Lawson Gh

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.

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.

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.

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.

Early Lesions of Proliferative Enteritis in Pigs and Hamsters

Gnotobiotic pigs and conventional hamsters were given suspensions of intestinal mucosa from a pig with proliferative hemorrhagic enteropathy and killed 10 or 21 days later. Affected animals had evidence of marked proliferation of immature enterocytes in the intestinal crypts. Numerous Campylobacter-like organisms were in the cytoplasm of enterocytes, and in some instances, bacteria were closely associated with enter-ocytes. Some intracellular bacteria lying below the microvillous border were within membrane-bound structures. Immunofluorescence and electron immunogold staining with specific antibodies indicated that these organisms were antigenically different from curved bacteria in the crypt lumen of early lesions. This study indicates that the life cycle of the intracellular organisms may involve entry into crypt enterocytes from the intestinal lumen with subsequent intracellular multiplication.

Some Features of Campylobacter sputorum subsp. mucosalis subsp. nov., nom. rev. and Their Taxonomic Significance

We studied strains of “Campylobacter sputorum subsp. mucosalis” isolated from intestinal adenomatosis from several sources. Our results supported the contention that this organism should be regarded as a distinct subspecies of Campylobacter sputorum. However, since the name of this organism was not included on the Approved Lists of Bacterial Names, we propose it as a revived name (i.e., Campylobacter sputorum subsp. mucosalis subsp. nov., nom. rev.). The type strain is FS253/72 (= NCTC 11000). The guanine-plus-cytosine content of the deoxyribonucleic acid of this organism supports its inclusion within the genus Campylobacter, but in its hydrogen dependence this organism shows clear similarities to the human oral vibrios and to Vibrio succinogenes. However, “C. sputorum subsp. mucosalis” possesses an unusual type c cytochrome, and in this way it appears to differ from V. succinogenes. It may be differentiated readily from other members of the species C. sputorum by salt and glycine tolerance tests or by serological techniques.

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 cosin-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.