Alzheimer type II astrocytes in the brains of pigs with salt poisoning (water deprivation/intoxication).

The finding of Alzheimer type II astrocytes, in addition to the pathognomonic combination of laminar cerebrocortical necrosis and eosinophil infiltration, in the brains of pigs is reported for the first time in cases of indirect salt poisoning following water deprivation.

Henipavirus: a review of laboratory animal pathology.

The genus Henipavirus contains two members-Hendra virus (HeV) and Nipah virus (NiV)-and each can cause fatal disease in humans and animals. HeV and Niv are currently classified as biosafety level 4, and NiV is classified as a category C priority pathogen. The aim of this article is to discuss the pathology of laboratory animal models of henipavirus infection and to assess their suitability as animal models for the development and testing of human therapeutics and vaccines. There has been considerable progress in the development of animal models for henipavirus disease. Suitable animal models include the golden hamster, ferrets, cats, and pigs, which develop disease resembling that observed in humans. Guinea pigs are a less reliable model for henipavirus disease, but they do develop henipavirus-induced encephalitis. Because human efficacy studies with henipaviruses are not permitted, animal studies are critical for the development of antiviral therapeutics and vaccines. Current research indicates that passive immunotherapy using monoclonal antibodies is protective of ferrets against NiV infection and that passive immunotherapy using NiV antibodies protects hamsters from HeV. Recombinant vaccines have been used to protect cats and pigs against NiV infection. Ribavirin and 6-aza-uridine were able to delay but not prevent NiV-induced mortality in hamsters. Further research is needed to develop a model and therapy for late-onset henipavirus encephalitis.

Topsin-M: the new benomyl for mycorrhizal-suppression experiments.

The fungicide benomyl was the most commonly used biocide for both field and greenhouse experiments in which arbuscular mycorrhizal fungal (AMF) suppression is desired. Unfortunately benomyl is no longer manufactured and therefore is not available for experimental use and no fungicide has been proposed as a successful alternative for experimentally suppressing mycorrhizal fungi. In this study we examined the potential for the fungicide Topsin M (topsin) to suppress mycorrhizal symbiosis in both field and greenhouse experiments. Topsin reduced AMF colonization of the obligately mycotrophic, warm-season grass Andropogon gerardii with a large and significant reduction in plant biomass production. Topsin reduced AMF colonization of the facultatively mycotrophic, cool-season grass Pascopyron smithii but did not significantly reduce biomass production. Fertilization with nitrogen and phosphorus was able to compensate for reductions in biomass due to the application of fungicide because biomass production of plants that received topsin fungicide was not significantly different from fertilized controls not receiving topsin. While we are not advocating that topsin fungicide is a universal mechanism for mycorrhizal-suppressed controls, in systems where benomyl was found to be successful topsin appears to be a useful, available and successful alternative.

Oral and sub-cutaneous vaccination of commercial pigs with a recombinant porcine adenovirus expressing the classical swine fever virus gp55 gene.

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55/E2 gene was administered to commercially available pigs via oral or subcutaneous routes and their susceptibility to oral and subcutaneous challenge with CSFV was determined. 100% of animals vaccinated and challenged subcutaneously were protected. In the groups of pigs vaccinated either orally or subcutaneously and then challenged orally, 60% of animals were protected. Before challenge, neutralising antibodies to CSFV were detected in 60% of pigs vaccinated subcutaneously, but in none of those given the vaccine orally. CSFV antigen was found in the spleens of surviving pigs that had been vaccinated orally. In contrast, subcutaneous vaccination was shown to preclude the presence of CSFV in the spleen of animals that survived challenge.

A guinea-pig model of Hendra virus encephalitis.

Subcutaneous inoculation, but not intradermal (footpad) or intranasal inoculation, with high doses of Hendra virus (HeV) consistently produced disease in guinea-pigs. Of 15 subcutaneously inoculated animals, 14 developed vascular disease with positive HeV immunohistochemical labelling in a range of tissues. A new observation was the presence of lesions, including syncytial cells, with immunolabelling in the transitional epithelium of the bladder. Virus isolation from the urine rather than from nasal, oral, rectal or conjunctival swabs, the other external sites, was consistent with previous epidemiological work in horses, indicating a limited possibility of transmission. The dose used (30 000 to 50 000 TCID(50)), which was higher than in previous studies, produced microscopical lesions of encephalitis in eight of the 15 subcutaneously inoculated guinea-pigs, with positive immunolabelling in blood vessels and neurons, especially in the medulla, cerebellum and thalamus. The virus was recovered from six of the encephalitic brains. Severe vascular degeneration in the centres of encephalitic lesions in six of the eight encephalitic guinea-pigs and positive immunolabelling in the choroid plexus of a further animal indicated that the virus entered the brain following virus-induced vascular injury and choroid plexus invasion. Guinea-pigs would appear to be suitable for the study of HeV encephalitis.

Vaccination of pigs with a recombinant porcine adenovirus expressing the gD gene from pseudorabies virus.

Five week old, commercially available large white pigs were vaccinated with either a single dose or two doses of a recombinant porcine adenovirus expressing the glycoprotein D gene from pseudorabies virus (PRV). Pigs were monitored for the development of serum neutralizing antibodies to PRV and challenged 3 weeks after final vaccination. Prior to challenge, pigs given 2 doses of the vaccine demonstrated boosted levels of antibody compared with those given a single dose, and all surviving pigs had increased neutralization titres over pre-challenge levels. Following challenge, pigs were monitored for clinical signs of disease, with blood and nasal swabs collected for virus isolation. All control animals became sick with elevated temperatures for 6 days post challenge, whereas; vaccinated animals displayed an increase in body temperature for only 2-3 days. Control pigs and those given a single dose all lost condition, but the group given 2 doses remained healthy. At postmortem, gross lesions of pneumonia only occurred in control animals and those given a single dose of vaccine. Histology carried out on the brains of all animals demonstrated a difference in severity of infection and frequency of immunohistochemical antigen detection between test animals, with control and single dose groups being most severely affected and pigs given 2 doses the least. Virus isolation studies demonstrated that no viraemia could be detected, but virus was found in nasal swabs from some animals in both groups of vaccinates following challenge.

A prime-boost vaccination strategy using naked DNA followed by recombinant porcine adenovirus protects pigs from classical swine fever.

Weaned pigs (6-week-old) and 7-day-old pre-weaned piglets were vaccinated with naked plasmid DNA expressing the gp55/E2 gene from classical swine fever virus (CSFV). Both groups of pigs were then given a booster dose of recombinant porcine adenovirus expressing the gp55 gene (rPAV-gp55). Following challenge with CSFV, 100% of weaned pigs and 75% pre-weaned piglets were protected from disease. Weaned pigs given a single dose of rPAV-gp55 were also protected, but showed a slight increase in temperature immediately post-challenge. However, weaned animals given a DNA prime before rPAV-gp55 showed no fluctuation in body temperature following challenge and no pathology in spleen or lymph nodes upon post-mortem. In addition, no CSFV could be re-isolated from the rPAV vaccinated group and from only one pig in the prime-boost group following challenge, suggesting that both vaccination regimes have the potential to reduce or prevent virus shedding following experimental challenge.

Experimental hendra virus infectionin pregnant guinea-pigs and fruit Bats (Pteropus poliocephalus).

Antibodies to Hendra virus (HeV) have been found in a high percentage of fruit bats (Pteropus spp.) in Australia, indicating a possible reservoir for the virus. The aim of the experiments reported here was to investigate transplacental infection as a possible mode of transmission of the virus in fruit bats and other animals. In a first experiment, 18 pregnant guinea-pigs in the mid-stage of gestation were inoculated with HeV, as an experimental model in a conventional laboratory animal. Nine developed HeV disease as confirmed by viral isolation, histopathology and immunohistochemistry. In five of the nine clinically affected guinea-pigs there was necrosis and strong positive immunostaining in the placentas in an indirect immunoperoxidase (IPX) test for HeV antigen. One of these five guinea-pigs aborted and HeV was isolated from its three fetuses, one of which was also positive to the IPX test. In three other sick guinea-pig dams, virus was isolated from fetuses, and there was positive immunostaining in two of the latter. In a second experiment, four fruit bats were inoculated with a similar dose of HeV. (A further four guinea-pigs inoculated at the same time developed severe disease, indicating adequate virulence.) Two bats were killed at 10 days post-inoculation and two were killed at 21 days. In these bats, no overt clinical disease was observed, but subclinical disease occurred, as indicated by viral isolation, seroconversion, vascular lesions and positive immunostaining. Transplacental transmission was indicated by positive immunostaining in two placentas and confirmed by isolation of virus from one of the associated fetuses.

Hendra and Nipah virus infections.

The most important clinical and pathological manifestation of Hendra virus infection in horses and humans is that of severe interstitial pneumonia caused by viral infection of small blood vessels. The virus is also capable of causing nervous disease. Hendra virus is not contagious in horses and is spread by close contact with body fluids, such as froth from infected lungs. Diagnosis should be based on the laboratory examination of blood, lung, kidney, spleen, and, if nervous signs are present, also of the brain. Evidence of infection with the more recently identified and related Nipah virus was found in the brain of one horse in which there was inflammation of the meningeal blood vessels. Fruit bats, especially Pteropus s., have been incriminated as the natural and reservoir hosts of both Hendra and Nipah viruses.

Immunohistochemistry in the identification of a number of new diseases in Australia.

Immunohistochemistry plays an important part in the diagnosis of some viral diseases. Demonstration of viral antigen in a lesion is an important contribution to diagnosis, either at the time of investigation or retrospectively. At the CSIRO Australian Animal Health Laboratory, the most frequent use of immunohistochemistry has been in the diagnosis of the important avian diseases, highly pathogenic avian influenza and Newcastle disease. The technology took key roles in the diagnoses of Hendra virus infections, and, later, an immunoperoxidase test gave the first indication of the existence of Australian bat lyssavirus. The test can often confirm that a virus isolated in an animal is the actual virus causing disease and not a coincidental isolation. Good examples of that in some more new diseases were the association of Wallal virus with blindness in kangaroos, and of the new porcine Menangle virus in natural and experimental cerebral disease in foetal piglets.

Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats.

OBJECTIVE: To determine the infectivity and transmissibility of Hendra virus (HeV). DESIGN: A disease transmission study using fruit bats, horses and cats. PROCEDURE: Eight grey-headed fruit bats (Pteropus poliocephalus) were inoculated and housed in contact with three uninfected bats and two uninfected horses. In a second experiment, four horses were inoculated by subcutaneous injection and intranasal inoculation and housed in contact with three uninfected horses and six uninfected cats. In a third experiment, 12 cats were inoculated and housed in contact with three uninfected horses. Two surviving horses were inoculated at the conclusion of the third experiment: the first orally and the second by nasal swabbing. All animals were necropsied and examined by gross and microscopic pathological methods, immunoperoxidase to detect viral antigen in formalin-fixed tissues, virus isolation was attempted on tissues and SNT and ELISA methods were used to detect HeV-specific antibody. RESULTS: Clinical disease was not observed in the fruit bats, although six of eight inoculated bats developed antibody against HeV, and two of six developed vascular lesions which contained viral antigen. The in-contact bats and horses did not seroconvert. Three of four horses that were inoculated developed acute disease, but in-contact horses and cats were not infected. In the third experiment, one of three in-contact horses contracted disease. At the time of necropsy, high titres of HeV were detected in the kidneys of six acutely infected horses, in the urine of four horses and the mouth of two, but not in the nasal cavities or tracheas. CONCLUSIONS: Grey-headed fruit bats seroconvert and develop subclinical disease when inoculated with HeV. Horses can be infected by oronasal routes and can excrete HeV in urine and saliva. It is possible to transmit HeV from cats to horses. Transmission from P poliocephalus to horses could not be proven and neither could transmission from horses to horses or horses to cats. Under the experimental conditions of the study the virus is not highly contagious.

Crystallization of the MS2 translational repressor alone and complexed to bromouridine.

The coat protein from the MS2 bacteriophage plays a dual role by encapsidating viral RNA and also by binding RNA as a translational repressor. In order to study the isolated dimer in a conformation not influenced by capsid interactions, a mutant molecule was crystallized that is defective in capsid assembly but is an active repressor. The unassembled dimer crystallized in the space group P21212 with a = 76.2, b = 55.7, and c = 28.4 A. In these crystals, monomers were related by twofold symmetry. When this dimer was co-crystallized with 5-bromouridine, crystals formed in space group R3 with a = b = 155.9 A, c = 29.9 A, gamma = 120 degrees; the dimer was the asymmetric unit.

Crystallization of the OP-G2 Fab fragment: a fibrinogen mimic with specificity for the platelet glycoprotein IIb/IIIa.

The OP-G2 monoclonal antibody binds to the platelet integrin, gpIIb/IIIa, in a mode that mimics fibrinogen binding. The specificity of this antibody is mediated by the third complementarity-determining region (CDR3) loop of the immunoglobulin heavy chain which contains a sequence (RYD) related to the RGD recognition sequence of fibrinogen. The OP-G2 Fab fragment has been crystallized by vapor diffusion from solutions containing polyethylene glycol and imidazole malate (pH 5.6). The crystals belong to space group P2(1)2(1)2 with a = 93.1, b = 83.8 and c = 53.7 A. One Fab molecule is present in the asymmetric unit. A complete data set to 2.0 A resolution has been collected.

Rheumatoid arthritis patients coping without drug therapy.

In view of the variable and unpredictable efficacy and toxicity of non-steroidal anti-inflammatory and disease-modifying anti-rheumatic drugs, it is not surprising that some patients with rheumatoid arthritis elect to cope without recourse to these medications. We describe four of these patients, consider the lessons to be learnt, and identify further research that needs to be done on this aspect of illness behaviour.

Affinity chromatography using protein immobilized via arginine residues: purification of ubiquitin carboxyl-terminal hydrolases.

4-(Oxoacetyl)phenoxyacetic acid (OAPA) forms a stable, covalent bond between its glyoxal group and the guanidino group of arginine and arginine derivatives [Duerksen, P. J., & Wilkinson, K. D. (1987) Anal. Biochem. 160, 444-454]. Studies were carried out to determine the chemical nature of this linkage, and the structure of the stable adduct between OAPA and methylguanidine was elucidated. The stable product results from an internal oxidation-reduction of the Schiff base adduct to form a cyclic alpha-aminoamide, 4-[4-(carboxymethoxy)phenyl]-2-(methylimino)-5-oxoimidazolidine. OAPA coupled to polyacrylamide beads was used to immobilize ubiquitin via its arginine residues, and the resulting affinity support was shown to specifically and reversibly bind a previously described enzyme, ubiquitin carboxyl-terminal hydrolase [Pickart, C. M., & Rose, I. A. (1985) J. Biol. Chem. 260, 7903-7910]. The resin was then used to isolate three newly identified ubiquitin carboxyl-terminal hydrolytic activities, which did not bind to ubiquitin immobilized via lysine residues. Significant purification was achieved in each case, and one isozyme was further purified to homogeneity.

Effects of antibiotic prophylaxis on women undergoing nonelective cesarean section in a community hospital.

A randomized, double-blind, placebo-controlled study was done of a short course of cefamandole administered intravenously after cord clamping as prophylaxis in women undergoing primary nonelective cesarean section in a community hospital. Duration of labor equal to or more than 14 hours was the only significant risk factor between patients who had postoperative infectious morbidity and those who had none. Four of 43 patients (9.3%) who received cefamandole, as opposed to 13 of 47 (27.7%) who received the placebo, developed infections (p less than 0.05). This difference was reflected totally in the difference in endomyometritis development between the groups. The use of intraoperative culturing predicted infection in 4 of 13 patients in the placebo group who developed infections postoperatively. No adverse side effects were noted, and there were no cases of delayed serious infection.

Severe chronic mucocutaneous candidiasis. Favourable response to oral therapy with ketoconazole.

Severe, early-onset, chronic mucocutaneous candidiasis was associated with bronchiectasis, oesophageal stricture, short stature and delayed puberty in a male aged 18 years. Topical treatment with antifungal agents, and several courses of intravenously administered transfer factor, amphotericin, and miconazole achieved only minor or transient improvement in the patient's condition. Correction of iron deficiency anaemia did not lead to alleviation of candidiasis. Skin reactivity to Candida antigen was absent and T-lymphocytes, which responded normally to phytohaemagglutinin (PHA), poke-weed mitogen and concanavalin A, had negative macrophage-inhibiting factor (MIF) and blastogenic responses to Candida antigen. Treatment with the orally effective imidazole derivative, ketoconazole, produced improvement within three days and clearing of mucosal lesions within five weeks. The patient then entered puberty spontaneously at the age of 20 years. After 18 months of treatment with ketoconazole, without side effects, the clinical manifestations of mucocutaneous candidiasis have not recurred. Because of the possibility of continuous, long-term administration, ketoconazole represents the best currently available agent for the treatment of chronic mucocutaneous candidiasis.