Steven McOrist

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.

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.

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.

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.

Some Viral and Protozool Diseases in the European Wildcat (Felis silvestris)

Ten European wildcats (Felis silvestris) were examined at necropsy and an additional 23 were examined clinically for evidence of viral diseases in Scotland. Two plasma samples taken from live free-living wildcats showed positive ELISA reactions to feline leukemia antigen. A feline leukemia virus of subgroup A was isolated from one of these samples, taken from a wildcat in north-western Scotland. No antibodies to feline coronavirus or feline immunodeficiency virus were detected in any sample. Three of the live wildcats and one of the dead had chronic mucopurulent rhinotracheitis suggestive of “cat flu.” One other dead wildcat had diffuse enlargement of anterior lymph nodes. The findings indicated that feline leukemia virus infection can occur in free-living Felis silvestris. It is possible that the disease exists as a sustained infection in some wildcat populations, although the close interaction between wildcat and the domestic cat means that the latter could act as a continual source of infection.

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.

PARASITISM OF WILD GOULDIAN FINCHES (ERYTHRURA GOULDIAE) BY THE AIR-SAC MITE STERNOSTOMA TRACHEACOLUM

Sixty-two percent of 26 wild caught Gouldian finches (Erythrura gouldiae) were infected with Sternostoma tracheacolum, a parasitic rhinonyssid mite. The intensity of infection was higher in adult finches than juveniles, and higher in juvenile females than juvenile males. Histopathological investigation of wild Gouldian Finches revealed bronchopneumonia and air sacculitis associated with mite infection. Although this mite may not have contributed to the decline of Gouldian finch populations in the wild during the past 20 yr, it may be suppressing the return of the finch to its former status.

Possible relationshio of proliferative enteritis in pigs and hamsters

Three- to six-week-old hamsters were orally inoculated with broths containing one of the following cultures: Campylobacter mucosalis; C. hyointestinalis; C. coli; C. jejuni, all of porcine proliferative enteritis origin, or else C. jejuni of hamster origin. Hamsters given the last of those organisms were shown to have colonisation of their intestines by C. jejuni and 36 of 40 developed an acute enteritis. Mild hyperplasia of enterocytes in ileal crypts was evident in one hamster 2 days after it was given C. coli. No other lesions were detected. Further 3-week-old hamsters were orally inoculated with homogenised intestinal mucosa collected from 4 pigs (A-D) affected by proliferative enteritis. Lesions of proliferative enteritis were detected in 7 of 41 hamsters necropsied 10-21 days after being dosed with mucosas B or D. Marked hyperplasia of ileal enterocytes, associated with numerous intracellular Campylobacter-like organisms, were invariably detected in experimentally affected hamsters. No particular Campylobacter sp. was consistently isolated. None of the controls had demonstrable lesions. The results suggested that cross-species transmission of proliferative enteritis was possible from pigs to hamsters. Therefore a common initiating or aetiological agent may be present. No specific organism was identified as filling this role by inoculation of hamsters with pure cultures.

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.

Clostridial enteritis in free-living lorikeets (Trichoglossus spp.).

Numerous free-living lorikeets (Trichoglossus spp.) in 19 flocks in eastern Australia either developed diarrhoea, depression and died or were found dead. Grossly, the duodenum and jejunum of affected birds were enlarged and haemorrhagic with yellow, necrotic mucusa. Histological examination of affected intestines revealed acute necrosis and oedema of villous structures, with vascular congestion, haemorrhage and a moderate infiltration of heterophils and lymphocytes. Gram-stained sections of affected intestines revealed numerous Gram-positive bacilli at the brush border. Anaerobic bacteriological culture of selected intestines revealed Clostridium species in profuse growths. Gas-liquid chromatography and biochemical tests revealed a complex of C. perfringens. Beta toxin was detected in intestinal contents and from half the C. perfringens cultures from affected birds.