Lawsonia Intracellularis Infection Research Articles

Serial use of serologic assays and fecal PCR assays to aid in identification of subclinical Lawsonia intracellularis infection for targeted treatment of Thoroughbred foals and weanlings

To assess the serial use of serum immunoperoxidase monolayer assays (IPMAs) and fecal PCR assays, combined with other diagnostic methods, to identify subclinical Lawsonia intracellularis infections for targeted treatment of Thoroughbred foals and weanlings at farms in which the pathogen was endemic or nonendemic. Evaluation study. 100 foals and weanlings (53 and 47 at farms in which L intracellularis was endemic and nonendemic, respectively). Serum was collected every 4 weeks and tested via IPMA, for antibodies against L intracellularis. Fecal samples were collected every 2 weeks and tested by use of an L intracellularis-specific PCR assay. When results for IPMAs or PCR assays were positive or clinical signs compatible with equine proliferative enteropathy (EPE) were detected, clinicopathologic testing was performed to determine treatment. No foals had positive results for the L intracellularis-specific IPMA until after weaning; 32 of 53 (60.4%) weanlings at the farm in which L intracellularis was endemic and 8 of 47 (170%) at the farm in which L intracellularis was nonendemic had positive IPMA results, whereas the number of weanlings that tested positive via fecal PCR assays at those farms was 6 and 0, respectively. Nineteen of 32 weanlings with positive IPMA results at the farm in which L intracellularis was endemic were treated for EPE; 5 of these had clinical signs of EPE. No weanlings at the nonendemic farm had clinical signs of or were treated for EPE. IPMA appeared to be a useful means of identifying weanlings exposed to L intracellularis.

Evaluation of Lawsonia intracellularis infection in a group of pigs in a subclinically affected herd from weaning to slaughter

The purpose of this study was to follow the course of a subclinical Lawsonia ( L.) intracellularis infection in a group of 60 pigs on a commercial farm from weaning to slaughter. From 6 to 16 and at 26 weeks of age, rectal faecal samples and blood samples were collected weekly from every pig for examination by PCR and blocking ELISA, respectively. At corresponding times starting at 8 weeks of age, pigs were randomly selected for necropsy ( n = 51). Intestinal tissues were examined histopathologically and by immunohistochemistry (IHC) for L. intracellularis antigen. Infection with L. intracellularis showed a mainly subclinical course. Shedding of L. intracellularis was detected by PCR in three pigs as early as 6 weeks of age and persisted up until 14 weeks of age. In most pigs shedding of L. intracellularis was seen only for 1–2 weeks followed by a rapid serum antibody response. More than 50% of pigs had seroconverted by week 10. At slaughter, 30.8% of investigated animals were still found to be seropositive by ELISA. Of the 60 study animals 39 were found positive by faeces PCR (65.0%), 49 animals were found positive by serology (81.7%), and 35 pigs (68.6%) had positive results by IHC at necropsy. All but one pig were found to be L. intracellularis infected by at least one of the three methods (98.3%). In conclusion, this is the first field study revealing the presence of prominent histological lesions characteristic for L. intracellularis infection and associated positive pathogen specific PCR and immunohistological results even in subclinically infected pigs. Although intestinal alterations disappeared after 3–4 weeks, L. intracellularis was detected by IHC for a longer time, especially in intestinal lymph nodes.

Application of a pig ligated intestinal loop model for early Lawsonia intracellularis infection

BackgroundPorcine proliferative enteropathy in pigs is caused by the obligate, intracellular bacterium Lawsonia intracellularis. In vitro studies have shown close bacterium-cell interaction followed by cellular uptake of the bacterium within 3 h post inoculation (PI). However, knowledge of the initial in vivo interaction between porcine intestinal epithelium and the bacterium is limited. The aims of the present study were to evaluate the usefulness of a ligated small intestinal loop model to study L. intracellularis infections and to obtain information on the very early L. intracellularis-enterocyte interactions.MethodsA ligated small intestinal loop model using three different L. intracellularis inocula was applied to 10-11-week-old pigs. The inocula were 1) wild type bacteria derived from overnight incubation of L. intracellularis bacteria from spontaneous disease, 2) crude vaccine bacteria (Enterisol® Ileitis Vet), and 3) vaccine bacteria propagated in cell culture. The bacteria-enterocyte interaction was visualised using immunohistochemistry on specimens derived 1, 3 and 6 h PI respectively.ResultsAlthough at a low level, close contact between bacteria and the enterocyte brush border including intracellular uptake of bacteria in mature enterocytes was seen at 3 and 6 h PI for the vaccine and the propagated vaccine inocula. Interaction between the wild-type bacteria and villus enterocytes was scarce and only seen at 6 h PI, where a few bacteria were found in close contact with the brush border.ConclusionsThe ligated intestinal loop model was useful with respect to maintaining an intact intestinal morphology for up to 6 h. Furthermore, the study demonstrated that L. intracellularis interacts with villus enterocytes within 3 to 6 h after inoculation into intestinal loops and that the bacterium, as shown for the vaccine bacteria, propagated as well as non-propagated, was able to invade mature enterocytes. Thus, the study demonstrates the early intestinal invasion of L. intracellularis in vivo.

Proliferative enteropathy (PE): Induced changes in galanin-like immunoreactivity in the enteric nervous system of the porcine distal colon

The aim of this study was to investigate the changes of galanin (GAL) like immonoreactivity in the porcine descending colon during proliferative entheropathy (PE). Accordingly, the distribution pattern of GAL - like immunoreactive (GAL-LI) nerve structures was studied by the immunofluorescence technique in the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexuses (ISP), as well as in the mucosal layer of the porcine descending colon under physiological conditions and during PE. In control animals GAL-LI perikarya have been shown to constitute 4.03 ± 0.1%, 6.67 ± 0.3% and 11.20 ± 0.5% in MP, OSP and ISP, respectively. PE caused changes in the GAL - like immunoreactivity, which differed in particular parts of the studied bowel segment. During PE the number of GAL-LI perikarya amounted to 2.90±0.5%, 8.42±1.0% and 21.72±1,4% within the MP, OSP and ISP, respectively. Moreover PE caused an increase in the number of GAL-LI nerve fibers in the colonic circular muscle and mucosal layers, as well as in all intramural plexuses, especially in ISP, where nearly every ganglion contained a very dense meshwork of the GAL-positive nerve fibers under the studied pathological factor. This study for the first time reports on changes in GAL-LI nerve structures of the porcine descending colon during Lawsonia intracellularis infection.

Comparative Evaluation of Diagnostic Methods for Lawsonia intracellularis Infection in Pigs, with Emphasis on Cases Lacking Characteristic Lesions

In this study the following methods for the diagnosis of Lawsonia intracellularis infection in pigs were compared in relation to a reference method (examination of ileal mucosal scrapings by the polymerase chain reaction [PCR]): Warthin-Starry (WS) staining of tissue sections, immunohistochemistry (IHC), in-situ hybridization (ISH), and PCR examination of faeces and of paraffin wax-embedded samples of ileum. Of 204 pigs examined, 32 were considered on the basis of the PCR to be infected. Gross and histopathological examination, including the use of WS staining, were of limited value. PCR examination of faeces proved to be the most sensitive (sensitivity 70%) of the methods used but, due to the occurrence of false positives, its specificity (95%) was the lowest. IHC (sensitivity 66%, specificity 99%) and ISH (sensitivity 54%, specificity 100%) were clearly superior to examination of WS-stained sections (sensitivity 34%, specificity 100%) for routine diagnosis; although less sensitive than the PCR, they indicated only cases of clinical relevance and, moreover, were capable of distinguishing different stages and levels of infection. Because examination of paraffin wax-embedded tissue by the PCR was shown to be associated with low sensitivity (41%), IHC was regarded as the method of choice for retrospective studies.

Distribution and Chemical Coding of Intramural Neurons in the Porcine Ileum During Proliferative Enteropathy

Enteric neurons are highly adaptive in their response to various pathological processes including inflammation, so the aim of this study was to describe the chemical coding of neurons in the ileal intramural ganglia in porcine proliferative enteropathy (PPE). Accordingly, juvenile Large White Polish pigs with clinically diagnosed Lawsonia intracellularis infection (PPE; n=3) and a group of uninfected controls (C; n=3) were studied. Ileal tissue from each animal was processed for dual-labelling immunofluorescence using antiserum specific for protein gene product 9.5 (PGP 9.5) in combination with antiserum to one of: vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptide Y (NPY) or galanin (GAL). In infected pigs, enteric neurons were found in ganglia located within three intramural plexuses: inner submucosal (ISP), outer submucosal (OSP) and myenteric (MP). Immunofluorescence labelling revealed increases in the number of neurons containing GAL, SOM, VIP and CGRP in pigs with PPE. Neuropeptides may therefore have an important role in the function of porcine enteric local nerve circuits under pathological conditions, when the nervous system is stressed, challenged or afflicted by disease such as PPE. However, further studies are required to determine the exact physiological relevance of the observed adaptive changes.

The microbiota of pigs influenced by diet texture and severity of Lawsonia intracellularis infection

Pigs with and without naturally occurring Lawsonia intracellularis infection were fed diets with different texture. In a previous study from 79 pig herds using a similar feeding on pelleted or non-pelleted form showed that the non-pelleted diet was associated with a reduced prevalence of L. intracellularis. In this study a mechanistic approach was taken for explaining and testing this observation by studying the microbiota and the occurrence of L. intracellularis in the distal ileum of 54 pigs by terminal restriction fragment length polymorphism (T-RFLP) analysis, Real-Time PCR and in situ hybridization. The texture of the diet influenced the microbiota, and from a quantitative discriminative analysis of the terminal restriction fragments (T-RFs) of ileum samples it was deduced that Clostridium spp. and Lactobacillus spp. were associated with the non-pelleted diet and Streptococcus spp. with the pelleted diet. In experimentally infected pigs it was verified that 89 bp and 90 bp sized T-RFs (HhaI) from ileum represented L. intracellularis. The non-pelleted diet seemed to reduce the relative amount of L. intracellularis in the total microbiota of the ileum, but the number of pigs detected positive with L. intracellularis by Real-Time PCR was not influenced. The five pigs with highest L. intracellularis content showed T-RFs that were not present in profiles from less or non-infected pigs, which may indicate that some bacterial species were associated with L. intracellularis infection.