Reprinted in the IVIS website with the permission of the meeting organizers

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1 Reprinted in the IVIS website with the permission of the meeting organizers

2 LAWSONIA INTRACELLULARIS INFECTIONS Connie Gebhart College of Veterinary Medicine, University of Minnesota St. Paul Minnesota 55108, U.S.A. Introduction Porcine proliferative enteropathy (PE), also known as ileitis, is a common infectious disease affecting weaned animals of various ages and species. The disease occurs worldwide and is of special economic importance in the swine industry as it causes diarrhea, stunted growth, and occasionally, sudden death (1). PE in pigs occurs as several different syndromes and all forms of PE share unique histological features, including the proliferation of the immature epithelial cells of the intestinal crypts, causing a thickening of the mucosa of the small and, sometimes, large intestine (2). Within these proliferating enterocytes are many intracellular, curved bacteria, identified as Lawsonia intracellularis, the etiology of PE. Identification and characterization of L. intracellularis as the etiology of PE has facilitated development of specific assays for diagnosis of PE and detection of the immune response in affected pigs. Further understanding of the pathogenesis and epidemiology of PE has and will continue to lead to advances in methods for control and prevention of the disease. Characteristics of proliferative enteropathy There are several different manifestations of PE in pigs, including the chronic form, porcine intestinal adenomatosis (PIA), and an acute form, proliferative hemorrhagic enteropathy (PHE) (2). A subclinical form of PE is more recently recognized in which L. intracellularis infections occur with no apparent clinical signs of the disease (3,4). PIA is generally seen in young growing pigs from 6 to 20 weeks of age, resulting in diarrhea and poor growth rate which persists for weeks. The most severely affected pigs may develop a severe thickening of the mucosa with necrotic lesions on the luminal surface. The PHE form tends to affect pigs from 4 to 12 months of age and results in bloody diarrhea with blood clots in the lumen of the intestine, and sudden death. Subclinical PE appears to be common in growing pigs, but is difficult to identify because there are no detectable clinical signs, just reduced performance parameters. All forms of PE in pigs and other affected animal species have the same unique histological features - proliferation of intestinal epithelial cells containing intracellular L. intracellularis bacteria (1). PE has been reported by various names in a number of other animal species, including hamsters, ferrets, rabbits, foxes, dogs, rats, horses, sheep, deer, emus, ostriches, primates, guinea pigs, and experimental mice. The disease is best described in pigs (2) and hamsters (4), but is of increasing importance in the horse industry (5). Characteristics of L. intracellularis L. intracellularis was identified recently as a new genus and species (7) and was formally named in 1995 in honor of Dr. G.H.K. Lawson, who was the discovering pioneer of the bacterium (8). Molecular taxonomic methods show that L. intracellularis is similar to the sulfate reducing bacteria, specifically, Desulfovibrio desulfuricans, and taxonomically distinct from other intracellular pathogens. The Lawsonia genus differs phenotypically from sulfate-reducers in that it is obligately intracellular, does not reduce sulfate, and is pathogenic. L. intracellularis is a small, Gram-negative, curvedshaped rod, which displays neither fimbriae nor spores. A long, single, unipolar flagellum has been observed by electron microscopy in various cell culture-grown isolates. Isolates demonstrate a darting motility in vitro upon escape from infected enterocytes. L. intracellularis divides transversely by septation and organisms are located free within the apical cytoplasm of infected enterocytes (1). Cultivation of L. intracellularis Strict environmental conditions are required for cultivation of L. intracellularis in vitro. Cell-culture systems are required to isolate and propagate the bacterium due to its intracellular location in vivo. Growth of L. intracellularis in vitro requires dividing eukaryotic cells, a reduced oxygen atmosphere, and a temperature of 37ºC (9). Growth occurs in either adherent or suspension tissue culture cells. Cell cultures are monitored daily by staining aliquots with Lawsoniaspecific stains. Cultures are then harvested or passed when the cell culture infection reaches 80 to 100 %, usually 5 to 7 days post-inoculation. Pathogenesis of L. intracellularis Comprehensive studies of lesion development and evolution have been conducted in hamsters and pigs. Morphological studies of early lesions in experimentally infected animals indicate that enterocyte hyperplasia is directly preceded by 48

3 the presence of the intracellular organism. In vivo, the onset of hyperplasia associated with PE follows an increase in numbers of intracellular L. intracellularis in enterocytes. Likewise, resolution of lesions is closely related to disappearance of the intracellular organisms, indicating a correlation between the two events (1). The means by which L. intracellularis produces hyperplasia is unknown. Few other cytopathologic effects on infected enterocytes are seen in vivo or in vitro. Inflammation is a factor only in later-stage lesions and is not characteristic of the primary lesion. Most studies on the pathogenesis of L. intracellularis have been conducted in vivo since the cellular proliferation that is characteristic of PE has not been reproduced in vitro. Reproduction of PE can be accomplished with either a pure culture or intestinal mucosal homogenate from previously infected pigs. The mucosal homogenate challenge has been successful in efficiently producing all forms of PE. A downside to this model is that it contains other microflora and, potentially, pathogens that may confound the challenge results. Also, mucosal homogenate represents a more severe challenge than occurs in typical field outbreaks of PE. A titrated mucosal homogenate model (10) showed that clinical disease severity was dose dependent and lower doses can be used to reproduce field-like clinical symptoms of PE. The pure culture model for PE provides a more quantifiable and defined reproduction of the disease. Unfortunately, this model requires isolation, cultivation, and virulence maintenance of L. intracellularis in vitro. A direct comparison of both challenge models (11) demonstrated that reproduction of clinical signs and lesions typical of PE was similar. In both experimental models, the incubation period is 7 to 14 days with early lesions appearing in the terminal ileum. Fecal shedding begins about 7 days post-challenge and animals seroconvert about 14 days post-challenge. The disease peak is about 21 days after infection. Clinical signs decrease and proliferative lesions begin to resolve after 28 days of infection, resulting in a 2-week delay in obtaining market weight. The greatest loss probably occurs due to increased numbers of pigs that fail entirely to reach market weight in the allotted time. Examination of the genesis of lesions during experimentally produced PE in pigs revealed L. intracellularis antigen in the intestine 5 days after inoculation. Microscopic lesions, consisting of enterocytes hyperplasia and reduced numbers of goblet cells, were observed 11 days after inoculation, and initial macroscopic lesions were detected 12 days after inoculation. Positive staining, but no gross or microscopic lesions, was detected on day 29, and pigs euthanized on day 35 post inoculation had no lesions and were negative by immunohistochemical staining. L. intracellularis antigen was not detected in any organ other than intestine, lymph node, and tonsil. Infection of enterocytes in the large intestine and rectum occurs late in the course of the disease and, consequently, the infection can be detected later in these locations. It appears that the small intestine is infected first, and the bacteria shed from those sites infect lower levels of the intestine (12). Diagnosis of proliferative enteropathy Clinical signs: Clinically, PE is difficult to diagnose because the signs are non-specific or even lacking entirely. In addition, the various forms of PE may mimic other enteric diseases, including salmonellosis, swine dysentery, colibacillosis, colonic spirochetosis, transmissible gastroenteritis, rotavirus infection, hemorrhagic bowel syndrome and porcine circovirus 2 infection. Furthermore, many of the above pathogens commonly occur concurrently with L. intracellularis and so confirmation of the presence of another agent does not rule out a PE diagnosis. One hallmark of PE is dramatic weight variation among pigs of the same age. Chronic PE is characterized by poor growth, uneven weight gain and a delay to market. Overall poor performance, gauntness or soft-to-watery stools may occur. Transient diarrhea often occurs, but is not always present. In acute PE, affected pigs may be pale and their feces black or bloody. An occasional pig may develop intestinal hemorrhage and die suddenly, followed by sporadic occurrences of pigs with bloody diarrhea (2). Postmortem diagnosis: Conventionally, PE has been diagnosed postmortem based on gross lesions seen at necropsy and microscopic tissue examination. Severe PE lesions are easily seen, however the more common moderate to mild lesions may be hard to detect. Gross lesions vary depending on the clinical manifestation of PE and may appear as hemorrhagic or chronic. If noted, they are usually seen in the ileum near the ileal-cecal junction and appear as a mucosal thickening. More chronic disease results in necrotic enteritis or a thickening of the outer muscular layer. In PHE, there is a large amount of undigested blood in the small intestinal lumen. A histological diagnosis of PE can be made by demonstrating the presence of proliferative enterocytes on routine H&E staining, but evaluating proliferation may be subjective; only cases with the more severe enterocyte proliferation can be diagnosed. Staining of histological sections using a silver stain reveals numerous intracellular organisms with a characteristic curved shape, usually in the apical cytoplasm of the crypt epithelial cells. However, this method is not specific for L. intracellularis and cannot always detect the organism in necrotic debris or autolyzed tissue. A more specific identification of L. intracellularis is achieved by immunohistochemistry staining of fixed tissues, which is more sensitive than the silver stain because it shows organisms within mononuclear cells in the lamina propria during recovery from PE. In addition, extracellular L. intracellularis can be identified in exudate or necrotic debris in superficial mucosa. In a comparative diagnostic study, immunohistochemistry staining detected nearly twice as many pigs with PE lesions compared to silver staining of formalin-fixed tissues (13). If immunohistochemistry is not 49

4 available, specific identification of L. intracellularis in the intestine can be achieved by PCR of the ileal mucosa. This technique is readily available in most diagnostic laboratories and, when applied to ileal mucosa rather than feces, is as specific as immunohistochemistry. Ante mortem diagnosis: More sensitive and specific diagnostic measures have become available for diagnosis of PE in the live pig, thus enabling studies of prevalence of the disease in pig herds. Culture from feces is not an option because the agent is an obligate intracellular bacterium and cultivation of L. intracellularis in intestinal cell lines is difficult. Other methods of ante mortem diagnosis include detection of L. intracellularis in feces by PCR or indirect antibody staining and serological assays for L. intracellularis antibodies using either an indirect fluorescent antibody (IFA) test, an immunoperoxidase monolayer assay (IPMA), or one of several ELISA tests recently developed (4). Several PCR assays have been developed that can detect L. intracellularis in feces. The sensitivity and specificity of PCR in fecal samples have been evaluated in many reports, which show variable sensitivity and consistently high specificity (about 97%). Sensitivity is affected by sample quality and the presence of inhibitory factors in feces. PCR appears to reliably demonstrate L. intracellularis in the feces of clinically affected pigs, but is not sensitive enough to routinely detect the organism in feces from subclinically affected animals. Recently developed real-time PCR methods offer the advantage over conventional PCR of quantitation of Lawsonia in samples as well as high throughput identification of positive samples. Demonstration of L. intracellularis in feces or mucosal homogenates can also be done with a specific antibody in an indirect antibody staining technique or an immunological method using immunomagnetic separation and ATP bioluminescence (14). However, expertise is required to evaluate the results and the availability of such techniques is limited by the need for a Lawsonia-specific antibody. Also, like PCR, these techniques may lack sensitivity for diagnosing subclinically affected animals. Current serological assays based on the detection of L. intracellularis antibodies in serum employ pure cultures of L. intracellularis, either used as a whole cell antigen on slides or plates or as a soluble antigen. Staining of bacteria is either by a fluorescent (IFA) or peroxidase-labeled (IPMA) secondary antibody. Recently, several groups have reported ELISA-based methods for detecting humoral IgG to L. intracellularis. These ELISA s have the advantage of high throughput sample testing, automated reporting of results and less chance of bias in sample evaluation. They include an indirect LPS-ELISA, a sandwich ELISA with LPS as the antigen, a blocking ELISA, and a sonicated whole cell ELISA. Agreements between tests vary and, unfortunately, all require cultivation of Lawsonia in vitro for antigen production. Two ELISA tests which utilize subunits of Lawsonia (15, 16) have been reported, but are not yet routinely used for pigs. Serologic assays have proven useful for routine diagnosis and determination of prevalence of PE in pig herds worldwide, although the humoral immune responses of pigs with the PIA and, especially, the subclinical forms of PE are often weak and short-lived. Furthermore, serology provides only historical information on exposure of the animal to L. intracellularis. Its use is highly suited to cross-sectional and serial profiling studies of humoral immune response for implementation of control and prevention measures in herds in which PE is endemic. Seroprofiling results will vary with any change in the immune status of the breeding herd or change in in-feed antimicrobial medication programs in a group of pigs. Immune response Challenge trials: Much of the information we have on the humoral immune response of pigs has been obtained through challenge trials using either mucosal homogenates from pigs affected with PE or virulent L. intracellularis grown in cell culture. In these trials, weaned pigs were challenged orally at about 5 weeks of age and were evaluated at weekly time points until necropsy. IgG titers of 1:30 to L. intracellularis appear in these settings 2 weeks after challenge. Up to 90% of pigs became seropositive 3 weeks after challenge, with a few showing titers of 1:480 or greater. At 4 weeks post-challenge, titers began to decay and a decreased percentage of the pigs were positive, though in one study serum IgG response was detectable up to 13 weeks post-challenge (12). Natural PIA outbreaks: Seroconversion in growing pigs during a natural field outbreak of PIA in the U.S. showed a slightly different pattern. Some nursing piglets showed L. intracellularis IgG titers of 1:30, which decayed within 3 to 4 weeks. These maternally derived antibodies may be important in protection against early infections in weaned piglets. In grow-finishers, titers of only 1:30 to 1:60 were detected beginning at about 12 weeks of age. The percent of pigs affected peaked around 24 weeks of age and then declined. For individual pigs, titers decayed within 3 to 4 weeks. Mild or no clinical signs of PE were noted in many of these outbreaks. Natural PHE outbreak: In contrast, high humoral IgG titers specific for L. intracellularis were obtained from pigs affected with PHE in a natural outbreak. Affected animals showed IgG titers as high as 1:1920. Titers decayed by about half every 3 weeks after peak and eventually were undetectable. Some gilts that had survived PHE had detectable 50

5 IgG titers to L. intracellularis at farrowing, suggesting that sows may confer passive immunity to piglets. Effectiveness and length of protection provided by passive immunity has not yet been established (12). Mucosal immunity: Local humoral immunity, in the form of IgA, is an important defense mechanism against enteric pathogens. Immunohistochemical studies of intestinal sections of PE-affected pigs demonstrated a large accumulation of IgA in the apical cytoplasm of proliferating enterocytes, however this response has not been shown to be specific to L. intracellularis. In a study conducted to observe the progression of infection through the course of the disease, IgA titers of 1:4 were found in intestinal gavages of infected pigs by day 15 post-challenge. Titers were detected up to 29 days post-challenge, with titers ranging from 1:4 to 1:16 in affected intestinal washings (12). Cell mediated immune response: The cell-mediated immune response is often an important feature of infections caused by intracellular organisms. A recent assay demonstrated production of specific interferon-gamma in the leukocytes of pigs experimentally challenged with L. intracellularis. Results of this assay parallel those of the humoral response in that specific interferon-gamma production begins about 2 weeks after challenge, peaks at 3 weeks and then begins to decay, though not as rapidly as the humoral response decays. Cell-mediated immune response was still detectable in some pigs 13 weeks post-exposure (10). Similarly, interferon-gamma played a role in limiting intracellular infection and increased cellular proliferation in experimentally infected mice (17). Therefore, L. intracellularis infection may stimulate interferon-gamma secreting lymphocytes involved in the natural clearance of infection. Delayed-type hypersensitivity: Experimentally challenged pigs also elicit a delayed-type hypersensitivity reaction (DTH), which was evaluated after intradermal injections of different concentrations of L. intracellularis antigen in pigs 20 days post-challenge infection. Challenged animals showed a dose-dependent DTH reaction that was more evident 24 hours after injection. Further studies will define other factors involved in immune response and its correlation to protective immunity in infected pigs (12). Epidemiology of proliferative enteropathy General considerations: Little is known about the epidemiology of L. intracellularis because of the lack of strain differentiation techniques. Isolation and cultivation of L. intracellularis is extremely difficult due to the obligate intracellular nature of this organism, making the identification of L. intracellularis subtypes by traditional methods unfeasible. Feces from infected pigs may provide the source of new infections for susceptible animals and pig-to-pig contact has been shown to be an important route of transmission of PE. Other possible mechanisms of transmission of L. intracellularis include mechanical and biological vectors. Information about survival and resistance of L. intracellularis in the environment is scarce. However, a unique investigation into this area showed that intestinal colonization of pigs by L. intracellularis was detected after the pigs had been orally inoculated with affected feces that had been stored for up to 2 weeks at 5 o C to 15 o C (18). Molecular epidemiology of L. intracellularis: Isolates of intracellular bacteria in PE lesions of a variety of animal species show >98% 16S-rDNA similarity to pig L. intracellularis isolates. Phenotypic characterization of outer membrane proteins and immunoblots of different L. intracellularis isolates using several antibodies demonstrate only minor differences among isolates. Standard subtyping methods have not proven to provide an acceptable level of discrimination for Lawsonia isolates. In contrast, genomic segments containing variable-number tandem repeats (VNTRs) have shown great variation and, hence, potential for demonstration of variation in Lawsonia isolates. Furthermore, PCR-based VNTR analysis does not require cultivation of L. intracellularis isolates for testing or molecular manipulation techniques beyond PCR. We have recently demonstrated the utility of multiple locus VNTR profiles of L. intracellularis as markers for differentiating isolates obtained from different animal species, geographical locations, and field outbreaks of PE. The results show that the L. intracellularis isolates in fecal samples from epidemiologically unrelated PE outbreaks had unique VNTR profiles. These data are consistent with the hypothesis that there are well-defined genetic differences at the VNTR loci in L. intracellularis isolates recovered from clinical sources. In contrast, L. intracellularis isolates from the same outbreak shared identical VNTR profiles, a finding consistent with the notion that VNTR profiles are stable over short time intervals. VNTR typing is likely to be of considerable utility in L. intracellularis isolate differentiation and epidemiological analyses of L. intracellularis outbreaks (19). Prevention and control of proliferative enteropathy Antimicrobial agents: PE is a widespread (96% of U.S. herds reported to be seropositive) and complex disease and its control is affected by the various management practices in use. Challenge exposure and controlled field evaluations of treatment and prevention measures in pigs have indicated that macrolides, tetracyclines, quinoxalines, lincosamides and pleuromutilins are the most effective antibiotics, when given at an adequate dosage rate per kg of bodyweight. Apparent medication failures with these drugs are most likely to occur in pigs with PE that are under dosed on a bodyweight basis or when pigs are medicated before the disease is present or after actual peaks of infection. Antimicrobial drugs now 51

6 known to be ineffective against L. intracellularis in clinical cases of PE include the aminoglycosides, bacitracins, and aminocyclitols. Various approaches to medication are possible, depending on the age and management style of pigs involved (2). Treatment of PHE outbreaks in herds requires a vigorous approach. In the U.S., the most common treatments include tylosin, tiamulin, lincomycin, chlortetracycline, and carbadox delivered orally in feed. Injectable antibiotics (such as tylosin and lincomycin) and in-water medications (such as tylosin, tiamulin and lincomycin) are preferred. For endemic PIA in growing pigs, common treatments include tylosin, tiamulin, lincomycin, chlortetracycline, carbadox and aivlosin in the feed. Severe chronic clinical disease manifested as wasting pigs with or without necrotic enteritis will require the use of in-water or injectable formulations. Controlled field trials suggest that incorporation of in-feed or water-soluble antibiotics for control achieves best results if given just prior to the peak period of L. intracellularis infection. Because infection and PE disease can vary in the time of onset on different farms and between groups on the same farm, in-feed antibiotics for treatment might be added too late to stop clinical signs and poor performance. Alternatively, if antibiotics are added too early, some pigs may not get the chance to develop active immunity to the disease and may remain naïve and susceptible to later severe acute PE (20). Vaccines: Development of protective immunity to Lawsonia has been demonstrated in pigs during controlled challenge trials and observed in sequential natural disease outbreaks, suggesting that a modified live vaccine may induce protective immunity. At the present time, there is one commercially available vaccine licensed for L. intracellularis (21). This is a modified live oral vaccine that has the ability to induce both humoral and cellular immune responses. Evaluations of the modified live vaccine in standard challenge models show that it effectively protects animals that are challenged with either homologous or heterologous Lawsonia isolates. These studies have shown reduction of productivity losses and clinical shedding, as well as reduced lesion development, in challenged pigs. This vaccine has rapidly grown to widespread usage where available, with no safety concerns identified. Inactivated, subunit, or other vaccine types are not yet available. Passive protection: Hyper-immunized chicken egg antibodies incorporated into swine diets have been used alone or in conjunction with antibiotics to control some enteric infections. Initially, a PE hamster challenge model was used to test the efficacy of Lawsonia-specific egg antibodies. There was a significant improvement in performance in animals given L. intracellularis egg antibodies versus no antibodies. In a subsequent pig study, animals that received 2kg of chicken egg anti-lawsonia antibodies had significant increases in performance parameters such as average daily gain and average daily feed intake compared to pigs that received the non-lawsonia egg product. No significant difference was detected between treatment groups regarding the amount of clinical signs, fecal shedding, and intestinal lesions characteristic of PE after the L. intracellularis challenge. Therefore, this challenge model demonstrated that feeding Lawsonia-specific egg antibodies can reduce production loss associated with PE (22). Summary All forms of PE in pigs as well as other animal species are caused by the obligately intracellular L. intracellularis. Lawsonia is a unique bacterium, which causes an unusual pathology in infected animals, including proliferation of the infected mucosal epithelial cells of the intestine. Advances in propagation of this organism in cell culture have allowed further studies on the pathogenesis of PE. Further improvement in diagnostics for PE has allowed more accurate determination of onset and prevalence of PE in pigs. Information from studies focusing on the humoral and, in particular, mucosal and cell-mediated immune responses against L. intracellularis infections may be used to evaluate the effectiveness of prevention and control strategies. Molecular methods for tracing specific isolates are now in place and will allow more rapid identification of the source and transmission patterns through epidemiological investigations. Newer methods of prevention and control are being evaluated that will be made available worldwide for eliminating production costs due to PE. References 1. Lawson, G.H.K., Gebhart, C. J. (2000) Proliferative enteropathy. Journal of Comparative Pathology 122, McOrist, S., Gebhart, C. J. (1999) Porcine proliferative enteropathy. Straw B, Mengeling W, D Allaire S and Taylor D (eds) Diseases of Swine, 8 th eds. Ames, IA, USA: Iowa State University Press, pp Guedes, R. M. C. (2004). Update on epidemiology and diagnosis of porcine proliferative enteropathy. Journal Swine Health and Production 12, Kroll, J.J., Roof, M.B., Hoffman, L.J., Dickson, J.S., Harris, D.L. (2005) Proliferative enteropathy: a global enteric disease of pigs caused by Lawsonia intracellularis. Animal Health Res. Rev. 6, Jacoby, R. O. (1978) Transmissible ileal hyperplasia of hamsters. American Journal of Pathology 91, Al-Ghamdi, G. M. (2003) Characterization of proliferative enteropathy in horses. PhD Thesis. University of Minnesota, St. Paul, MN 52

7 7. Gebhart, C. J., Barns, S., McOrist, S., Lin, G., Lawson, G.H. K. (1993) Ileal symbiont intracellularis, an obligate intracellular bacterium of porcine intestines showing a relationship to Desulfovibrio species. Int. J. System. Bacteriol. 43, McOrist, S., Gebhart, C. J., Boid, R. Barns, S. (1995) Characterization of Lawsonia intracellularis gen. nov., sp.nov., the obligately intracellular bacterium of porcine proliferative enteropathy. Int. J. Syst. Bacteriol. 45, Lawson, G., McOrist, S., Jansi, S., Mackie, R. (1993) Intracellular bacteria of porcine proliferative enteropathy: cultivation and maintenance in vitro. Journal of Clinical Microbiology 31, Guedes, R. M. C., Gebhart, C. J. (2003) Onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or an attended vaccine strain of Lawsonia intracellularis. Veterinary Microbiology 91, Guedes, R. M. C., Gebhart, C. J. (2003) Comparison of the intestinal mucosa homogenate and pure culture of the homologous Lawsonia intracellularis isolate in reproducing proliferative enteropathy in swine. Veterinary Microbiology 93, Guedes, R. M. C. (2002) Porcine proliferative enteropathy: diagnosis, immune response, and pathogenesis. PhD Thesis, University of Minnesota, St. Paul, MN 13. Guedes, R.M.C., Gebhart, C.J., Winkelman, N.L., Mackie-Nuss, R., Marsteller, T.A., Deen, J. (2002) Comparison of different methods for diagnosis of porcine proliferative enteropathy. Can. J. Vet. Res. 66, Watarai, M., Yamato, Y., Murakata, K., Kim, S., Omata, Y., Furuoka, H. (2005) Detection of Lawsonia intracellularis using immunomagnetic beads and ATP bioluminescence. Journal of Veterinary Medical Science 67, Watarai, M., Yamato, Y., Horiuchi, N., Kim, S., Omata, Y., Shirahata, T., Furuoka, H. (2004) Enzyme-linked immunosorbent assay to detect Lawsonia intracellularis in rabbits with proliferative enteropathy. Veterinary Medicine and Science 66, Wattanaphansak, S. (2006) Diagnosis of proliferative enteropathy. MS Thesis, University of Minnesota, St. Paul, MN 17. Smith, D. G. E., Lawson, G. H. K. (2001) Lawsonia intracellularis: Getting inside the pathogenesis of proliferative enteropathy. Veterinary Microbiology 82, Collins, A., Love, R., Pozo, J., Smith, S., McOrist, S. (2000) Studies on ex vivo survival of Lawsonia intracellularis. Swine Health and Production 8, Beckler, D. C., Kapur, V., Gebhart, C. J., (2004) Molecular epidemiologic typing of Lawsonia intracellularis. Conference for Research Workers in Animal Diseases, pp McOrist, S. (2005) Defining the full costs of endemic porcine proliferative enteropathy. The Veterinary Journal 170, Kroll, J. J., Roof, M. B., McOrist, S. (2004) Evaluation of protective immunity in pigs following oral administration of an avirulent live vaccine of Lawsonia intracellularis. American Journal of Veterinary Research 65, Winkelman, N. L., Kinsley, K., Gebhart, C. J., Scherbring, E. (2004) Effectiveness of Lawsonia intracellularis specific chicken egg antibody to control ileitis in a swine disease challenge model. Proceedings of the 18 th International Pig Veterinary Society Congress, Hamburg, Germany, pp