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G H Lawson

Publications and source records attributed to G H Lawson.

At least 19 recordsLinked to original sources
PubMed2001

Lawsonia intracellularis: getting inside the pathogenesis of proliferative enteropathy.

Although proliferative enteropathy (PE) has been recognised for several decades, Lawsonia intracellularis, the aetiological agent, was identified formally in only 1995. This organism is both highly fastidious and obligately intracellular bacterium, characteristics which have inevitably restricted investigations in all aspects of its biology. Despite these limitations, advances have been made in characterising and understanding L. intracellularis-host interaction both in vivo and in vitro. Based upon evidence provided by mainly pathological and histological investigations conducted to date, we review salient features of our current understanding of processes involved throughout the course of infection by this unique pathogen.

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PubMed1997

In-vitro interactions of Lawsonia intracellularis with cultured enterocytes.

Strains of the obligately intracellular bacterium Lawsonia intracellularis, the etiologic agent of porcine proliferative enteropathy, were co-cultured in rat enterocyte cell cultures (IEC-18) and examined ultrastructurally. No regular surface arrays typical of surface or S-layers were visible on any bacterial strain, with or without Triton-X-100 detergent treatment. In separate experiments, there was no difference in the ability of L. intracellularis to attach and enter enterocytes with or without the presence of added bovine plasma fibronectin, or the peptide Arg-Gly-Ser. Interestingly, there was an increase in the invasiveness of L. intracellularis in the presence of the peptide Arg-Gly-Asp (RGD), in a dose-related manner. A reduction was observed in the ability of L. intracellularis to invade enterocytes in the presence of monovalent fragments of IgG monoclonal antibodies to an outer surface component of L. intracellularis. This neutralization showed an antibody concentration-dependent titration effect and was not apparent with co-cultures incorporating control antibodies. The exact nature of ligand and cell receptor interactions for L. intracellularis remain to be determined.

Animals↗
PubMed1996

Developed and resolving lesions in porcine proliferative enteropathy: possible pathogenetic mechanisms.

Proliferative enteropathy, caused by Lawsonia intracellularis, offers the opportunity to examine bacterial mechanisms that influence epithelial cell proliferation. Ultrastructural features of developed and resolving lesions included the presence of enlarged intestinal crypts containing undifferentiated immature epithelial cells and an absence of goblet cells. Numerous intracytoplasmic bacteria, identified as L. intracellularis, were consistently present within affected cells. In recovering intestinal tissue, additional features were (1) the common presence of pale, swollen, protruding epithelial cells, (2) shrunken, degenerate epithelial cells, (3) apoptotic bodies in both epithelial cells and macrophages, (4) the reappearance of normal goblet cells, and (5) reduced numbers of L. intracellularis within lesions. Bacteria were released from cells via cytoplasmic and cellular protrusions into the intestinal lumen. It is speculated that the presence of the intracytoplasmic bacterium, L. intracellularis, may disrupt normal processes of cell growth, differentiation or apoptosis in the intestinal epithelium.

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PubMed1995

Entry of the bacterium ileal symbiont intracellularis into cultured enterocytes and its subsequent release.

Separate suspensions of two strains of ileal symbiont (IS) intracellularis, an obligate intracellular bacterium and the causative agent of porcine proliferative enteropathy, were added to 40 or 80 per cent confluent monolayers of established cultures of rat (IEC-18) or pig enterocytes (IPEC-J2). Peak numbers of intracellular organisms were detected within the enterocytes six days later, but no cytopathic effects were evident. After an initial close association with the cell membrane of the enterocytes, single bacteria were internalised after three hours within membranes-bound vacuoles. The formation of an electron-dense projection between cell membranes and external bacteria was only evident if the bacterial suspensions were centrifuged on to the monolayers. The release of internalised bacteria into the cytoplasm, with the breakdown and loss of membrane-bound vacuoles, was also evident three hours after infection. Internalised bacteria were associated with, but not observed within, coated membrane pits. Mitochondria were closely associated with internalised vacuoles and with released bacteria. Two to six days after infection, multiplication of the bacteria free in the cytoplasm was frequently observed. In infected cells six days after the inoculation of monolayers, groups of bacteria were found within large, balloon-like, cytoplasmic protrusions, and the subsequent release of bacteria from the monolayer provided a means of bacterial exit from the cells. Many events in the in vitro culture model closely resembled events observed at the cellular level in animals infected with IS intracellularis and the model provides a useful basis for investigating the pathogenetic mechanisms of this bacterium.

Animals↗
PubMed1995

Infection of cultured rat enterocytes by Ileal symbiont intracellularis depends on host cell function and actin polymerisation.

The mechanisms of entry of Ileal symbiont intracellularis into IEC-18 rat enterocyte cells and subsequent bacterial proliferation were examined in centrifuge-assisted and static infections. Live, oxygen or neomycin damaged, and formalin killed bacteria, each rapidly entered viable cells. Live or damaged bacteria did not enter cells nor proliferate within cells after static infection of cells cooled to 5 degrees C. Infection of cells was greatly reduced at 20 degrees or 32 degrees compared to infection at 37 degrees C. Centrifuge-assisted infection was also reduced by chilling the cells. Cytochalasin D but not B inhibited the entry process indicating an actin-dependent infection, although other pathways may also be involved in centrifuge-assisted infections. Drugs capable of modifying cell membrane charge, heparin receptors or trypsin-labile proteins were all inactive in preventing or enhancing infection. We therefore conclude that infection of enterocytes by IS intracellularis is dependent on host cell activity and actin polymerization, but is independent of bacterial viability.

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PubMed1995

Antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy.

Proliferative enteropathy is caused by the microaerophilic obligate intracellular bacterium ileal symbiont (IS) intracellularis. Treatment of this disease is problematic because of the lack of in vivo or in vitro data on the activities of antimicrobial agents. A new procedure for determining the susceptibility of IS intracellularis was developed by using a tissue culture system which promotes the in vitro multiplication of this organism. Nineteen antimicrobial agents were evaluated in triplicate cultures for their intracellular and extracellular activities against up to three IS intracellularis strains isolated from pigs with proliferative enteropathy. The MIC was defined as the lowest concentration which prevented multiplication of 99% of the IS intracellularis isolates. Penicillin, erythromycin, difloxacin, virginiamycin, and chlortetracycline were the most active compounds tested, all with MICs of < or = 1 microgram/ml. Tiamulin and tilmicosin were the next most active compounds, with MICs of < or = 4 micrograms/ml. The MICs of aminoglycosides were generally > 32 micrograms/ml. Both lincomycin and tylosin were relatively inactive against the IS intracellularis strains tested, with MICs of 32 and 64 micrograms/ml, respectively. These results indicate that some compounds capable of intracytoplasmic accumulation and blocking bacterial protein synthesis were active against IS intracellularis strains isolated from pigs with proliferative enteropathy. The in vitro cultivation system shows promise as a method for studying the interaction between IS intracellularis and antimicrobial agents and for screening new antibiotics for use in therapy.

Animals↗
PubMed1994

Polymerase chain reaction for diagnosis of porcine proliferative enteropathy.

A polymerase chain reaction (PCR) assay for detection of the intracellular bacteria, ileal symbiont intracellularis of porcine proliferative enteropathy is described. The test is based on specific DNA primers and gave positive PCR product from samples of preserved intestinal mucosa and faeces from affected pigs. Mucosa and faeces from normal pigs gave no positive PCR products. The identity of the PCR product was confirmed by DNA-DNA hybridization with a probe, pCLO78, specific for IS intracellularis. Positive results were only observed in animals with active lesions of proliferative enteropathy. PCR is probably the most useful method for diagnosis of proliferative enteropathy that is currently available for live animals.

Animals↗
PubMed1994

Experimentally induced proliferative enteritis in hamsters: an ultrastructural study.

Hamsters, three weeks old, were dosed orally with suspensions of intracellular bacteria grown in rat enterocyte tissue culture cells IEC-18, which had been infected with suspensions of intracellular bacteria derived from the lesions of proliferative haemorrhagic enteropathy occurring naturally in two pigs. Each bacterial strain, identified as Ileal symbiont intracellularis, was passaged in the cell lines once, twice or five times, collected with the cells and used as inocula. Ten of 16 hamsters dosed with 916/91 passaged one or five times developed lesions of proliferative enteritis. In these 10 hamsters, marked hyperplasia of ileal enterocytes associated with numerous intracellular curved bacteria was detected. An ultrastructural study of epithelial cells in the ileum of affected hamsters showed numerous intracellular bacteria in the cytoplasm. Similar bacteria were not seen in unaffected animals. Intracellular bacteria were usually seen in groups and could appear as electron dense or in a more electron lucent form. These bacteria were clearly seen to enter cells from the intestinal lumen, via endocytic vacuoles at the brush border. There was rapid breakdown of the entry vacuoles, leaving bacteria free in the cytoplasm where division was usually observed. These bacteria were often seen in close association with normal or distended mitochondria and rough endoplasmic reticulum.

Animals↗
PubMed1994

Reproduction of proliferative enteritis in hamsters with a pure culture of porcine ileal symbiont intracellularis.

Hamsters, three weeks old, were inoculated orally with suspensions of intracellular bacteria, grown in tissue culture cells, IEC-18, rat enterocytes. Cells had been infected with suspensions of intracellular bacteria derived from the lesions of proliferative haemorrhagic enteropathy occurring naturally in two pigs 916/91 and 1482/89. Infected cell lines containing each separate strain, 916/91 and 1482/89, were passaged one, two or five times and pure cultures of intracellular bacteria, identified as ileal symbiont intracellularis by immunological means, were collected from the cells and used as inocula. Ten of sixteen hamsters dosed with 916/91 passaged one or five times, developed lesions of proliferative enteritis evident as necropsy three weeks after inoculation. Hamsters inoculated with 1482/89 passaged twice and stored frozen, or IEC-18 cells alone or those left uninoculated, failed to develop lesions of proliferative enteritis. Campylobacter jejuni infection occurred throughout, in all groups. Marked hyperplasia of ileal enterocytes, associated with numerous intracellular curved bacteria was invariably detected in experimentally affected hamsters. Immunofluorescence reactions with specific antibodies indicated that these intracellular bacteria were also ileal symbiont intracellularis. The results suggested that proliferative enteritis could be reproduced in hamsters with a pure culture of an agent derived from pigs. We concluded that the reproduction of the disease with our inocula containing a single agent clarifies the aetiology of proliferative enteritis in both hamsters and pigs.

Animals↗
PubMed1994

Specific in situ hybridization of the intracellular organism of porcine proliferative enteropathy.

The identity of the intracellular bacteria found in the enterocytes of pigs with proliferative enteropathy was investigated using specific DNA probes to various Campylobacter species and to a novel organism, ileal symbiont intracellularis. The ilea from pigs (Nos. 1-7) that were diagnosed by routine histopathology as having proliferative enteropathy were used. Diagnosis was made on the basis of proliferation of the enterocytes on hematoxylin and eosin-stained sections and the presence of large numbers of intracellular curved organisms on Warthin-Starry silver-stained sections. Four of these pigs (Nos. 1-4) had the chronic form of the disease, porcine intestinal adenomatosis, and three (Nos. 5-7) had the acute form, proliferative hemorrhagic enteropathy. An additional three normal pigs (Nos. 8-10) were obtained from three separate farms with no history of proliferative enteropathy. Frozen ileal sections were examined by in situ hybridization with DNA probes specific for ileal symbiont intracellularis and the three porcine intestinal Campylobacter species, C. coli, C. hyointestinalis, and C. mucosalis. In all seven pigs with either the intestinal adenomatosis or hemorrhagic enteropathy form of the disease, a DNA probe specific for ileal symbiont intracellularis hybridized to localized foci in the apical cytoplasm of ileal enterocytes. These hybridization sites corresponded to the location of intracellular bacteria in silver-stained sections of adjacent tissue. Sections from the three normal pigs tested with this probe and from all pigs tested with the Campylobacter species-specific DNA probes showed no specific hybridization reactions. The identity of the intracellular organism in these diseased pigs is ileal symbiont intracellularis.

Animals↗
PubMed1993

Interactions of porcine lymphocytes with Campylobacter-like organism membranes purified from proliferative enteropathy.

Peripheral blood mononuclear cells prepared from 6 healthy pigs and 6 pigs apparently affected by proliferative enteropathy were incubated with dilutions of a sonicate of purified Campylobacter-like organisms 872/87 prepared from a separate case of proliferative enteropathy. Incubations were carried out in RPMI 1640 medium, supplemented with foetal calf serum, L-glutamine, sodium pyruvate and 2-mercaptoethanol for 5 days, with relevant concanavilin A and medium controls. Marked stimulation of lymphocyte proliferation was clearly evident in samples prepared from the pigs apparently affected by proliferative enteropathy (peak stimulation index 4.4-13.2) compared to the healthy pigs (peak stimulation index 3.0). Diagnosis of proliferative enteropathy in these naturally derived cases was confirmed by a specific IgM indirect immunofluorescence assay. The results suggested that a specific antigen-related mitogenesis of blood lymphocytes occurs in pigs affected by proliferative enteropathy. This may have implications for immunization regimes.

Animals↗
PubMed1993

Ileal symbiont intracellularis, an obligate intracellular bacterium of porcine intestines showing a relationship to Desulfovibrio species.

A new genus and species of obligate intracellular-bacteria found in porcine intestines are described. Growth on any bacteriological medium deprived of living cells has not been demonstrated. The organism has been grown intracellularly in cell culture. The 16S rRNA gene sequence data, DNA probe results, and microscopic observations provide evidence that these bacteria differ from those in other described genera and that they belong to the delta subdivision of the class Proteobacteria. We have amplified and sequenced the 16S ribosomal DNA of four preparations of the intracellular bacterium from pigs. For this, intracellular organisms were released and purified from the infected cells without culture techniques. After DNA purification, the polymerase chain reaction with primers complementary to highly conserved eubacterial sequences was used to amplify regions of 16S ribosomal DNA which were subsequently cloned (in some cases) and sequenced directly by standard techniques. The sequences obtained from each preparation were identical and were most similar to that of a sulfate-reducing proteobacterium, Desulfovibrio desulfuricans ATCC 27774 (91% similarity). An oligonucleotide probe complementary to a hypervariable region of the 16S rRNA sequence of the bacterium hybridized with intracellular organisms obtained from porcine intestines. The bacterium is a gram-negative, curved rod with tapered ends. It multiplies intracellularly in the cytoplasm of ileal epithelial cells by septation. The vernacular name Ileal symbiont (IS) intracellularis is proposed for this bacterium. The type strain of IS intracellularis is strain 1482/89 grown in cell culture from a pig affected by proliferative enteropathy. It is deposited in the National Collection of Type Cultures, Colindale, London, as NCTC 12656.

Animals↗
PubMed1993

Reproduction of porcine proliferative enteropathy with pure cultures of ileal symbiont intracellularis.

Porcine proliferative enteropathy is consistently associated with the presence of intracellular curved bacteria in epithelial cells in affected portions of intestine. Two strains of these intracellular bacteria were cultured in a cell culture system with rat enterocytes (IEC-18) and passaged several times and used as oral inocula for 14 gnotobiotic and 8 conventional pigs. DNA and immunological studies had identified these bacteria as belonging to a new taxon, Ileal symbiont (IS) intracellularis. Conventional pigs dosed with approximately 3.7 x 10(6) of these organisms passaged six times in cell culture developed severe lesions of proliferative enteropathy in the ileum. Other conventional pigs dosed with a lower titer or with organisms passaged 13 times developed moderate and minor lesions, respectively. All gnotobiotic pigs dosed with organisms failed to develop lesions. Control pigs, eight conventional and two gnotobiotic, dosed with diluent, uninfected cell material or left undosed failed to develop lesions also. Reisolation of IS intracellularis and demonstration of the organism in mucosal and fecal samples only occurred in conventional pigs dosed with organisms. Gnotobiotic pigs lacking a normal intestinal flora have not been shown to be colonized by the organism. Seroconversion to IS intracellularis or mucosal infiltration by inflammatory cells was not observed in experimentally affected pigs, confirming the weak immune response characteristic of the natural disease. These results support the identification of IS intracellularis as an etiological agent of proliferative enteropathy in pigs.

Animals↗
PubMed1993

Intracellular bacteria of porcine proliferative enteropathy: cultivation and maintenance in vitro.

An obligate intracellular bacterium was isolated from the intestines of all 10 cases of porcine proliferative enteropathy from four different pig farms. The organism grew in a rat enterocyte cell line (IEC-18) and was maintained over 20 passages. The growth of the bacteria was assessed by immunostaining of cells exposed to infection. Infection was not associated with morphological cell change, and growth was confined to cells infected at the time of each transfer of infection and the progeny of these cells. The bacterium is a microaerophilic, cell dependent, curved or rod-shaped, gram-negative bacillus that multiplies freely in the enterocyte cytoplasm. Cell cultures containing the intracellular bacteria appear to be free of other microorganisms, including chlamydiae and viruses.

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