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.

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.

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.

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.

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.

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.

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.

The effect of Pasteurella haemolytica and the leukotoxin of Pasteurella haemolytica on bovine lung explants.

Bovine lung explants were used in a study designed to compare the pathogenic effects of Pasteurella haemolytica type 1, a nonpathogenic organism Neisseria subflava, or the crude leukotoxin of P. haemolytica on alveolar macrophages and lung parenchymal cells. Concentrated, purified peripheral blood neutrophil suspensions were added with the bacteria to some explants. Duplicate pairs of cultures from each treatment group were fixed at regular intervals up to 24 hours after seeding and morphological changes were assessed by light and electron microscopy. Pasteurella haemolytica caused deterioration of alveolar macrophages within one hour but did not affect parenchymal cells for more than 12 hours. Neisseria subflava did not affect alveolar macrophages initially, but caused an accelerated deterioration after four hours. After 24 hours, bacterial overgrowth caused similar deterioration of all cells in explants seeded with either bacterium. Alveolar macrophages phagocytosed large numbers of N. subflava but rarely ingested P. haemolytica. Added neutrophils did not have any discernible effect on any of the explants and did not potentiate bacterial effects. Addition of crude leukotoxin of P. haemolytica to the culture medium significantly accelerated alveolar macrophage deterioration without apparent effect on parenchymal cell survival. These results support the hypothesis that the severe tissue destruction of fulminant pneumonic pasteurellosis is not a direct result of bacterial infection.

Leukomyelitis in the goat: a report of three cases.

Three cases of focal myelitis in the spinal cords of young goats are described. The clinical findings and pathological changes were similar to those reported for viral leukoencephalomyelitis of goats. There were granular structures in a few cells in malacic areas, which on electron micrographs appear to be clumps of chromatin in the nuclei of gemistocytic astrocytes. They may represent mitotic figures.

Polyserositis and meningitis associated with Escherichia coli infection in piglets.

Two piglets which had a history of anorexia and weakness were examined pre and postmortem. Other piglets in the same herd had died within 24 hours of the onset of similar signs. The two piglets examined had a fibrinous polyserositis. Grossly, the pleura, peritoneum and joints were affected and an acute meningitis was noted on microscopic examination of the brains. Pure cultures of Escherichia coli were recovered from all but one of the organs and exudates cultured.

A review of insecticide poisonings among domestic livestock in southern Ontario, Canada, 1982-1989.

From 1982 to 1989, inclusive, 20 poisonings were investigated by the Ontario Ministry of Agriculture and Food following ingestion by domestic livestock of granular insecticides including terbufos (13 poisonings), disulfoton (two poisonings), fonofos (two poisonings), phorate (two poisonings), and carbofuran (one poisoning); all are used for rootworm (Diabrotica spp.) control in corn. A further three poisonings of livestock occurred following the ingestion of the foliar insecticide, endosulfan (two poisonings), and the seed protectant insecticides diazinon plus lindane (one poisoning). There were six poisoning cases as a result of excessive topical applications of the three insecticides coumpahos, fenthion, and lindane as dusts or sprays to control external parasites. Together, these events caused the deaths of 258 domestic animals of which 200 were cattle, 23 were swine, and 35 were sheep. Not all deaths are reported to the Ministry and the cases reported here may only represent 30-50% of the actual deaths over the period. Based on total populations of livestock, the percent losses were very small but they represent serious losses to individual growers. The economic loss is estimated at $160,000 over the eight years, or $20,000 per annum, and this does not include veterinary costs.Some of the poisoned animals died within as little as three to four hours of ingestion while others were sick but survived for several days. Lethal doses of insecticide were found in the rumen, abomasum, or stomach of dead animals. Signs typical of cholinesterase inhibition caused by organophosphorus poisoning were observed in most cases. Cholinesterase readings were found to be zero in dying animals. Necropsy findings were rarely more than pulmonary edema or myocardial hemorrhage. Where organochlorine insecticides were ingested, convulsions were the major manifestation.Contamination of feed was most often accidental, and chemical analysis was most helpful in identifying both potent and minor sources, thus facilitating cleanup procedures.

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.

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.