Ligand-induced conformational change in penicillin acylase.

The enzyme penicillin acylase (penicillin amidohydrolase EC 3.5.1. 11) catalyses the cleavage of the amide bond in the benzylpenicillin (penicillin G) side-chain to produce phenylacetic acid and 6-aminopenicillanic acid (6-APA). The enzyme is of great pharmaceutical importance, as the product 6-APA is the starting point for the synthesis of many semi-synthetic penicillin antibiotics. Studies have shown that the enzyme is specific for hydrolysis of phenylacetamide derivatives, but is more tolerant of features in the rest of the substrate. It is this property that has led to many other applications for the enzyme, and greater knowledge of the enzyme's structure and specificity could facilitate engineering of the enzyme, enhancing its potential for chemical and industrial applications. An extensive study of the binding of a series of phenylacetic acid derivatives has been carried out. A measure of the relative degree of inhibition of the enzyme by each of the compounds has been obtained using a competitive inhibition assay, and the structures of a number of these complexes have been determined by X-ray crystallography. The structures reveal a clear rationale for the observed kinetic results, but show also that some of the ligands cause a conformational change within the binding pocket. This change can generally be understood in terms of the size and orientation of the ligand within the active site.The results reveal that ligand binding in penicillin acylase is facilitated by certain amino acid residues that can adopt two distinct, energetically favourable positions in order to accommodate a variety of compounds within the active site. The structures of these complexes provide evidence for conformational changes in the substrate-binding region that may act as a switch in the mechanism of autocatalytic processing of this enzyme.

Clinical and pathological responses of weaned pigs to atmospheric ammonia and dust.

Nine hundred and sixty weaned pigs were exposed for five weeks to controlled concentrations of atmospheric ammonia and dust in a single, multifactorial experiment. The treatments were a mean dust concentration of either 1.2, 2.7, 5.1 or 9.9 mg/m3 (inhalable fraction) and a mean ammonia concentration of either 0.6, 10.0, 18.8 or 37.0 ppm, concentrations representative of commercial conditions. The experiment was carried out over two years and the pigs were used in eight batches, each consisting of five lots of 24 pigs. Each treatment combination was replicated once, and an additional control lot (nominally 0 mg/m3 dust and 0 ppm ammonia) was included in each batch. The dust concentration was the same in the other four lots in each batch in which the four concentrations of ammonia were used; thus, the split-plot design was more sensitive to the effects of ammonia than dust. The groups of pigs were kept separately in five rooms in a purpose-built facility, and the pollutants were injected continuously into the air supply. Ammonia was supplied from a pressurised cylinder, and the endogenous dust in each room was supplemented by an artificial dust manufactured from feed, barley straw and faeces, mixed by weight in the proportions 5:1:4; its ingredients were oven-dried, milled and mixed, and then resuspended in the air supply. The health of the pigs was assessed in terms of general pathology, respiratory tract pathology, and the microbiology of the nasal cavity, trachea and lung. In each batch, postmortem examinations were carried out on 40 pigs after five weeks' exposure to the pollutants and on 30 pigs two weeks later to test for carryover and recovery--a total of 560 pigs. These examinations revealed minimal gross pathology and widespread minor pathological changes of little significance. The pigs' turbinate and lung scores were low and unaffected by exposure to pollutants. All the putative bacterial pathogens, with the exception of toxigenic Pasteurella multocida type D, were isolated from the respiratory tract of the pigs of both ages, but there were no differences between the effects of the different concentrations of pollutants.

Isolation of a Lelystad virus-like agent from British pigs and scanning electron microscopy of infected macrophages.

Six, one-week-old gnotobiotic piglets were inoculated with tissues or sera collected from field cases of porcine reproductive and respiratory syndrome. The piglets showed little or no illness, and two that were necropsied at 8 and 9 days post infection appeared grossly normal. However, a Lelystad virus-like agent was isolated from most of the inoculated pigs using porcine alveolar macrophage cultures. Seroconversion to the Lelystad virus was observed and some animals developed microscopically detectable interstitial pneumonias. Scanning electron microscopy was used to study the in vitro cytopathic effect of the Lelystad virus on porcine alveolar macrophages.

Congenital infection of pigs with ruminant-type pestiviruses.

Congenital infections of pigs were induced with two ruminant-type pestiviruses isolated from pigs. One of the viruses was bovine viral diarrhoea virus-like and the other border disease virus-like. Both produced symptoms similar to those observed with low virulence strains of classical swine fever virus. A striking effect of persistent virus infection in post-natal life was stunting in viraemic animals. It was also shown that a congenitally infected pig shed virus for 2.5 years and in sufficient quantity to infect other pigs, even by indirect contact. Unlike ruminants, congenitally infected pigs sometimes had persistent viraemia but eventually eliminated the virus. Clearance of virus from the blood was related to the appearance of neutralizing antibodies. However, clearance from the tissues sometimes took as much as 5 months longer than from the blood.

Scrapie associated fibril detection on decomposed and fixed ovine brain material.

Samples of cerebral cortex from eight scrapie affected sheep and two unaffected control sheep were stored for up to nine days at temperatures ranging from 18 degrees C to 29 degrees C. Scrapie associated fibrils (SAF) could be detected in proteinase K treated brain extracts from all the eight scrapie affected animals after five days storage and in six out of the eight after nine days storage. SAF could not be detected in any brain extracts from the two control animals. Formol saline fixed brain material from a further six scrapie affected and two clinically normal sheep, were also subjected to an extraction technique used to detect fibrils. No characteristic SAF were observed in any of these fixed samples. Long filamentous structures were observed in four of the fixed scrapie affected brain extracts and in one of the fixed unaffected control brain extracts.

Solanum malacoxylon poisoning in pigs.

Solanum malacoxylon was given orally to four pigs. The animals were examined clinically and subjected to post mortem examination. Macroscopic lesions were not seen with the exception of a small calcified plaque in the endocardium of one animal. Microscopic examinations revealed slight calcification of elastic fibres in the soft tissues. The pathological changes in the bones were extensive and are described in detail. The pigs showed minimal lesions at dose levels which cause considerable systemic calcification in cattle and sheep.

Failure to induce neoplasia in nude mice by injection of disrupted cells from continuous cell lines.

Nude mice were injected subcutaneously with disrupted cells from three continuous cell lines (HeLa, NCTC clone 2472 and Morris hepatoma). No tumours were observed at either the inoculation site or in other organs taken at 35 or 90 days after injection. Live cells from one line (HeLa) produced tumours at the inoculation site after eight to 12 days.

Environmental factors affecting the severity of pneumonia in pigs.

The variable manifestations of respiratory problems in finishing pigs have led to the concept of a multiple-factor aetiology for swine respiratory disease and in particular for enzootic pneumonia. The primary and secondary agents of the disease produce their most detrimental economic effects and the highest levels of mortality and morbidity during the finishing period, when the economics of production necessitate indoor housing and intensification. This paper considers the contribution of four main groups of environmental factors to the high levels of clinical disease and lesions which are found whenever large numbers of pigs are examined at slaughter. They are meteorological factors, population and social factors, management factors and airborne pollution.

Citrinin as a possible cause of the pruritus, pyrexia, haemorrhagic syndrome in cattle.

An outbreak of the pruritus, pyrexia, haemorrhagic syndrome affected eight of a herd of 175 cows which was divided into two groups of 115 and 60 according to yield. There was no difference in management between them but citrus pulp pellets were fed only to the larger group in which the eight cows were affected. Silage, which had been made without the use of additives, was also fed to both groups. The citrus pulp was visibly mouldy and contained 30 to 40 parts per billion of citrinin. Signs of the syndrome occurred within three days of the cows starting to ingest the citrus pulp, which was fed for 21 days, and the last case occurred six days after the feeding of citrus pulp ceased. Five calves whose dams had been fed citrus pulp were subsequently born with superior prognathism. In contrast to the eight cows that developed the syndrome only one out of 68 heifers which were fed larger quantities of citrus pulp for 10 days developed mild signs of the syndrome and then recovered, suggesting that older animals may be more susceptible. The clinical signs, gross pathology and histopathology are described and compared with those of previous outbreaks. Mycotoxins, particularly citrinin, were strongly implicated as the cause of this outbreak.

Porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression.

Porcine reproductive and respiratory syndrome (PRRS) was first known as blue-eared pig disease in the United Kingdom and the causative agent as 'Lelystad virus'. The disease is characterised by very variable clinical signs, including reproductive failure and respiratory disease. The respiratory syndrome is often associated with severe infection with secondary bacterial agents including Pasteurella multocida, Haemophilus parasuis and Streptococcus suis. However, some seropositive herds show no clinical signs of disease. The secondary infections may be facilitated by the destruction of circulating lymphocytes, by the destruction of the mucociliary clearance system and, most importantly, by a large reduction in the numbers of alveolar macrophages. The clinical syndrome observed in a herd may therefore depend in part upon the other diseases present.

Thrombocytopenic purpura in lambs given bovine colostrum.

Newborn lambs which had been given colostrum from a single cow developed thrombocytopenia and seven died. The clinical signs included purpura, haemoglobinuria, melaena, dullness and lethargy. The lambs died at one to two days old. Post mortem examination revealed widespread haemorrhages throughout the body. Lambs given small amounts of colostrum developed a transient thrombocytopenia but did not die.

Concurrent maedi-visna virus infection and pulmonary adenomatosis in a commercial breeding flock in East Anglia.

The seroprevalence of maedi-visna virus infection in thin potential cull ewes aged over two years in a flock in East Anglia increased from 3.7 per cent in August 1985 to 39.0 per cent in September 1987 and 93.3 per cent in May 1989. This increase coincided with the first appearance of sheep pulmonary adenomatosis in the flock. Four emaciated ewes which were dyspnoeic were necropsied between 1987 and 1989. Maedi and pulmonary adenomatosis were confirmed histologically in one of these ewes and pulmonary adenomatosis was confirmed in the other three.

Natural scrapie in goats: neuropathology.

The brains of the 20 goats affected with natural scrapie received at the Central Veterinary-Laboratory, Weybridge, since 1975 were examined microscopically. Lesions of a spongiform encephalopathy were found in the brainstem, cerebellum, diencephalon, corpus striatum, and also in the neopallium or cerebral cortex. The lesions in the neopallium have not previously been reported in natural scrapie in goats. Deposits of amyloid were present in the thalamus in three of the 20 goats.

Infection of pigs and cattle with bovine viral diarrhoea virus on a farm in England.

Deaths within a litter of sucking pigs led to a suspicion of hog cholera, but pestiviruses isolated from both dead and live piglets appeared to be bovine viral diarrhoea virus. Persistent viraemia with bovine viral diarrhoea virus was demonstrated in living littermates and also in a bought-in calf, housed in a nearby pen on the same farm. Only two of the littermates survived, both of which had been virus negative and seropositive from the outset of testing. Porcine and bovine virus isolates grew well in calf testicular cells and were neutralised equally by sera collected at the farm from cattle and pigs. However, a comparison by means of their reactivity to monoclonal antibodies showed that they were similar but not identical, and only the porcine isolates grew well in a porcine kidney cell line.

Natural scrapie: detection of fibrils in extracts from the central nervous system of sheep.

Extracts from the cervical spinal cord and from the medulla, thalamus, cerebellum and cerebral cortex of the brains of 10 sheep, histopathologically confirmed as cases of scrapie, were examined by electron microscopy for the presence of scrapie-associated fibrils. Characteristic fibrils were observed in all the extracts except for that from the thalamus of one sheep. No fibrils were found in any extracts from three control sheep. A comparison of these results with a similar study of 22 cases of bovine spongiform encephalopathy (BSE) suggests that in cases of scrapie the area of the brain chosen for the detection of fibrils is less critical than in cases of BSE, in which fibrils are more readily extracted from areas of the brain stem.

Effect of sequential porcine reproductive and respiratory syndrome and swine influenza on the growth and performance of finishing pigs.

Sows and gilts on a breeding and finishing unit seroconverted to porcine reproductive and respiratory syndrome (PRRS) virus between February and March 1992. The signs of the disease progressed through the breeding herd to the piglets, weaners, growers and finally the finishing pigs. Forty-eight finishing pigs, on a nutritional experiment in which the individual pigs' food intake was recorded daily, showed signs of PRRS and later of swine influenza virus infection. Blood samples collected at slaughter from 47 of the pigs were seropositive to PRRS virus but seronegative to swine influenza virus. However, an immunocytochemical examination of a representative sample of the lungs of the pigs showed that swine influenza virus was present, despite the negative serological results. The infection of the finishing pigs with PRRS virus was associated with recurrent periods of inappetence and a decline in growth rate and performance. The total financial loss from the sequential infection with the two viruses, in a herd without high health status, was estimated at approximately 7 pounds per pig.

Detachment of the ischial tuberosity sows.

The clinical and pathological features of five cases of detachment of the ischial tuberosity are described. In two cases, the detachment occurred unilaterally; one was a gilt showing severe lameness which precluded mating and the other was a uniparous sow which showed only slight lameness. Three gilts which were affected bilaterally developed progressive lameness during pregnancy and were unable to stand after farrowing. One of these gilts died and two had to be slaughtered. The diagnostic features of the epiphyseal detachment were shortening of the caudal extremity of the ischium, palpation of the displaced ischial tuberosity and crepitation elicited by pressing the displace tuberosity against the ischium.

Neuropathology of scrapie: a study of the distribution patterns of brain lesions in 222 cases of natural scrapie in sheep, 1982-1991.

Detailed neuropathological findings in 222 cases of naturally occurring scrapie from Great Britain are described. The material consisted of formalin-fixed brain from eight breeds of sheep submitted between 1982 and 1991. Paraffin-embedded histological sections were made from several specified brain areas, including the medulla oblongata, cerebellum, pons, mesencephalon, diencephalon, septal area, basal ganglia and frontal cortex. Sections were examined by conventional and polarised light microscopy and the type and distribution of the lesions were recorded. Histologically, the lesions included vacuolation of neuronal perikarya and grey matter neuropil, neuronal degeneration (especially "dark' neurons) and loss, a reactive glial (predominantly astrocytic) response and amyloidosis. Vacuolar lesions were present in the cerebral cortex of 37 per cent of cases, centred around the superior frontal gyrus. Vacuolar lesions were detected in the neocortex for as long as sections have been taken from the superior frontal gyrus and are thus probably not a new feature of the disease. The distribution of vacuolation in the grey matter neuropil could be classified into seven patterns. Data from individual breeds of sheep showed that in some breeds there were significant differences in the age at which animals with different patterns of vacuolation died from scrapie.

The natural occurrence of scrapie in moufflon.

Six cases of scrapie were confirmed in two separately maintained flocks of moufflon, in both of which the disease appeared to be endemic. The clinical signs and histopathology were indistinguishable from those observed in scrapie-affected domesticated sheep. The pathology included lesions in the cerebral cortex which, although commonly present in scrapie-affected sheep, have not previously been described in the natural disease.

Aleutian disease in domestic ferrets: diagnostic findings and survey results.

Aleutian disease was diagnosed as the cause of posterior ataxia and paresis in domestic ferrets. Six serologically positive animals (four clinically affected and two unaffected) were investigated in detail and seven other clinically affected ferrets were also identified. The diagnostic findings included hypergammaglobulinaemia, histological lesions in the central nervous system and parvovirus-like particles in mesenteric lymph nodes. A wider serological survey of 446 animals owned by members of a ferret club revealed an incidence of 8.5 per cent seropositive animals.