Porcine Proliferative Enteropathy Research Articles

Taxonomy of Campylobacter, Arcobacter, Helicobacter and related bacteria: current status, future prospects and immediate concerns.

The taxonomy of the genus Campylobacter has changed dramatically since its inception in 1963. At that time the genus comprised just two species. At present, taxa that were once assigned to Campylobacter may belong to one of over 50 species distributed among six genera. Most of these taxa belong to a phylogenetically distinct group referred to as either ribosomal RNA (rRNA) superfamily VI or the epsilon division of the class Proteobacteria. The taxonomic diversity of the group is matched by the diverse habitats in which they may be found, and by the wide range of diseases that they are associated with. Recognition of their clinical and economic importance has resulted in intense interest in the group, and the application of increasingly sophisticated isolation, detection and chemotaxonomic methods continues to elucidate new aspects of their biodiversity. However, despite the advances in new bacterial systematics, there remain a number of important issues concerning the classification of various campylobacterial taxa that require careful consideration. Ultimately, these issues are relevant to many working in the field of applied microbiology, including clinicians, veterinarians, epidemiologists and taxonomists. The purpose of this article is briefly to review the major developments in the taxonomy of Campylobacter from its inception to the present day; summarize the most recent changes in the field; analyse current topical issues of special relevance to applied microbiologists, including identification of the bacteria; and speculate on future prospects for campylobacterial taxonomy.

Treatment and control of porcine proliferative enteropathy using different tiamulin delivery methods

Objective: To determine the clinical and pathological effects when pigs infected with Lawsonia intracellularis and showing clinical signs of gastrointestinal disease are treated with tiamulin hydrogen fumarate (THF) administered in feed or water. Methods: All pigs in Study 1 (47 pigs) and Study 2 (48 pigs) were inoculated with a virulent pure culture of L intracellularis. After clinical signs became apparent, treated pigs were medicated for 28 days with THF administered in the feed at 35 g per ton (Study 1), or for 5 days with THF administered in the drinking water at 60 mg per liter (Study 2). Necropsies were performed on remaining control and treated pigs in Study 1 on Day 37 post-inoculation, and in Study 2 on Day 23 post-inoculation. Results: Medication in the feed or water significantly reduced development of gross and microscopic lesions, severity of clinical signs, and prevalence of fecal shedding of L intracellularis in treated pigs. Feed treatment with THF (2.0 mg per kg daily) had no effect on seroconversion, whereas water treatment (7.3 mg per kg daily) significantly reduced prevalence of seroconversion against L intracellularis. Implications: Tiamulin hydrogen fumarate in the feed (35 g per ton) or water (60 mg per liter) was effective in treating and controlling PPE. Timing of medication relative to time of infection with Lintracellularis may affect the immune response (seroconversion), fecal shedding, and development of lesions. Antibiotic dosage may also affect the immune response to L intracellularis infection, and may reduce fecal shedding, interfering with polymerase chain reaction tests for Lintracellularis.

Prevalence of porcine proliferative enteropathy and its control with tylosin in Korea

Porcine proliferative enteropathy(PPE) is an enteric disease been caused by Lawsonia intracellularis. It has become one of the critical problems in the pig industry. To investigate the prevalence of PPE in Korea, serum samples of 828 pigs from 65 herds were tested using indirect immunofluorescence antibody technique(IFA). The infection rate in individual pigs varied from 44 to 69%, whereas 100% in pig farms. The infection frequency was 57, 44.9, and 59.4% according to age respectively. Administration of tylosin in feed at a concentration of 110 ppm for 14 days reduced the infection rate of the farms. These data indicated that the high prevalence of PPE may be controlled by tylosin.

Improved diagnosis of porcine proliferative enteropathy caused by Lawsonia intracellularis using polymerase chain reaction-enzyme-linked oligosorbent assay (PCR-ELOSA)

Proliferative enteropathy (PE) caused by Lawsonia intracellularis is a major diarrheal disease affecting swine worldwide. Routine laboratory diagnosis of PE is done by amplification of L. intracellularis -specific DNA sequences by PCR followed by agarose gel electrophoresis and staining of PCR products with ethidium bromide. We report the development of an enzyme-linked oligosorbent assay (ELOSA) for specific identification of chromosomal L. intracellularis 328-bp PCR amplified products. The ELOSA involved determination of optical density value at 450 nm (OD450) after hybridization of biotin-labelled PCR products with an amine-modified internal oligonucleotide capture probe immobilized in microwell plates, and avidin-biotin-peroxidase complex. A positive ELOSA cut-off value of ≥0·375 was established using the mean OD450of negative control specimens plus three times the standard deviation. Using this value, the detection limit of PCR amplified L. intracellularis -specific products by ethidium bromide-stained agarose gel electrophoresis, Southern blot, and ELOSA were estimated to be 6·1 ng, between 0·8 and 3·0 ng, and 0·8 ng of DNA, respectively. Comparison of ethidium bromide-stained agarose gel analysis with ELOSA for detection of L. intracellularis -specific PCR products from 315 clinical specimens revealed 78% sensitivity, 100% specificity and 94% accuracy. The ELOSA produced a spectrophotometric signal that confirmed the authenticity of PCR products without subjective Íinterpretation of ethidium bromide-stained PCR products after agarose gel electrophoresis.

Therapeutic efficacy of water-soluble lincomycin-spectinomycin powder against porcine proliferative enteropathy in a European field study

Controlled clinical trials to a standardised protocol were conducted into the effect of a water-soluble antibiotic on proliferative enteropathy and its causative agent (Lawsonia intracellularis) on commercial pig farms at...

Specific Detection of Lawsonia intracellularis in Porcine Proliferative Enteropathy Inferred from Fluorescent rRNA In Situ Hybridization

Fluorescent in situ hybridization targeting 16S ribosomal RNA was used for specific detection of the obligate intracellular bacterium Lawsonia intracellularis in enterocytes from pigs affected by proliferative enteropathy. A specific oligonucleotide probe was designed and the specificity of the probe was determined by simultaneous comparison with indirect immunofluorescence assay for detection of L. intracellularis in formalin-fixed tissue samples from 15 pigs affected by porcine proliferative enteropathy. We used 10 tissue samples from pigs without proliferative mucosal changes as negative controls. The results showed that the oligonucleotide probe is specific for L. intracellularis and that fluorescent in situ hybridization targeting ribosomal RNA is a suitable and fast method for specific detection and histological recognition of L. intracellularis in formalin-fixed tissue.

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.

Development of persistent intestinal infection and excretion of Lawsonia intracellularis by piglets

Challenge experiments using Lawsonia intracellularis as oral inocula have established its aetiological role in porcine proliferative enteropathy. Thirty piglets, in four groups, were weaned at 21 days of age and inoculated orally at 24 days. Six piglets were challenged with 1·0 × 10 8 L intracellularis strain 916/91 (NCTC 12657) passaged 12 times in vitro, six with 5·0 × 10 8 of the same strain, seven with 3·0 × 10 8 L intracellularis strain LR189/5/83, passaged nine times, and 11 controls were dosed with sucrose-potassium glutamate buffer. An immunofluorescence assay for L intracellularis was applied to faecal smears and a polymerase chain reaction (PCR) incorporating L intracellularis-specific primers was applied to extracts of bacterial DNA derived from the faeces samples. Up to five pigs in each challenge group excreted detectable L intracellularis in faeces, in samples taken between two and 10 weeks after challenge. Some of the pigs had up to 7 × 10 8 L intracellularis g −1 faeces. The average weight gains of the higher dose challenge groups were moderately below those of the control pigs between three and nine weeks after challenge; diarrhoea was also observed in six pigs, two to four weeks after challenge. Numerous L intracellularis were detected in the intestines of all the pigs challenged with strain LR189/5/83 and two of the pigs challenged with 916/91, but not in other tissues.

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.

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.

Ulcerative colitis and porcine proliferative enteropathy: A common bacterial etiology?: [formula omitted]∗, M. Goddard†, S. McOrist§, J.H. Cummings∗. MRC Dunn Clinical Nutrition Centre, Cambridge∗, Addenbrooke's Hospital, Cambridge†; Royal (Dick) School of Veterinary Studies, University of Edinburgh§, U.K.

Ulcerative colitis and porcine proliferative enteropathy: A common bacterial etiology?: [formula omitted]∗, M. Goddard†, S. McOrist§, J.H. Cummings∗. MRC Dunn Clinical Nutrition Centre, Cambridge∗, Addenbrooke's Hospital, Cambridge†; Royal (Dick) School of Veterinary Studies, University of Edinburgh§, U.K.

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 testis 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 prolifrative enteropathy that is currently available for live animals.

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.

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.

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.

DNA analysis of intracellular campylobacter-like organisms associated with the porcine proliferative enteropathies: novel organism proposed

Intracellular Campylobacter-like organisms are a consistent feature of the porcine proliferative enteropathies. The relationship between these organisms and known Campylobacter sp. previously associated with the disease was studied using restriction enzyme analysis and DNA-DNA blot hybridization techniques. BglII restriction enzyme fragment patterns of DNA of the Campylobacter-like organisms were fundamentally different from those of C. mucosalis, C. hyointestnalis, C. jejuni, and C. coli. Crude DNA preparations from Campylobacter-like organisms hybridized strongly with homologous preparations, weakly with porcine DNA and not at all with DNA from Campylobacter sp. Fragment specific DNA probes prepared from Campylobacter-like organisms only hybridized with homologous preparations. This work suggests that the intracellular Campylobacter-like organisms are not one of the known Campylobacter sp. It is possible that they are a novel, uncultured organism worthy of a new name, such as HC. intracellulare'.

DNA analysis of intracellular campylobacter-like organisms associated with the porcine proliferative enteropathies: novel organism proposed

Intracellular Campylobacter-like organisms are a consistent feature of the porcine proliferative enteropathies. The relationship between these organisms and known Campylobacter sp. previously associated with the disease was studied using restriction enzyme analysis and DNA-DNA blot hybridization techniques. BglII restriction enzyme fragment patterns of DNA of the Campylobacter- like organisms were fundamentally different from those of C. mucosalis, C. hyointestinalis, C. jejuni, and C. coli Crude DNA preparations from Campylobacter-like organisms hydridized strongly with homologous preparations, weakly with porcine DNA and not at all with DNA from Campylobacter sp. Fragment specific DNA probes prepared from Campylobacter-like organisms only hybridized with homologous preparations. This work suggests that the intracellular Campylobacter-like organisms are not one of the known Campylobacter sp. It is possible that they are a novel, uncultured organism worthy of a new name, such as ‘ C. intracellulare’.

Antigenic analysis of Campylobacter species and an intracellular Campylobacter-like organism associated with porcine proliferative enteropathies.

Whole-cell and outer membrane preparations of Campylobacter mucosalis, C. hyointestinalis, C. jejuni, and C. coli isolated from porcine intestines were compared with preparations of intracellular Campylobacter-like organisms extracted directly from the lesions of pigs with proliferative enteropathy. By gradient polyacrylamide gel electrophoresis, outer membrane and total protein profiles of C. mucosalis, C. hyointestinalis, C. jejuni, and C. coli were significantly different from each other and from those of the Campylobacter-like organisms. Immunoblotting of these preparations with rabbit antisera or monoclonal antibodies prepared against the intracellular Campylobacter-like organisms showed strong reactions only with a 25,000- to 27,000-molecular-weight component of preparations of the intracellular organisms. Antisera to cultivable Campylobacter species isolates did not react with preparations of intracellular organisms. Isoelectric focusing of sonicated preparations showed protein profile differences and an immune-reactive component in the intracellular organisms with a pI of 4.5. This study suggests that the intracellular Campylobacter-like organism associated with proliferative enteropathy may be a novel bacterium with significant antigenic differences from the Campylobacter species previously associated with the disease.

Serological diagnosis of the porcine proliferative enteropathies: implications for aetiology and epidemiology.

Campylobacter mucosalis and C hyointestinalis have been associated with the proliferative enteropathies of pigs. An examination of the antibody response to these organisms and to the intracellular campylobacter-like organism was undertaken. Antibody to the campylobacter-like organism was predominantly IgM, short lived, and could be detected by an immunofluorescence test using bacteria released from lesions as antigen. The majority (75 per cent) of pigs with proliferative enteropathy at necropsy were antibody positive and a small number (4 per cent) of pigs in which lesions were not observed were found to have antibody. Antibody appeared to be correlated with the presence of lesions rather than with exposure to infection and was independent of the presence of antibody to C mucosalis or C hyointestinalis. In natural outbreaks of the disease antibody to the campylobacter-like organism was more prevalent than clinical signs in the affected animals.

Infection of established cell lines with Campylobacter mucosalis

Campylobacter mucosalis (CM) and other Campylobacter spp. mainly from cases of the porcine proliferative enteropathies were used to infect cell line cultures. CM attached to and persisted intracellularly in PK (15), MDBK, BHK, HeLa, INT-407 and other cells; in PK (15) this infection resulted in a marked cytopathic effect and total destruction of the cell culture in 10 days. MDBK, INT-407, BHK and HeLa cultures were more resistant to the effects of infection. CM did not adhere to Vero, LLCMK 2 or CEF cells and no cytopathic changes took place. The other Campylobacter spp tested did not attach to cell cultures and cell death occurred within 4 days. Attempts to passage CM were unsuccessful due to a decrease in the number of bacteria at each passage.