Proliferative Haemorrhagic Enteropathy Research Articles

Recent antimicrobial susceptibility of Lawsonia intracellularis field isolates from pigs with proliferative hemorrhagic enteropathy in Korea

Recent antimicrobial susceptibility of Lawsonia intracellularis field isolates from pigs with proliferative hemorrhagic enteropathy in Korea

Systemic cytokine response in pigs infected orally with a Lawsonia intracellularis isolate of South Korean origin.

In the swine industry, Lawsonia intracellularis is one of the main enteric pathogens; it causes acute intestinal hemorrhage (proliferative hemorrhagic enteropathy) in naïve adult pigs and a wasting disease (proliferative enteropathy) in growing pigs. Among many kinds of cytokines, interferon-γ (IFN-γ) has previously been reported to play a significant role in limiting intracellular infection and increasing cellular proliferation associated with L. intracellularis. However, the levels of various circulating inflammatory cytokines, including IFN-γ, in animals infected with L. intracellularis is still an area of considerable interest for understanding immunity against this bacterium. In addition, there has been no information on cytokine response in animals infected with any L. intracellularis isolate of South Korean origin or Asian origin. To determine the relationship between the changes in the systemic inflammatory cytokine response in the peripheral blood of the host after L. intracellularis infection, we measured the levels of some pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IFN-γ), anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor-β (TGF-β)), and a chemokine (IL-8) in pigs infected with L. intracellularis isolated from South Korea. This study demonstrated that a L. intracellularis isolate of South Korean origin induced cytokine (TNF-α, IL-6, and IFN-γ) responses in infected animals within 15 days post-infection although the circulating levels of IL-4, IL-10, IL-8 and TGF-β were induced relatively late.

Serum folate, cobalamin, homocysteine and methylmalonic acid concentrations in pigs with acute, chronic or subclinical Lawsonia intracellularis infection

Lawsonia intracellularis is the causative agent of porcine proliferative enteropathy. The clinical presentation can be acute (i.e. proliferative hemorrhagic enteropathy, PHE), chronic (i.e. porcine intestinal adenomatosis, PIA) or subclinical. In humans with chronic enteropathies, low serum folate (vitamin B9) and cobalamin (vitamin B12) concentrations have been associated with increased serum concentrations of homocysteine and methylmalonic acid (MMA), which reflect the availability of both vitamins at the cellular level. The aim of this study was to evaluate serum folate, cobalamin, homocysteine and MMA concentrations in serum samples from pigs with PHE, PIA or subclinical L. intracellularis infection, and in negative controls. Serum folate, cobalamin, homocysteine and MMA concentrations differed significantly among pigs in the PHE, PIA, subclinical and negative control groups. Serum folate concentrations in the PHE and PIA groups were lower than in the subclinical and negative control groups, while serum cobalamin concentrations were lower in the PIA group than in other groups. Serum concentrations of homocysteine were higher in the PHE, PIA and subclinical groups than in the negative control group. Serum concentrations of MMA were higher in the subclinical and PIA groups than in the control group. These data suggest that pigs infected with L. intracellularis have altered serum cobalamin, folate, homocysteine and MMA concentrations.

An Episode of Proliferative Hemorrhagic Enteropathy Associated with Lawsonia Intracellularis in a Pig Farm from Romania

Introduction: In swine, Lawsonia intracellularis is known to be responsible for porcine proliferative enteropathy. The syndrome can be divided in an acute intestinal hemorrhage (proliferative hemorrhagic enteropathy) affecting naive adult pigs and a wasting disease (porcine intestinal adenomatosis) in growing pigs. As new diagnostic techniques develop, there is increased number of pig farms where L. intracellularis is being identified worldwide. Aims: There are few reports of L. intracellularis outbreaks in Romania. We aimed to describe the clinical signs, treatment, outcome, gross necropsy and histopathological lesions from an episode of proliferative hemorrhagic enteropathy associated with L. intracellularis in a farm from Transylvania. Materials and methods: A farm of 4000 pigs (TOPIGS line) from Bistrita-Nasaud County. The microscopical examination was composed of Hematoxilin and Eosin exam, Warthin-Starry silver stain and immunohistochemistry for Multi-Cytokeratin. Results: Over a period of 6 days, a total number of 10 pigs dyed (from 2000 animals in the age group 90-120 days), and 1 pig dyed (from 1000 animals in the age group 60-90 days) with pallor, anorexia or with no clinical signs. Gross lesions were represented by pallor of the carcass and were restricted to the ileum. The intestinal wall was thicker (cerebriform aspect) and turgid. A mixture of blood and fibrin was present in the ileum, impregnating the faeces in the large intestine. Histologically the mucosa was thicker due to epithelial proliferation (Multi-Cytokeratin positive), with few Goblet cells, with severe erosion, necrosis and haemorrhage. Curved rod-shaped bacteria with morphology consistent with L. intracellularis were observed in the apical cytoplasm of epithelial cells using the Warthin-Starry silver stain. The food was medicated with chlortetracycline (20 - 40 mg a.s./kg b.w./day) and the pigs remained healthy, with no further mortality. Conclusion: We report here an outbreak of proliferative hemorrhagic enteropathy associated with L. intracellularis in a pig farm from Romania, emphasizing the importance of early diagnostic and control measures for this disease.

Antimicrobial Susceptibility Testing of Two Lawsonia intracellularis Isolates Associated with Proliferative Hemorrhagic Enteropathy and Porcine Intestinal Adenomatosis in South Korea

This study represents the first published data on antimicrobial susceptibility of Asian isolates of Lawsonia intracellularis. We assessed MICs of 16 antimicrobials for two isolates of L. intracellularis recovered from diseased pigs in South Korea, one from a finisher pig with acute proliferative hemorrhagic enteropathy in 2002 and the other from a grower pig with porcine intestinal adenomatosis in 2010. Tylosin and tilmicosin were found to be the most active against L. intracellularis both intracellularly (MICs, 0.25 to 0.5 μg/ml and 0.125 μg/ml, respectively) and extracellularly (MICs, 0.25 to 0.5 μg/ml and 1 μg/ml, respectively).

Microarray and cytokine analyses of field cases of pigs with diarrhoea

This field study explored the cytokine expression in intestinal tissue and serum from 19 diarrhoeic and 9 healthy pigs in herds with a long-time history of Lawsonia intracellularis-infection. The disease, proliferative enteropathy (PE), is associated with diarrhoea and poor performance in growers and haemorrhagic diarrhoea and sudden death in finisher pigs, but the immunopathology is poorly understood. Histopathology, demonstration of L. intracellularis and porcine circovirus type 2 (PCV2) in intestinal tissue by PCR, and detection of serum antibodies to L. intracellularis, were performed. The presence of IL-1β, IL-6, IL-10, IFN-α, IFN-γ, TNF-α and TGF-β in sera was determined by immunoassays, and intestinal mRNA expression of these cytokines plus IL-12p40 was determined by qPCR. Intestinal specimens from pigs with intestinal adenomatosis ( n = 2), proliferative haemorrhagic enteropathy or swine dysentery ( n = 2), and controls ( n = 2) were analysed by a genome wide porcine microarray. The clinical signs of PE were not always supported by the subsequent analyses, and the presence of PCV2 may have contributed to an increased mRNA expression for IFN-γ in intestinal specimens from some pigs. The limited gene expression in the microarray analyses and the limited expression of cytokines in both sera and intestines, indicate that the immune response is poorly activated in the initial course of an infection with L. intracellularis. However, the gene encoding for insulin-like growth factor binding protein 3 (IGFBP-3) was up-regulated in two pigs with prominent mucosal proliferation.

The effect of porcine proliferative enteropathy on the introduction of gilts into recipient herds

Porcine proliferative enteropathy (PPE) caused by Lawsonia intracellularis (LI) is one of the most important enteric diseases in growing swine. The acute form of this disease, proliferative hemorrhagic enteropathy (PHE), has become a common problem when replacement breeding stock are introduced into a herd. In the cases described, animals of different immune status were moved between supply and recipient herds. An LI-free breeding-stock herd (Herd A) supplied gilts to four herds, one of which was LI-free (Herd B). During a 1-year period, PHE was observed in replacement animals after they entered the LI-infected herds (Herds C, D, and E); however, no problems were reported in Herd B. After an outbreak of PHE occurred in Herd A, replacement animals from this herd no longer developed PHE in the three LI-infected recipient herds. However, an outbreak of PHE occurred in Herd B when LI-infected replacement gilts were introduced.

Detection of Lawsonia intracellularis in the tonsils of pigs with proliferative enteropathy

The presence of Lawsonia intracellularis, the obligate intracellular bacterium causing proliferative enteropathy (PE), in the tonsils of pigs as a locus for infection or extraintestinal occurrence of the bacterium was investigated by PCR and immunohistochemistry. Tonsillar occurrence of L. intracellularis could be part of the pathogenesis of PE and an important risk factor in the spread of the disease. L. intracellularis was detected by only PCR in the tonsils of 2/32 pigs without PE at necropsy but with a clinical history of diarrhoea and detection of the bacterium in faeces 1 to 3 weeks prior to necropsy but not in four pigs with moderate PE lesions. However, L. intracellularis was detected in the tonsils of 4/9 pigs with PE complicated with necroses and in 4/4 pigs with proliferative haemorrhagic enteropathy in which L. intracellularis antigen also was demonstrated in tonsillar macrophages and as intact bacteria in the lumen of the crypts. The results show that L. intracellularis is detectable in the tonsils of pigs and that the tonsillar presence of L. intracellularis appears to be correlated to the severity of the intestinal lesions possibly as a result of local retention and not as part of the pathogenesis of PE.

Simulation of the economic impact of proliferative enteritis on pig production in Australia.

The economic impact of proliferative enteritis (PE) on an 'average' pig farm was calculated using the AUSPIG decision support system. Inputs were modelled on actual cases of PE, in which affected herds suffered from depressed growth rate, decreased feed efficiency and stock losses. The costs associated with non-haemorrhagic PE and proliferative haemorrhagic enteropathy ranged from $15/sow/yr to $141/sow/yr, respectively, depending on the clinical severity of the disease, incidence of infection and the type of medication strategy used to treat and control the disease.

Outbreaks of proliferative haemorrhagic enteropathy on two pig farms

Clinical signs of proliferative haemorrhagic enteropathy (PHE) including anaemia, dysentery and sudden death were observed in finisher pigs and young breeding stock on 2 farms. On farm A, PHE occurred 12 months after repopulation of the farm. Other outbreaks of PHE occurred after the withdrawal of therapeutic concentrations of in-feed antibacterial agents (farm A), or after monensin sodium (100 g/t) replaced olaquindox (100 g/t) in feed (farm B). The outbreaks, the possible sources of contamination and the role of antibacterial feed additives in the treatment and control of PHE are described.

Prevalence of proliferative enteritis on pig farms in Australia

Three surveys, undertaken to assess the prevalence of proliferative enteritis (PE) on pig farms in Australia and to investigate risk factors associated with clinical disease, indicated that PE was a common disease in pig farms. Forty of the 71 (56%) randomly-selected producers had either observed PE or had a veterinarian diagnose the disease in their herd during 1988 to 1990. A relatively low prevalence of the disease was recorded at veterinary diagnostic laboratories, and this suggested that diagnoses of PE were often not confirmed by histopathological examination of the intestines of affected pigs. Non-haemorrhagic PE occurred most often in six- to 24-week-old pigs, but was also reported in 52-week-old pigs. Proliferative haemorrhagic enteropathy usually affected pigs over 16 weeks of age, but was also reported in pigs as young as six weeks and as old as four years of age. A survey of pig-specialist veterinarians indicated that most veterinarians diagnosed PE based on clinical and gross pathological examination of affected pigs, without laboratory confirmation. There were difficulties associated with measuring the prevalence of PE among herds, including the effectiveness of antibacterials for its prevention and control, its subclinical nature and probable mis-diagnoses. This study highlighted the need for an ante-mortem diagnostic test to measure the prevalence of PE more accurately.

Enzyme-linked immunosorbent assay for measuring ileal symbiont intracellularis-specific immunoglobulin G response in sera of pigs

Proliferative enteritis (PE) is a common intestinal disease on pig farms. The disease is caused by ileal symbiont (IS) intracellularis (Campylobacter-like organisms) bacteria. An enzyme-linked immunosorbent assay (ELISA) was developed to measure IS intracellularis-specific immunoglobulin G (IgG) response in the sera of pigs. The antigen used in the ELISA was filtered, percoll gradient-purified IS intracellularis extracted from the intestines of pigs affected with proliferative hemorrhagic enteropathy. The antibody responses of pigs challenged with intestinal homogenates from pigs affected with proliferative hemorrhagic enteropathy containing IS intracellularis or percoll-gradient purified IS intracellularis were low and variable. The low IgG titers measured in challenged pigs support previous findings that IgG plays a minor role in the immune response of pigs to IS intracellularis. On a farm in which infection was endemic, pigs seroconverted at between 7 and 24 weeks of age. High IgG titers, indicative of maternally acquired antibody, were present in 3-week-old pigs. The IgG titers in piglets were lowest at 6 weeks of age, which approximates the age of onset of clinical disease. These results suggest that IgG plays a role in determining the susceptibilities of pigs to natural infection. Measurements of seroconversion by the ELISA might aid in epidemiological investigations of PE in naturally infected herds. However, the variable antibody responses in experimentally challenged pigs would seem to limit its usefulness as an antemortem diagnostic test for PE.

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 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 occuring 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 at 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.

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.

Immunocytological responses in porcine proliferative enteropathies.

The ileum, colon, and mesenteric lymph nodes of pigs naturally affected by either of the two major forms of proliferative enteropathy, namely, intestinal adenomatosis or hemorrhagic enteropathy, were examined for immunocytological responses to infection by immunocytochemistry, using antibodies directed against elements of the porcine immune system. In both forms, there was mucosal proliferation of immature enterocytes which lacked substantial major histocompatibility complex class II expression and a marked accumulation of immunoglobulin A (IgA) at the apical cytoplasm of affected enterocytes in association with intracellular Campylobacter-like organisms. In intestinal adenomatosis, there was only a mild infiltration of CD8+ and CD25+ T cells in the intestinal lamina propria. In hemorrhagic enteropathy, there was a moderate infiltration of CD8+ and CD25+ T cells and IgM+ B cells in the lamina propria. In rats and humans, villous enterocytes are thought to act as antigen-presenting cells, with major histocompatibility complex class II molecules present on their surface, capable of initiating a T-cell response (particularly of CD8+ T cells) in response to bacterial antigens. Therefore, the selection of immature crypt cells by the intracellular Campylobacter-like organisms for entry and multiplication may represent a remarkable microbial adaptation associated with local immunomodulation and enhanced bacterial survival. The accumulation of IgA within affected enterocytes may represent a reduced capability of the cells to process nonspecific IgA or an accumulation of specific IgA.

Problems of interpretation of ileal lesions in New Zealand slaughter pigs

Abstract The term proliferative enteritis (PE) is a collective one used to describe a group of related diseases characterised by adenomatous intestinal epithelium frequently seen to contain intra-cellular campylobacter-like organisms. It embraces four related disease syndromes, porcine intestinal adenomatosis (PIA), necrotic enteritis (NE). regional ileitis and proliferative haemorrhagic enteropathy (PHE), all affecting mainly the terminal ileum.

Cloned DNA probes specific for the intracellular Campylobacter-like organism of porcine proliferative enteritis

Although the etiology of porcine proliferative enteritis is not understood, the consistent presence of intracellular Campylobacter-like organisms (CLOs) in proliferating pig intestinal epithelial cells suggests that the organism is involved in the disease process. In order to obtain information about this organism, we generated and characterized specific DNA probes to the intracellular CLO which was purified without culturing. Intracellular CLOs were isolated from mucosa by homogenization, filtration, and absorption to wheat germ agglutinin-Sepharose. The DNA was purified, and a CLO genomic library was constructed. The specificity of recombinant plasmids was confirmed by both dot blot hybridization and Southern analysis of normal and diseased mucosa, as well as of a variety of Campylobacter species. Several of the CLO-specific probes hybridized with porcine mucosa obtained from pigs with proliferative enteritis but not with nondiseased mucosa. The probes hybridized equally with mucosa or DNA obtained from each of the two clinical forms of proliferative enteritis, i.e., proliferative hemorrhagic enteropathy and porcine intestinal adenomatosis. The CLO-specific probes failed to hybridize with any of the commonly isolated porcine Campylobacter species, including Campylobacter hyointestinalis, C. mucosalis, and C. coli. Therefore, the intracellular CLO of porcine proliferative enteritis may be an as yet unidentified or uncultured species.

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 enterocytes. 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.

Reproduction of proliferative enteritis in gnotobiotic pigs

Gnotobiotic pigs dosed orally with filtrates (0.8 and 0.65 micron) of intestinal mucosa from a pig affected by proliferative haemorrhagic enteropathy developed lesions of proliferative enteritis, affecting mainly the ilea. Other piglets dosed with filtrates of affected mucosa from the same source and from other proliferative haemorrhagic enteropathy or intestinal adenomatosis mucosae, did not develop lesions. All inocula contained numerous campylobacter-like organisms evident in stained smears, Campylobacter coli and C mucosalis. C coli colonised the intestines of all the pigs, C hyointestinalis (which was not detected in the inocula) did so in some affected and unaffected pigs while C mucosalis was not recovered from any of the intestines. Although other explanations are possible the number and viability of the intracellular campylobacter-like forms is likely to be the critical factor in infectivity. In affected intestines the crypts were colonised by campylobacter-like organisms, and their attachment and entry into enterocytes was associated with cellular proliferation. Immunofluorescence reactions suggested that the intracellular campylobacter-like organisms were antigenically distinct from the known Campylobacter species. It is possible, therefore, that porcine proliferative enteritis is caused by a further unidentified Campylobacter species, or that there is a marked antigenic change of C hyointestinalis or C coli on entry into porcine enterocytes.