Detection of Lawsonia intracellularis using immunomagnetic beads and ATP bioluminescence

Lawsonia intracellularis is an obligate intracellular pathogenic bacterium that causes proliferative enteropathy in various animals. The detection of L. intracellularis in clinical and environmental samples is necessary for the diagnosis of infection and epidemiological investigations. For the detection of L. intracellularis in fecal samples, we have developed an immunological method using immunomagnetic separation and ATP bioluminescence. Magnetic beads were coated with an anti-Lawsonia surface antigen (LsaA) antibody in order to capture the L. intracellularis in fecal samples from infected rabbits and the bacteria captured by the immunomagnetic beads were assayed by means of ATP bioluminescence. Our results showed that L. intracelluraris was detected by immunomagnetic separation of bacteria-holding magnetic beads and ATP-based bioluminescence, suggesting that our methods could be useful for the diagnosis of proliferative enteropathy.     

Colonisation and shedding of Lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms

Lawsonia intracellularis is an intracellular bacterium causing proliferative enteropathy in various animal species, and is considered an economically important pathogen of pigs. Rats and mice have been implicated as external vectors for a wide range of pig pathogens, including L. intracellularis. Previous studies have demonstrated L. intracellularis infection and proliferative enteropathy in rodents, but did not show the duration of shedding or the number of L. intracellularis shed by infected rodents, and therefore the infection risk that rodents pose to pigs. In this study, the number of L. intracellularis shed in the faeces and intestinal mucosa of wild rats trapped on pig farms was determined by a quantitative real time polymerase chain reaction assay. The prevalence of L. intracellularis in wild rats trapped on pig farms with endemic proliferative enteropathy (PE) was very high (≥ 70.6%), and large numbers of L. intracellularis were shed (10(10)/g of faeces) in a small proportion of wild rats. The duration of colonisation in laboratory rats and mice challenged with porcine isolates of L. intracellularis was also shown. Faecal shedding of L. intracellularis persisted for 14-21 days in rats and mice that were mildly affected with histological lesions of PE. The humoral immune response to L. intracellularis persisted for 40 days in both species. This study demonstrates that rodents may be an important reservoir of L. intracellularis on piggeries, and hence rodent control is important in disease eradication programs on pig farms.     

Cultivation and characterization of Lawsonia intracellularis isolated from rabbit and pig

Lawsonia intracellularis is an obligate intracellular pathogenic bacterium that causes proliferative enteropathy in domestic and experimental animals. In this study, we improved the in vitro cultivation method of L. intracellularis to increase the passage efficiency and showed that L. intracellularis isolated from a rabbit and a pig have different antigenic properties. Bacteria should be recovered from infected cells before cell death due to infection to obtain higher bacterial passage efficiency, and measurement of LDH activity in the cell culture medium was useful for determining the timing of bacterial passage. L. intracellularis isolated from the rabbit and pig showed different band patterns in immunoblotting. Our results should be helpful in the development of serological diagnosis and epidemiological investigation methods.     

Enzyme-linked immunosorbent assay to detect Lawsonia intracellularis in rabbits with proliferative enteropathy

Lawsonia intracellularis is an obligate intracellular pathogenic bacterium that causes proliferative enteropathy in domestic and experimental animals. Antiserum against synthetic peptides of the Lawsonia surface antigen (LsaA) well recognized L. intracellularis in infected ileum by immunohistochemistry. The synthetic peptides in LsaA showed strong reaction with serum from rabbits infected with L. intracellularis by enzyme-linked immunosorbent assay. These results suggest that ELISA used synthetic peptides in LsaA and anti-LsaA serum might be useful to diagnose for proliferative enteropathy.     

Isolation of Lawsonia intracellularis in Korea and reproduction of proliferative enteropathy in pigs and hamsters

Lawsonia intracellularis (L. intracellularis) was isolated from a Korean pig suffering acute proliferative enteropathy. In vitro culture conditions of L. intracellularis were established in McCoy cells. Pigs and hamsters experimentally infected with the pure culture of L. intracellularis reproduced clinical signs and intestinal lesions of proliferative enteropathy. The presence of L. intracellularis in the intestinal lesions was confirmed by immunohistochemistry with L. intracellularis-specific monoclonal antibodies.     

Immunohistochemistry and polymerase chain reaction for the detection of Lawsonia intracellularis in porcine intestinal tissues with proliferative enteropathy

Detection method of Lawsonia intracellularis was studied in formalin-fixed paraffin-embedded intestinal tissues from 5 naturally infected pigs by immunohistochemistry with a monoclonal antibody against outer membrane protein of L. intracellularis. Warthin-Starry silver stain revealed clusters of argyrophilic, slightly curved rod-shaped organisms in the apical cytoplasm of enterocytes. Immunohistochemical staining with a L. intracellularis-specific monoclonal antibody confirmed the presence of the organism in the apical cytoplasm of hyperplastic enterocytes. The presence of L. intracellularis in the ileum of pig with proliferative enteropathy was confirmed by polymerase chain reaction (PCR) further on the basis of amplification of 319 base pair products specific for porcine L. intracellularis chromosomal DNA. Immunohistochemistry and PCR may be a complementary method to confirm the diagnosis of L. intracellularis infection in pigs.     

In vitro cultivation and partial characterization of Lawsonia intracellularis from a Japanese field case of porcine proliferative enteropathy

Lawsonia intracellularis isolated from a Japanese field case of porcine proliferative enteropathy (PPE) was cultivated and partially characterized. The bacterial cells isolated from the intestinal mucosa of a pig suffering from the acute form of PPE were used to inoculate rat small intestine cells (IEC-18) and human epithelial cells (HEp-2). Infected foci, which were stained with L. intracellularis-specific rabbit antiserum, were observed in the cell culture at 5 days post inoculation. The DNA sequence of several genes in the Japanese isolate had high similarity with those of the L. intracellularis type strain, suggesting the genetically close relationship of the two strains. This is the first report describing the cultivation and partial characterization of L. intracellularis originated in Japan.     

Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen

Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic purposes (by immunohistochemistry) and for bacterial characterization. Several antibody producing hybridomas were established by fusion of mouse myeloma with spleen cells from BALB/c mice immunized with mucosa scrapings of the intestinal mucosa from a L. intracellularis infected pig. A monoclonal antibody (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT). Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This antigen was released from L. intracellularis by mild heat treatment and was resistant to proteinase K digestion, suggesting it to be non-protein, e.g., lipopolysaccharide (LPS). This suggestion was supported by its presence in the aqueous phase of a phenol-water extract. The inhibitory effect of periodate oxidation on the antigen-antibody binding confirmed the participation of a carbohydrate epitope. The new mAb was tested highly specific for L. intracellularis by applying in situ hybridization with a L. intracellularis specific probe targeting 16S ribosomal RNA simultaneously with the IFAT.     

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.     

Lawsonia intracellularis proliferative enteropathy in a filly

Proliferative enteropathy (PE) caused by the obligate intracellular bacterium Lawsonia intracellularis is a disease of high economic impact in swine worldwide. In most other species the disease occurs as a sporadic infection. This paper reports a PE caused by L. intracellularis in a 9-month-old Pura Raza Espa?ola filly with a history of profuse diarrhoea. Pathological lesions consisted of a severe proliferative enteritis associated with argyrophilic bacteria in the apical cytoplasm of proliferating crypt epithelium. Characteristic PCR products confirmed the presumptive diagnosis of L. intracellularis infection. To our knowledge this is the first report of PE in a horse in Europe caused by L. intracellularis.     

胞内劳森菌人工感染猪的病理学观察

将猪源胞内劳森菌GXNN株在兔体传代,用感染兔肠道黏膜及内容物攻毒试验猪,成功复制出猪增生性肠炎,病猪表现为生长缓慢,腹泻,攻毒后12 d~17 d体温升高,肠系膜淋巴结肿大,回肠黏膜皱褶增厚等;病理组织学观察可见回肠有丰富的集合淋巴小结,回肠腺窝杯状细胞消失,部分腺窝上皮细胞增生,淋巴结内的淋巴小结数目增多。     

Characterization of the interferon gamma response to Lawsonia intracellularis using an equine proliferative enteropathy challenge (EPE) model

Lawsonia intracellularis is the etiological agent of infectious intestinal hyperplasia for which several clinical diseases have been described including proliferative enteropathy (PE), intestinal adenomatosis, and ileitis. While initially recognized as the causative agent of PE in pigs, L. intracellularis is now viewed as an emerging cause of intestinal hyperplasia in a wide range of mammalian species, including horses. Equine proliferative enteropathy (EPE) has been reported worldwide though definitive diagnosis is difficult and the epidemiology of the disease remains poorly understood. Weanlings, in particular, appear to be most at risk for infection, though the reasons for their particular susceptibility is unknown. Using an infectious challenge model for EPE, we demonstrate that EPE, like porcine proliferative enteropathy, can exhibit three clinical forms: classical, subclinical and acute. Out of six pony weanlings, one developed signs of classic EPE, one developed acute EPE, and two developed subclinical EPE. Attempts to induce pharmacological stress through the use of dexamethasone failed to have any effect on outcome. Peripheral blood cells collected from those weanlings that developed clinical EPE exhibited decreased expression of interferon-gamma (IFN-γ) following in vitro stimulation with L. intracellularis. By contrast, those weanlings that did not develop clinical disease generated a robust IFN-γ response. These results indicate IFN-γ likely plays a significant role in protection from disease caused by L. intracellularis in the equid.     

Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces

The course of naturally acquired Lawsonia intracellularis infection was studied in 41 pigs by testing blood and faeces samples collected four to seven times from before weaning to slaughter 5 months old. At slaughter, a sample of ileum was taken for histopathology. In the first sampling when the pigs were 2-4 weeks old maternally derived IgG against L. intracellularis was demonstrated by immunofluorescence antibody test in nine pigs whereas the bacterium was detected by PCR in faeces from six pigs. The maternally derived antibodies did not prevent pigs from becoming infected as seven pigs later on shed and/or were seropositive for L. intracellularis. The lowest prevalence of L. intracellularis was observed in 6-13 weeks old pigs and it seemed as though L. intracellularis in early infected pigs only activates a minor antibody response. At slaughter 66% of the pigs were found positive by immunofluorescence antibody test compared to 24% by immunohistochemistry on ileal samples. Thus, applied at the time of slaughter the antibody test appeared to be a highly sensitive ante-mortem diagnostic tool for identifying L. intracellularis exposed pigs with or without current proliferative enteropathy.     

Immunohistochemical detection of Lawsonia intracellularis in tissue sections from pigs

The aim of the present study was to develop an immunohistochemical method (IHC) for detection of Lawsonia intracellularis (L. intracellularis) in formalin-fixed, paraffin embedded sections of intestines from pigs and to implement this method in differential diagnosis of swine diseases with diarrhea in postweaning pigs. The study was conducted on 165 sections of intestines (ileum, caecum and colon) collected from 76 pigs, representing 42 Polish pig farms. The animals included in the analysis suffered from diarrhea, with bloody or grey to brown feces, and were suspected of porcine proliferative enteropathy (PPE). Sections of intestines were analyzed for the presence of L. intracellularis by polymerase chain reaction (PCR) and IHC. Among 165 intestinal samples from pigs with diarrhea, L. intracellularis DNA was detected by PCR in 33 (20.0%) samples. In this group, 30 samples (18.2% of all the samples tested) were also found positive in IHC, while only 3 (1.8%) were IHC-negative. One hundred thirty-two (80.0%) samples were negative in both tests. The PCR- and IHC-positive samples originated from 11 pigs, 4- to 20-week old, from 8 farms. L. intracellularis antigen was visualized by IHC mostly in intestinal crypts and/or in mononuclear cells of the lamina propria). The positive signal in epithelial cells was observed close to the luminal borders, creating typical specifically stained rims around the crypt lumina. The results of the present study further confirm the usefulness of IHC in the detection of L. intracellularis antigen in the intestinal tissues.     

The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces

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.     

Lawsonia intracellularis infection in horses: 2005-2007

Background: Lawsonia intracellularis is an emerging equine pathogen that is a cause of equine proliferative enteropathy (EPE).
Objective: To describe the signalment, month of presentation, common clinical signs, clinicopathologic values, diagnostic tests used, antimicrobial use, and survival status in horses affected with EPE; to evaluate how affected horses sold at public auction as yearlings; and to determine results of fecal polymerase chain reaction (PCR) and serum immunoperoxidase monolayer assay (IPMA) results in age matched, clinically normal herdmates.
Animals: The study group was 57 horses treated for disease associated with L. intracellularis infection between August 2005 and January 2007.
Methods: Retrospective study examined horses exhibiting evidence of infection with L. intracellularis and testing positive for fecal PCR or serum IPMA.
Results: Horses ranged in age from 2 to 8 months with a median age of 6 months, and all were examined between August and January. Ventral edema was present in 81% of horses and hypoalbuminemia occurred in all horses. Only 50% of horses tested positive on both PCR and IPMA. Ninety-three percent of horses survived, and survival was unrelated to antimicrobial administered. Affected horses sold as yearlings an average of 68% less than other yearlings by the same sire. Age matched, clinically normal herdmates also tested positive for L. intracellularis on fecal PCR (6%) and IPMA (33%).
Conclusion: L. intracellularis infection should be considered in young horses with ventral edema and hypoalbuminemia that are examined between August and January. Both fecal PCR and serum IPMA are needed to help determine disease status. Treated animals usually survive, although they do not sell for as high a price at public auction as other yearlings by the same sire. Age matched, clinically normal herdmates also test positive for L. intracellularis on fecal PCR and serum IPMA.     

Lawsonia intracellularis proliferative enteropathy in a foal

A weanling foal was diagnosed with proliferative enteropathy caused by Lawsonia intracellularis based on history, clinical findings of depression, anorexia, weight loss, colic, diarrhea, and ventral edema, and a combination of serology and fecal PCR. An epidemiological investigation on the premises revealed that many of the other foals and adult horses were seropositive for L. intracellularis, despite being clinically normal, and identified a dog as a potential carrier and source of infection for the foal.The foal was successfully treated with a combination of azithromycin and rifampin.     

Prevalence of Lawsonia intracellularis, Salmonella spp. and Eimeria spp. in healthy and diarrheic pet rabbits

A total of 170 fresh fecal samples (healthy; n=137, diarrheic; n=33) were collected from pet rabbits. By using PCR and formol-ether concentration method, a total 13/137 healthy rabbit feces were positive for L. intracellularis, 6/137 for Salmonella, and 13/137 for Eimeria. On the other hand, a total 17/33 diarrheic rabbit fecal samples were positive for L. intracellularis, 10/33 for Salmonella, and 21/33 for Eimeria. From these results, more than 20% of clinically normal and 97% of diarrheic rabbits were positive for single or concurrent infection of three pathogens. To the best of our knowledge, this is the first report to describe the prevalence of the microorganisms L. intracellularis, Salmonella and Eimeria in pet rabbits.     

Comparison of intestinal mucosa homogenate and pure culture of the homologous Lawsonia intracellularis isolate in reproducing proliferative enteropathy in swine

Little information is available on reproduction of proliferative enteropathy (PE) using a virulent pure culture of Lawsonia intracellularis. Reproduction of the disease using PE-diseased mucosa homogenates, however, is well-characterized. The aims of this study were to evaluate and compare clinical signs, growth performance and the severity of lesions in pigs inoculated with intestinal mucosa homogenate or pure culture of the homologous L. intracellularis isolate. Five-week-old pigs were inoculated with pure culture of L. intracellularis (isolate PHE/MN1-00; n=10), PE-diseased mucosa (n=10), or control media (n=4). The L. intracellularis isolate PHE/MN1-00 used in the pure culture inoculum was extracted from a fragment of the same intestine used to prepare the mucosa homogenate. Clinical signs and growth performance were evaluated throughout the study. Fecal shedding was evaluated in all animals weekly during the experiment. All animals were euthanized 22 days post-inoculation, the intestines were examined grossly and histologically. Results showed that both the infection procedures reproduced clinical disease, macroscopic and histologic lesions typical of PE. Fecal shedding was detected in animals in both challenge groups. In conclusion, the L. intracellularis isolate PHE/MN1-00 reproduces typical clinical signs and lesions of PE similar to the homologous infection with an intestinal mucosa homogenate.     

Reproductive performance of gilts following an outbreak of acute proliferative enteropathy due to Lawsonia intracellularis

After a sudden outbreak of clinical porcine proliferative enteropathy (PPE) in a Croatian indoor breeding unit, the farm experienced decreased reproductive performance of Lawsonia intracellularis positive gilts. Conception-, farrowing-, and adjusted farrowing rates were lower (P<0.001) in gilts with positive L. intracellularis status. The number of live born and the total born litter size were significantly lower (P<0.001) in L. intracellularis positive gilts compared to seronegative ones. No differences were observed in numbers of stillborn and mummified pigs between the L. intracellularis positive and negative gilts.