oxygen availability) confirmed that LAPS had been applied in a manner representative of the commercial process. All of the organs observed were structurally intact for both treatment groups. No lesions were observed in the external ears, oral cavity, tracheal lumen, crop and air sacs of birds from either treatment group. There was no difference between treatments in the wingtips, nasal turbinates, thymus, biceps femoralis and colon. Haemorrhagic lesions were observed in the calvaria, brains, hearts and lungs of both treatment groups, but lesions in these areas were more severe in the LAPS treatment group. It was not possible to distinguish between pathological changes induced by decompression or recompression. In the barbiturate group, more severe haemorrhagic lesions were observed in the superficial pectoral muscles as well as greater congestion of the infraorbital sinuses, liver, spleens, duodenum, kidneys and gonads. These findings provide evidence that LAPS did not result in distension of the intestines and air sacs sufficient to cause changes, which were grossly visible on postmortem examination. There was also no evidence of barotrauma in the ears and sinuses. The pathological changes observed in the barbiturate treatment were as expected based on barbiturate toxicity. Low atmospheric pressure stunning appears to produce pathological changes by a variety of well-established mechanisms, and while these pathological data have limited value as welfare indicators, the results confirm that organ integrity was not compromised by the process.

Associations between membership of farm assurance and organic certification schemes and compliance with animal welfare legislation.

Animal health (AH) defines the outcome of their inspections of livestock holdings as full compliance with the legislation and welfare code (A), compliance with the legislation but not the code (B), non-compliance with legislation but no pain, distress or suffering obvious in the animals (C) or evidence of unnecessary pain or unnecessary distress (D). The aim of the present study was to investigate whether membership of farm assurance or organic certification schemes was associated with compliance with animal welfare legislation as inspected by AH. Participating schemes provided details of their members, past and present, and these records were matched against inspection data from AH. Multivariable multilevel logistic binomial models were built to investigate the association between compliance with legislation and membership of a farm assurance/organic scheme. The percentage of inspections coded A, B, C or D was 37.1, 35.6, 20.2 and 7.1 per cent, respectively. Once adjusted for year, country, enterprise, herd size and reason for inspection, there was a pattern of significantly reduced risk of codes C and D compared with A and B, in certified enterprises compared with the enterprises that were not known to be certified in all species.

Calf mortality as a welfare indicator on British cattle farms.

The objective of this study was to test calf mortality as an indicator of on-farm welfare and its use for welfare targeted surveillance. Calf mortality data were retrieved for three UK counties to estimate calf mortality rates at holding and county level. A selection criterion based on upper quartiles of calf mortality for the county of concern was defined. Its predictive ability was tested in a field study. The death risk of calves less than 6 months of age in 2002 was 1.76% in Inverness, 5.83% in Cheshire and 4.8% in Norfolk. Fifty-two welfare inspections matched by parish were conducted between October 2004 and January 2005. The positive predictive value was 26.92% and the negative predictive value was 65.38%. The addition of herd type, county and membership to an assurance scheme improved the predictive value. This study shows that calf mortality can be the starting point to design targeted welfare inspections in countries with centralized animal data recording systems.

Action pattern and substrate specificity of the hyaluronan lyase from group B streptococci.

The hyaluronan lyase of group B streptococci rapidly cleaves hyaluronan by an elimination mechanism to yield the unsaturated disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose. Additionally, it has been shown that the enzyme has limited specificity for achondroitin sulphate and cleaves the chain at unsulphated sites [Baker,Yu, Morrison, Averett and Pritchard (1997) Biochem. J. 327,65-71]. In the present extension of that study it was found that 6-sulphated regions of chondroitin sulphate are also susceptible to cleavage by this hyaluronan lyase. Of the four 6- and/or 4-sulphated tetrasaccharides which can be isolated from testicular hyaluronidase digests of chondroitin sulphate, only those two tetrasaccharides with a6-sulphated disaccharide at the reducing end were cleaved. From thisand other data, a model is proposed for the cleavage specificity of hyaluronan lyase on a chondroitin sulphate. Evidence is presented in support of an action pattern for hyaluronan lyase which involves aninitial random endolytic cleavage followed by rapid exolytic and processive release of unsaturated disaccharide. Since the on lyoligosaccharides which tend to accumulate in near-complete digests of hyaluronan are unsaturated, it is argued that the processive cleavage occurs from the non-reducing to the reducing end of a hyaluronan chain. This detailed knowledge of substrate specificity contributes to our understanding of the enzyme's role in Group B streptococcal pathogenesis. In addition, the hyaluronan lyase may find application in sequence studies of chondroitin sulphates.

Characterization of the active site of group B streptococcal hyaluronan lyase.

Hyaluronan lyase is secreted by most strains of the human pathogen, group B streptococcus. Site-directed mutagenesis of the enzyme identified three amino acid residues important for enzyme activity, H479, Y488, and R542. These three residues are in close proximity in the putative active site of a homology model of group B streptococcal hyaluronan lyase. The homology model was based on the crystal structure of another related glycosaminoglycan lyase, chondroitin AC lyase, which exhibits different substrate specificity. Two asparagine residues in the active site groove, N429 and N660, were also found to be essential for enzyme activity. In addition, conversion of two adjacent tryptophan residues in the groove to alanines abolished activity. All amino acids found to be essential in GBS hyaluronan lyase are conserved in both enzymes. However, several amino acids in the active site groove of the two enzymes are not conserved. In the 18 cases in which one of these amino acids in GBS hyaluronan lyase was replaced with its corresponding amino acid in chondroitin AC lyase, no major loss of activity or change in substrate specificity was observed.

Specificity of the hyaluronate lyase of group-B streptococcus toward unsulphated regions of chondroitin sulphate.

The purification and properties of a hyaluronate lyase secreted by Streptococcus agalactiae, which is believed to facilitate the invasion of host tissues by the organism, have been described previously [Pritchard, Lin, Willingham and Baker (1994) Arch. Biochem. Biophys. 315, 431-436]. The specificity of the limited cleavage of chondroitin sulphate by the enzyme is the subject of this report. To simplify the task, a chondroitin sulphate from the Swarm rat chondrosarcoma, which contains only 4-sulphated and unsulphated disaccharide repeats, was used in this study. Tetrasaccharides from an ovine testicular hyaluronidase digest of the chondroitin sulphate were isolated, identified and tested as substrates of the streptococcal hyaluronate lyase. Only tetrasaccharides with an unsulphated disaccharide at the reducing end were cleaved (by elimination at the N-acetylgalactosaminidic bond). Thus chondroitin sulphate chains are cleaved by the action of this lyase at every unsulphated disaccharide repeat, but release of unsaturated unsulphated disaccharides only occurs from sites where two or more sequential unsulphated disaccharide repeats are present. Analysis of the chondrosarcoma chondroitin sulphate showed that of approximately five unsulphated disaccharide repeats per chain, two are clustered. The ability of group-B streptococcal hyaluronate lyase to cleave chondroitin sulphate may allow the organisms to invade tissues more efficiently. The demonstrated specific and highly limited cleavage of chondroitin sulphate by this bacterial lyase promises to be a useful tool in the determination of chondroitin sulphate structure and variability.

Identification of a histidine residue essential for enzymatic activity of group B streptococcal hyaluronate lyase.

Hyaluronate lyase produced by group B streptococci (GBS) degrades hyaluronan completely to unsaturated disaccharide units and also cleaves unsulfated regions of chondroitin sulfate. The enzyme is rapidly inactivated by diethyl pyrocarbonate and enzymatic activity is restored by treatment with hydroxylamine, suggesting that a histidine residue is present in the active site. Amino acid sequence comparisons of GBS hyaluronate lyase and four other related enzymes revealed that one of the 16 histidine residues of the enzyme (His-479) is present in a highly conserved region. Conversion of His-479 to a glycine by site-directed mutagenesis resulted in a complete loss of enzymatic activity of the modified protein. We propose that His-479 is in the active site of GBS hyaluronate lyase and participates in the initial abstraction of hydrogen ions from the glucuronic acid residues of hyaluronan.

Characterization of PepB, a group B streptococcal oligopeptidase.

Group B streptococci were recently reported to possess a cell-associated collagenase. Although the enzyme hydrolyzed the synthetic collagen-like substrate N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala, we found that neither the highly purified enzyme nor crude group B streptococcal cell lysate solubilized a film of reconstituted rat tail collagen, an activity regarded as obligatory for a true collagenase. We cloned and sequenced the gene for the enzyme (pepB). The deduced amino acid sequence showed 66.4% identity to the PepF oligopeptidase from Lactococcus lactis, a member of the M3 or thimet family of zinc metallopeptidases. The group B streptococcal enzyme also showed oligopeptidase activity and degraded a variety of small bioactive peptides, including bradykinin, neurotensin, and peptide fragments of substance P and adrenocorticotropin.

Cloning and expression of the gene for group B streptococcal hyaluronate lyase.

Group B streptococci (GBS) are a major cause of serious human perinatal infections. Most clinical isolates of GBS secrete hyaluronate lyase, and production of high levels of the enzyme has been associated with strain virulence. Degenerate oligonucleotide primers, designed on the basis of the amino acid sequences of tryptic peptides prepared from the purified enzyme, permitted the polymerase chain reaction amplification from GBS chromosomal DNA of a 363-base pair internal DNA fragment of the GBS hyaluronate lyase gene (hylB). This DNA fragment was used as a probe to screen a lambda phage library of GBS chromosomal DNA fragments. Sequence analysis of positive clones identified an open reading frame capable of coding for a 111-kDa protein. Since no single clone was found to contain the entire gene it was necessary to reconstruct the gene from two plasmids containing inserts with suitable overlapping sequences. When this reconstructed gene was transformed into Escherichia coli, high level expression of hyaluronate lyase activity was obtained.

Characterization of the group B streptococcal hyaluronate lyase.

Hyaluronate lyase is one of several proteins secreted by group B streptococci which are believed to contribute to strain virulence. Characterization of the purified enzyme revealed that it degrades hyaluronan by a mechanism different from that of other previously studied hyaluronidases. Instead of randomly cleaving hyaluronan chains leading to a continuous decrease in average chain size, the group B streptococcal enzyme initially yields primarily unsaturated disaccharides. The observation that most of the free reducing ends generated during group B streptococcal hyaluronate lyase digestion are present in the unsaturated disaccharide units supports the conclusion that they are released primarily from the ends of the hyaluronan chains. Furthermore, the experimental evidence is consistent with a mode of action by which the enzyme initially makes a random cut in a hyaluronan chain and then processively moves along the chain releasing disaccharide units. Group B streptococcal hyaluronate lyase also slowly degrades chondroitin sulfate, and its desulfation greatly increases the reaction rate. A preferential cleavage of unsulfated residues is consistent with the observed extensive release of free chondroitin sulfate chains following very limited digestion of aggrecan from bovine nasal cartilage.

Group B streptococcal neuraminidase is actually a hyaluronidase.

The extracellular group B streptococcal enzyme described in numerous reports as a neuraminidase is really a hyaluronidase. Over the past 25 years, the enzyme was routinely assayed with bovine submaxillary mucin as the substrate and by the thiobarbituric acid procedure to measure released sialic acid. Characterization of the actual compound released by the enzyme revealed it to be an alpha,beta-unsaturated derivative of hyalobiuronic acid that was derived from hyaluronic acid contaminating the mucin preparation. Previous reports describing an association of elevated levels of extracellular neuraminidase with virulent strains of group B streptococci must be reevaluated with the recognition that the enzyme is really a hyaluronidase.

Capsular type-specific polysaccharide partially inhibits group B Streptococcus-induced pulmonary hypertension.

Capsular type-specific polysaccharide is thought to be an important pathogenetic factor in Group B streptococcus (GBS) sepsis. To determine the effects of capsular type-specific polysaccharide on GBS-induced hemodynamic responses, anesthetized infant piglets were infused for 3 h with three related GBS Type lb strains that express different amounts of capsular type-specific polysaccharide. A larger capsule strain and a smaller capsule strain were isolated from an infected infant and its mother, respectively. A capsule-deficient mutant was then made from the larger capsule strain by transposon insertion mutagenesis. The smaller capsule strain and capsule-deficient mutant caused similar elevations in mean pulmonary artery pressure and pulmonary vascular resistance index and reductions in cardiac index. The larger capsule strain caused moderate pulmonary hypertension, but this response was smaller than for the other two GBS strains. Further comparisons in responses between the large capsule strain and its capsule-deficient mutant were then performed using unanesthetized piglets. The mutant caused significantly greater pulmonary hypertension and arterial plasma thromboxane B2 levels than the large capsule strain. The pulmonary hypertension induced by both strains was reversed by dazmegrel, a thromboxane A2 synthase inhibitor. These results suggest that (1) capsular type-specific polysaccharide is not an essential component in the generation of acute hemodynamic responses; (2) expression of large amounts of capsular type-specific polysaccharide on the organism surface partially inhibits GBS-induced pulmonary hypertension; and (3) the inhibition of the pulmonary responses is due to reduced thromboxane A2 release.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine monoclonal antibodies to type Ib polysaccharide of group B streptococci bind to human milk oligosaccharides.

The chemical structures of the repeating units of the type Ib polysaccharide of group B streptococci and of the desialylated form of this antigen are almost identical to those of some oligosaccharides in human milk and certain fetal antigens. The structural similarities suggested that the molecules may be immunologically cross-reactive. Mouse monoclonal antibodies to the sialylated and nonsialylated forms of the type Ib polysaccharide were produced and tested for their ability to bind to immobilized human milk oligosaccharides. One antibody, SMB19, reacted specifically with the sialylated form of the type Ib polysaccharide and was also bound by an affinity column containing immobilized sialyllacto-N-tetraose a. The antibody was eluted from the affinity column with EDTA, since its binding to the antigen was calcium dependent. A second monoclonal antibody. SIbD2, bound specifically to the nonsialylated form of the type Ib polysaccharide and also to immobilized lacto-N-tetraose. The antibody was eluted from the affinity column at an acidic pH and retained immunologic activity. These results further extend our previous observations that certain antibodies raised against group B streptococci can also react with normal human glycoconjugates.

Role of capsule in pulmonary hypertension induced by group B streptococcus.

The type-specific polysaccharide capsule of group B streptococcus (GBS) is thought to be an important factor in the pathogenesis of disease. We used an acutely instrumented piglet model to assess the hemodynamic effects of rapid infusions of two heat-killed GBS type Ib strains isolated from the spinal fluid of an infant with late-onset meningitis and from the vaginal culture of his mother. These strains expressed different amounts of capsule, as determined by buoyant density centrifugation and electron micrographs, and they produced different hemodynamic effects in the piglets. The mother's strain, which had a smaller capsule, caused significantly higher increases in pulmonary artery pressure and vascular resistance than did the infant's strain, which had a larger capsule. Transposon mutants were then made from the infant's isolate to further study the role of capsule in pulmonary hypertension. Two mutants lacking detectable capsular type-specific polysaccharide were compared with the original isolate and with an isogenic mutant containing transposons but having a large capsule. The nonencapsulated mutants caused significantly higher changes in pulmonary artery pressure and resistance than did the encapsulated strains. Pulmonary hypertension may play a role in the pathophysiology of GBS sepsis, but the presence of a large capsule may partially cloak the hemodynamically active component(s) of the bacteria. The lower initial host response to heavily encapsulated GBS may play a role in pathogenesis by helping the organisms avoid host defense mechanisms.

Structure of the type-specific polysaccharide antigen of Streptococcus rattus.

The structure of the type-specific polysaccharide antigen of Streptococcus rattus was determined by methylation analysis, periodate oxidation, and by 2D-1H- and 13C-n.m.r.-spectroscopy. The polysaccharide was found to possess the trisaccharide repeating unit----3)-alpha-L-Rhap-(1----2)-[alpha-D-Galp-(1----3)]-alpha-L-+ ++Rhap- (1----.

Effect of fibronectin on IgA-mediated uptake of type III group B streptococci by phagocytes.

Previous studies have shown that a type-specific IgA monoclonal antibody alone or in combination with fibronectin (Fn) enhances protective efficacy in two animal models of group B streptococcal infection. To investigate the mechanisms by which IgA mediates protection, the effects of Fn on phagocytosis of group B streptococci (GBS) opsonized with a type III-specific IgA monoclonal antibody were examined. Specific IgA alone or in combination with Fn did not promote the phagocytosis of GBS by polymorphonuclear leukocytes (PMNL). Fibronectin also had no significant effect on phagocytosis of IgA-opsonized GBS by monocytes. Specific IgA alone promoted phagocytosis of GBS by culture-derived macrophages in a dose-dependent fashion. Fibronectin enhanced macrophage uptake of the GBS opsonized in a suboptimal concentration of specific IgA (phagocytic index = 2.32 +/- 0.56 vs. 3.26 +/- 0.48 with Fn; P less than .05). These data suggest that protection against GBS in neonatal rats by a combination of Fn and specific IgA is mediated by macrophages rather than by PMNL or monocytes. Fibronectin may have a critical role in host defense at sites where IgA and macrophages predominate.

Mucosal and systemic responses to an oral liposome-Streptococcus mutans carbohydrate vaccine in humans.

Purified polysaccharide antigens are often poorly immunogenic, especially when given by the oral route. However, it has been shown in experimental animals that liposomes can greatly increase the immunogenicity of certain polysaccharide antigens. Here we report the induction of immune responses in humans to an oral vaccine consisting of liposomes containing purified serotype carbohydrate antigen of Streptococcus mutans, the primary etiological agent of dental caries. Four volunteer subjects swallowed enteric coated gelatin capsules containing liposomal-antigen for seven consecutive days. Pre- and post-immunization samples of saliva and plasma were analyzed for antibody activity to S. mutans carbohydrate by ELISA. Salivary anticarbohydrate IgA responses were detected in all four subjects between day 21 and day 32. Upon second and third immunizations, subjects experienced salivary responses earlier than following the first immunization. Early (day 4-7) plasma IgA responses to the carbohydrate were found in three subjects which consisted of both polymeric and monomeric forms. Variable levels of plasma IgG anti-carbohydrate antibody activity were seen in three individuals. These results show that orally administered liposomal-S. mutans serotype carbohydrate antigen induces a salivary IgA response in humans and provides evidence for the efficacy of liposomal antigen delivery system in the induction of a protective mucosal immune response against microbial pathogens.

An IgA monoclonal antibody directed against type III antigen on group B streptococci acts as an opsonin.

We have investigated the mechanisms by which a murine IgA mAb directed against the type III Ag (IgA anti-III mAb) of group B streptococci (GBS) protects neonatal rats from lethal infection with these organisms. Purified IgA anti-III mAb enhanced phagocytosis of type III GBS by rat peritoneal macrophages in vitro by fourfold compared with phagocytosis of buffer-treated GBS. In the absence of antibody, neonatal rat serum did not promote phagocytosis, but addition of neonatal rat serum to GBS opsonized with IgA anti-III led to a sevenfold increase in phagocytosis. Heat inactivation of C destroyed the ability of neonatal rat serum to enhance phagocytosis in the presence of IgA. C3 deposition was observed when GBS coated with IgA anti-III mAb were incubated in untreated neonatal rat serum or in serum treated with Mg/EGTA. This latter observation suggested that C3 deposition occurred through activation of the alternative pathway. The control IgA mAb MOPC 315 did not enhance GBS ingestion or C3 deposition on GBS. Depletion of C in vivo by using cobra venom factor abolished the protective effect of IgA anti-III mAb in the neonatal rat model. These data suggest that the ability of this IgA to activate C further enhances its opsonic activity and may be essential for its protective effect in vivo.