Correlations between the biochemistry and mechanical states of a sea-urchin ligament: a mutable collagenous structure.

Mutable collagenous tissues (MCTs) of echinoderms can be regarded as intelligent and dynamic biomaterials, due to their ability to reversibly change their mechanical properties in a short physiological time span. This mutability phenomenon is nervously mediated and involves secreted factors of the specialized 'juxtaligamental' cells, which, when released into the extracellular matrix (ECM), change the cohesive forces between collagen fibrils. MCTs exist in nature in several forms, including some associated with echinoderm autotomy mechanisms. Since the molecular mechanism of mutability is still incompletely understood, the aim of this work was to provide a detailed biochemical analysis of a typical mutable collagenous structure and to identify possible correlations between its biochemistry and mechanical states. A better understanding of the mutability phenomena is likely to provide a unique opportunity to develop new concepts that can be applied in the design of dynamic biomaterial for tissue regeneration, leading to new strategies in regenerative medicine. The MCT model used was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was analyzed in different mechanical states, mimicking the mutability phenomenon. Spectroscopic techniques, namely Fourier transform infrared (FT-IR) and confocal Raman microscopy, were used to identify the specific molecular components that contribute to the CDL biochemical microenvironment and to investigate the possibility that remodelling/synthesis of new ECM components occurs during the mutability phenomenon by analogy with events during pregnancy in the uterine cervix of mammals (which also consists mainly of mechanically adaptable connective tissues). The results demonstrate that CDL ECM includes collagen with biochemical similarities to mammalian type I collagen, as well as sulphated glycosaminoglycans (GAGs). CDL mutability seems to involve a molecular rearrangement of the ECM, without synthesis of new ECM components. Although there were no significant biochemical differences between CDLs in the various mechanical states were observed. However, subtle adjustments in tissue hydration seemed to occur, particularly during stiffening.

Pasteurella multocida: diseases and pathogenesis.

Pasteurella multocida is an enigmatic pathogen. It is remarkable both for the number and range of specific disease syndromes with which it is associated, and the wide range of host species affected. The pathogenic mechanisms involved in causing the different syndromes are, for the most part, poorly understood or completely unknown. The biochemical and serological properties of some organisms responsible for quite different syndromes appear to be similar. Thus, the molecular basis for host predilection remains unknown. The recent development of genetic manipulation systems together with the availability of multiple genome sequences should help to explain the association of particular pathological conditions with particular hosts as well as helping to elucidate pathogenic mechanisms.

The mechanically adaptive connective tissue of echinoderms: its potential for bio-innovation in applied technology and ecology.

Echinoderms possess unique connective tissues, called mutable collagenous tissues (MCTs), which undergo nervously mediated, drastic and reversible or irreversible changes in their mechanical properties. Connective tissue mutability influences all aspects of echinoderm biology and is a key-factor in the ecological success of the phylum. Due to their sensitivity to endogenous or exogenous agents, MCTs may be targets for a number of common pollutants, with potentially drastic effects on vital functions. Besides its ecological relevance, MCT represents a topic with relevance to several applied fields. A promising research route looks at MCTs as a source of inspiration for the development of novel biomaterials. This contribution presents a review of MCT biology, which incorporates recent ultrastructural, biomolecular and biochemical analyses carried out in a biotechnological context.

Physiological and immunocytochemical evidence that glutamatergic neurotransmission is involved in the activation of arm autotomy in the featherstar Antedon mediterranea (Echinodermata: Crinoidea).

The crinoid echinoderm Antedon mediterranea autotomises its arms at specialised skeletal joints known as syzygies that occur at regular intervals along the length of each arm. Detachment is achieved through the nervously mediated destabilisation of ligament fibres at a particular syzygy. The aim of this investigation was to identify neurotransmitters that are involved in the autotomy response. Physiological experiments were conducted on isolated preparations of syzygial joints, which can be induced to undergo autotomy-like fracture by applying stimulatory agents such as elevated [K(+)](o). Initial experiments with elevated [K(+)](o) showed that the autotomy threshold (the minimum amount of stimulation required to provoke autotomy) is lowest in syzygies at the arm base and rises distally. Of a range of neurotransmitter agonists tested, only l-glutamate invoked syzygial destabilisation, as did its analogues l-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate, but not l-(+)-2-amino-4-phosphonobutyrate (l-AP4) or N-methyl-d-aspartate (NMDA). The implication that l-glutamate stimulates syzygial fracture through AMPA/kainate-like receptors was supported by the finding that the action of l-glutamate was inhibited by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Acetylcholine depressed the response of syzygial preparations to l-glutamate, suggesting a possible mechanism by which the autotomy threshold could be varied constitutively and facultatively. An immunocytochemical method employing a polyclonal antibody against l-glutamate conjugated to glutaraldehyde revealed l-glutamate-like immunoreactivity in all components of the putative neural pathway controlling the autotomy reflex, including the epidermis, brachial nerve, syzygial nerves and cellular elements close to the syzygial ligaments. We conclude that it is highly probable that l-glutamate acts as an excitatory neurotransmitter in the activation of arm autotomy in A. mediterranea.

Cardiac valvular and vascular disease in Bull Terriers.

The hearts of 27 Bull Terriers and 6 control dogs were evaluated. Heart murmurs were auscultated in 14 (52%) Bull Terriers. At necropsy, 25 Bull Terriers (93%) had myxomatous degeneration of the mitral valve or abnormalities of the left ventricular outflow tract. Small vessel arteriosclerosis in the myocardium and fibrosis of cardiac conduction tissue were common histologic findings in Bull Terriers with clinical cardiac disease. These lesions were also detected in dogs without clinical evidence of cardiac disease and only mild murmurs or structural valvular disease.

The pathology of bronchointerstitial pneumonia in young foals associated with the first outbreak of equine influenza in Australia.

REASONS FOR PERFORMING STUDY: The first outbreak of equine influenza virus (EIV) infection was confirmed in Australia in 2007. Some EIV-positive young foals died with bronchointerstitial pneumonia, an rare disease process in this age group that is often postulated to be caused by viral infection. OBJECTIVES: The aim of this study was to describe post mortem lesions in EIV-infected foals. METHODS: Post mortem examinations were conducted on 11 young foals (age 2-12 days) submitted to the Scone Veterinary Hospital, NSW over a 2-month period in 2007. The foals had presented with or developed fatal pneumonia, and were known or suspected to be EIV-positive. Equine influenza virus nucleic acid was detected in tissue specimens using an Influenza A group reactive real-time reverse transcriptase PCR assay. RESULTS: Grossly there was diffuse or extensive pulmonary consolidation. Histological changes included: bronchiolar and alveolar necrosis; neutrophilic infiltration; hyaline membrane formation; and hyperplasia and squamous metaplasia of airway epithelium. Tissues for 10 foals were EIV-positive, with a positive nasal swab from the remaining animal. CONCLUSIONS: This is the first detailed pathological description of bronchointerstitial pneumonia associated with EIV infection in young foals. It is also the first series of such cases in which a causative agent has consistently been detected. POTENTIAL RELEVANCE: Given the findings in this outbreak, and a previous outbreak in the UK in 1965 involving a similarly naive population, veterinary clinicians and pathologists should be aware that EIV can cause fatal bronchointerstitial pneumonia in young foals that do not have maternal immunity. The lesions did not differ from those previously reported in foals of various ages with bronchointerstitial pneumonia of other or undefined causes, indicating that this is most likely a stereotypical response to a variety of insults. Therefore, tissue specimens should be obtained from cases of pneumonia in young foals for virological and bacteriological testing.

Mechanical adaptability of a sponge extracellular matrix: Evidence for cellular control of mesohyl stiffness in Chondrosia reniformis Nardo.

The marine sponge Chondrosia reniformis Nardo consists largely of a collagenous tissue, the mesohyl, which confers a cartilaginous consistency on the whole animal. This investigation was prompted by the incidental observation that, despite a paucity of potentially contractile elements in the mesohyl, intact C. reniformis stiffen noticeably when touched. By measuring the deflection under gravity of beam-shaped tissue samples, it was demonstrated that the flexural stiffness of the mesohyl is altered by treatments that influence cellular activities, including [Ca2+] manipulation, inorganic and organic calcium channel-blockers and cell membrane disrupters, and that it is also sensitive to extracts of C. reniformis tissue that have been repeatedly frozen then thawed. Since the membrane disrupters and tissue extracts cause marked stiffening of mesohyl samples, it is hypothesised that cells in the mesohyl store a stiffening factor and that the physiologically controlled release of this factor is responsible for the touch-induced stiffening of intact animals.

Pathogenicity of Vietnamese enterotoxigenic Escherichia coli strains in colostrum-deprived one-day-old piglets.

Preweaning colibacillosis is a major cause of economic loss to the swine industry in Vietnam. The aim of this study was to examine the enteropathogenicity of representative enterotoxigenic Escherichia coli (ETEC) strains obtained during an earlier epidemiologic survey conducted in five provinces in North Vietnam. This included isolates belonging to serotype O8 that produced heat-stable and heat-labile enterotoxins but did not produce any of the recognized fimbriae (F4, F5, F6, F41, F18). In vitro hemagglutination (unique mannose-resistant hemagglutination activity with guinea pig, sheep, human, and chicken red blood cells at 37 degrees C, but not at 18 degrees C) and enterocyte brush border attachment assays suggested that the F- ETEC strains produced an unidentified colonization factor that promoted adherence to the intestinal epithelium. Colostrum-deprived 1-day-old piglets challenged with an F- strain (1-2 x 10(9) bacteria) developed acute watery diarrhea within 4 hours of inoculation and suffered up to 20% weight loss, with comparable severity to piglets challenged with conventional F4 and F5 strains. At necropsy, viable counts and histopathologic examination of intestinal sections demonstrated colonization of the duodenum, jejunum, and ileum by F4-positive strains. In comparison, the F- and F5-positive strains attached exclusively to the ileum. Transmission electron micrographs of negatively stained F- cells grown at 37 degrees C demonstrated the presence of fimbriae. These results confirm the presence of a potentially new pathogenic ETEC fimbrial type in piggeries in Vietnam, with a unique hemagglutination property and attachment characteristics similar to ETEC bearing F5 fimbriae.

Mutable collagenous tissue: overview and biotechnological perspective.

The mutable collagenous tissue (MCT) of echinoderms can undergo extreme changes in passive mechanical properties within a timescale of less than 1 s to a few minutes, involving a mechanism that is under direct neural control and coordinated with the activities of muscles. MCT occurs at a variety of anatomical locations in all echinoderm classes, is involved in every investigated echinoderm autotomy mechanism, and provides a mechanism for the energy-sparing maintenance of posture. It is therefore crucially important for the biology of extant echinoderms. This chapter summarises current knowledge of the physiology and organisation of MCT, with particular attention being given to its molecular organisation and the molecular mechanism of mutability. The biotechnological potential of MCT is discussed. It is argued that MCT could be a source of, or inspiration for, (1) new pharmacological agents and strategies designed to manipulate therapeutically connective tissue mechanical properties and (2) new composite materials with biomedical applications.

Is muscle involved in the mechanical adaptability of echinoderm mutable collagenous tissue?

The mutable collagenous tissue (MCT) of echinoderms has the capacity to change its mechanical properties in a time scale of less than 1 s to a few minutes under the influence of the nervous system. Although accumulating evidence indicates that the mechanical adaptability of MCT is due primarily to the modulation of interactions between components of the extracellular matrix, the presence of muscle in a few mutable collagenous structures has led some workers to suggest that contractile cells may play an important role in the phenomenon of variable tensility and to call for a re-evaluation of the whole MCT concept. This contribution summarises present information on MCT and appraises the argument implicating muscle in its unique mechanical behaviour. It is concluded that there is no evidence that the variability of the passive mechanical properties of any mutable collagenous structure is due to muscle.

Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A.

We have constructed a defined acapsular mutant in Pasteurella multocida X-73 (serogroup A:1) by disrupting the hexA gene through the insertion of a tetracycline resistance cassette. The genotype of the hexA::tet(M) strain was confirmed by PCR and Southern hybridization, and the acapsular phenotype of this strain was confirmed by electron microscopy. The hexA::tet(M) strain was attenuated in both mice and chickens. Complementation of the mutant with an intact hexAB fragment restored lethality in mice but not in chickens. In contrast to the results described previously for P. multocida serogroup B (J. D. Boyce and B. Adler, Infect. Immun. 68:3463-3468, 2000), the hexA::tet(M) strain was sensitive to the bactericidal action of chicken serum, whereas the wild-type and complemented strains were both resistant. Following inoculation into chicken muscle, the bacterial count of the hexA::tet(M) strain decreased significantly, while the wild-type and complemented strains both grew rapidly over 4 h. The capsule is thus an essential virulence determinant in the pathogenesis of fowl cholera.

Autotomy as a prelude to regeneration in echinoderms.

'Autotomy' refers to the adaptive detachment of animal body parts where this serves a defensive function, is achieved by an intrinsic mechanism, and is nervously mediated. With regard to each echinoderm class, this article itemises those structures that are autotomous, evaluates the extent to which autotomy precedes regeneration in natural populations, reviews current knowledge of the morphology of autotomy planes and mechanisms that effect fracture at autotomy, and comments on autotomy-related issues arising from studies of the cellular events of regeneration. Each autotomy plane can be regarded as an assemblage of breakage zones traversing the individual anatomical components of the autotomous structure. In any one autotomy plane some breakage zones are permanent sites of weakness that are fractured by external forces and some are potential sites of weakness that undergo a loss of tensile strength only at the time of autotomy. The latter occur predominantly in mutable collagenous structures, although there are a few examples of muscles that undergo an endogenous rupturing process. Available evidence indicates that autotomy is by far the commonest proximate cause of structural loss in echinoderms. Most echinoderm regeneration is therefore necessitated by autotomy and proceeds from the retained side of a fractured autotomy plane. Due to a lack of relevant research there is as yet little evidence for or against the presence of specific regeneration-promoting adaptations at autotomy planes, although it is argued that an autotomy plane designed primarily to effect rapid detachment would by itself increase regenerative efficiency.

The demonstration of Pasteurella multocida in the alimentary tract of chickens after experimental oral infection.

A selective medium containing polymyxin B, crystal violet, thallous acetate, bacitracin and cycloheximide in 10% sheep blood dextrose starch agar, and a modified Pasteurella multocida-specific polymerase chain reaction (PCR) assay were developed for the respective isolation and detection of P. multocida from chicken alimentary tract. The selective medium and the PCR assay were highly sensitive, detecting 100 cfu from colon contents. These techniques were used to follow the localisation of an orally administered virulent P. multocida in chickens. Pasteurellae could be isolated from the crop of some birds up to 30 h, occasionally from other sites after 28 h. It was concluded that the crop was a likely site for colonisation and that infection was most likely to occur through the mucosa of the jejunum or ileum.

The virulence and protective efficacy for chickens of pasteurella multocida administered by different routes.

The relative virulence for chickens of five strains of Pasteurella multocida was evaluated. Twenty groups, each of ten chickens, were inoculated with a standard dose of 10(5) of each of five strains by the intramuscular (I.m.), intravenous (I.v.), intratracheal (I.tr.) or conjunctival (Co) routes. The highest mortality occurred in the groups dosed I.m. and I.v., followed by I.tr. inoculation. The relative virulence of each strain did not change when inoculated by the different routes. The most virulent strain, VP161, caused 100% mortality by all except the Co route. The least virulent strain, VP17, caused a single mortality by the I.v. route, but gave a high level of protection to birds inoculated by both the I.m. and I.v. routes, when challenged by intramuscular injection with (VP161). There was no protection against I.m. challenge in the birds inoculated by the I.tr. or Co routes. Serum antibody levels measured by ELISA correlated with the level of protection against virulent challenge for groups inoculated I.m. or I.v., but not I.tr. Western blots of pooled sera from each group did not show any specific antigen recognition that might explain the observed differences in protection. Inoculation with strain VP17, (both I.m. and I.tr.) also gave a high level of protection to birds challenged with strain VP161 by intratracheal instillation.

PCR detection and analysis of Pasteurella multocida from the tonsils of slaughtered pigs in Vietnam.

A total of 36 tonsil swab samples were collected from healthy swine prior to slaughter at the abattoirs in Can tho and Tien giang provinces of Southern Vietnam. The presence of Pasteurella multocida in these samples was detected by the combination of direct cultivation and isolation, mouse inoculation and the polymerase chain reaction (PM-PCR). P. multocida was detected in 16 samples by PCR, with 17 strains ultimately isolated. All samples were negative for serogroup B by HSB-PCR and conventional serotyping, with isolates identified as A:3, D:1 or D:3. In addition, all samples were determined to be negative for the P. multocida toxin (PMT). Characterisation of isolated P. multocida by REP-PCR and biotyping revealed nine distinct REP profiles and seven biotypes among the 17 isolates. Some correlation was seen with P. multocida isolated from a previous Australian outbreak of acute swine pasteurellosis, and those isolated from fowl cholera outbreaks in Vietnamese poultry.

Overexpression and immunogenicity of the Oma87 outer membrane protein of Pasteurella multocida.

The outer membrane protein of Oma87 from Pasteurella multocida A:1 has significant similarity to the D15 protective antigen of Haemophilus influenzae (Ruffolo and Adler, 1996). Four fragments of Oma87 from a P. multocida serotype D strain were cloned into a pGEX expression vector and transformed into E. coli JM105. Western blot analysis revealed that convalescent chicken sera reacted with only GST-F1 fusion protein which contained amino acids 18 through to 130 of Oma87 fused to the GST protein. Vaccination with the GST-F1 protein failed to protect chickens against challenge with a virulent P. multocida serotype A.

Acute septicaemic pasteurellosis in Vietnamese pigs.

Sixteen isolates of Pasteurella multocida were cultured from cases diagnosed as acute septicaemic pasteurellosis in Vietnamese pigs. The HSB-PCR assay provided rapid presumptive determination of 10 isolates of P. multocida identified as haemorrhagic septicaemia (HS) causing type B cultures (B:2, B:5, B:2,5). Serological designation using the Carter and Heddleston typing systems confirmed these findings, and identified the six HSB-PCR negative cultures as either A:1, A:3 or D:3,4. Biochemical fermentation and REP-PCR revealed phenotypic and genotypic identity between P. multocida type A:1 isolated from Vietnamese pigs and poultry. Marked homogeneity was also demonstrated among HSB-PCR positive swine isolates, which were shown to possess genotypic identity with P. multocida type B:2 from buffaloes diagnosed with HS.