Effect of Tetracycline Dose and Treatment Mode on Selection of Resistant Coliform Bacteria in Nursery Pigs.

This study describes the results of a randomized clinical trial investigating the effect of oxytetracycline treatment dose and mode of administration on the selection of antibiotic-resistant coliform bacteria in fecal samples from nursery pigs. Nursery pigs (pigs of 4 to 7 weeks of age) in five pig herds were treated with oxytetracycline for Lawsonia intracellularis-induced diarrhea. Each group was randomly allocated to one of five treatment groups: oral flock treatment with a (i) high (20 mg/kg of body weight), (ii) medium (10 mg/kg), or (iii) low (5 mg/kg) dose, (iv) oral pen-wise (small-group) treatment (10 mg/kg), and (v) individual intramuscular injection treatment (10 mg/kg). All groups were treated once a day for 5 days. In all groups, treatment caused a rise in the numbers and proportions of tetracycline-resistant coliform bacteria right after treatment, followed by a significant drop by the time that the pigs left the nursery unit. The counts and proportions of tetracycline-resistant coliforms did not vary significantly between treatment groups, except immediately after treatment, when the highest treatment dose resulted in the highest number of resistant coliforms. A control group treated with tiamulin did not show significant changes in the numbers or proportions of tetracycline-resistant coliforms. Selection for tetracycline-resistant coliforms was significantly correlated to selection for ampicillin- and sulfonamide-resistant strains but not to selection for cefotaxime-resistant strains. In conclusion, the difference in the dose of oxytetracycline and the way in which the drug was applied did not cause significantly different levels of selection of tetracycline-resistant coliform bacteria under the conditions tested.IMPORTANCE Antimicrobial resistance is a global threat to human health. Treatment of livestock with antimicrobials has a direct impact on this problem, and there is a need to improve the ways that we use antimicrobials in livestock production. We hypothesized that antibiotic resistance development following treatment of diarrhea in nursery pigs could be reduced either by lowering the dose of oxytetracycline or by replacing the commonly used practice of flock treatment with individual or small-group treatments, since this would reduce the number of pigs treated. However, the study showed no significant difference between treatment groups with respect to the number or proportion of tetracycline-resistant coliforms selected. The most important conclusion is that under practical field conditions, there will be no added value, in terms of lowering resistance development, by exchanging flock treatment for individual or small-group treatment of nursery pigs. The reason for the lack of an effect of single-animal treatment is probably that such animals share the environment with treated animals and take up resistant bacteria from the environment.

Recent advances in understanding the pathogenesis of Lawsonia intracellularis infections.

Proliferative enteropathy is an infectious disease caused by an obligate intracellular bacterium, Lawsonia intracellularis, and characterized by thickening of the intestinal epithelium due to enterocyte proliferation. The disease is endemic in swine herds and has been occasionally reported in various other species. Furthermore, outbreaks among foals began to be reported on breeding farms worldwide within the past 5 years. Cell proliferation is directly associated with bacterial infection and replication in the intestinal epithelium. As a result, mild to severe diarrhea is the major clinical sign described in infected animals. The dynamics of L. intracellularis infection in vitro and in vivo have been well characterized, but little is known about the genetic basis for the pathogenesis or ecology of this organism. The present review focuses on the recent advances regarding the pathogenesis and host-pathogen interaction of L. intracellularis infections.

Detection of enteric pathogens in Turkey flocks affected with severe enteritis, in Brazil.

Twenty-two flocks of turkeys affected by enteric problems, with ages between 10 and 104 days and located in the Southern region of Brazil, were surveyed for turkey by PCR for turkey astrovirus type 2 (TAstV-2), turkey coronavirus (TCoV), hemorrhagic enteritis virus (HEV), rotavirus, reovirus, Salmonella spp., and Lawsonia intracellularis (Li) infections. Eleven profiles of pathogen combination were observed. The most frequently encountered pathogen combinations were TCoV-Li, followed by TCoV-TAstV-2-Li, TCoV-TastV-2. Only TCoV was detected as the sole pathogen in three flocks. Eight and 19 flocks of the 22 were positive for TAstV-2 and TCoV, respectively. Six were positive for Salmonella spp. and L. intracellularis was detected in 12 turkey flocks. Reovirus and HEV were not detected in this survey. These results throw new light on the multiple etiology of enteritis in turkeys. The implications of these findings and their correlation with the clinical signs are comprehensively discussed, illustrating the complexity of the enteric diseases.

Comparative genome sequencing identifies a prophage-associated genomic island linked to host adaptation of Lawsonia intracellularis infections.

Lawsonia intracellularis is an obligate intracellular bacterium and the causative agent of proliferative enteropathy (PE). The disease is endemic in pigs, emerging in horses and has also been reported in a variety of other animal species, including nonhuman primates. Comparing the whole genome sequences of a homologous porcine L. intracellularis isolate cultivated for 10 and 60 passages in vitro, we identified a 18-kb prophage-associated genomic island in the passage 10 (pathogenic variant) that was lost in the passage 60 (non-pathogenic variant). This chromosomal island comprises 15 genes downstream from the prophage DLP12 integrase gene. The prevalence of this genetic element was evaluated in 12 other L. intracellularis isolates and in 53 infected animals and was found to be conserved in all porcine isolates cultivated for up to 20 passages and was lost in isolates cultivated for more than 40 passages. Furthermore, the prophage region was also present in 26 fecal samples derived from pigs clinically affected with both acute and chronic forms of the disease. Nevertheless, equine L. intracellularis isolates evaluated did not harbor this genomic island regardless of the passage in vitro. Additionally, fecal samples from 21 clinically affected horses and four wild rabbits trapped in horse farms experiencing PE outbreaks did not show this prophage-associated island. Although the presence of this prophage-associated island was not essential for a virulent L. intracellularis phenotype, this genetic element was porcine isolate-specific and potentially contributed to the ecological specialization of this organism for the swine host.

Genotyping of Equine Lawsonia intracellularis Sampled in Japan by Using Multilocus Variable-Number Tandem Repeat Analysis.

The incidence of equine proliferative enteropathy, caused by Lawsonia intracellularis, is increasing around the world. To investigate the relationships of variable-number tandem repeat (VNTR) patterns with host species and clinical status in horses, multilocus VNTR analysis (MLVA) was applied to 98 L. intracellularis samples collected from horses, seven from pigs, seven from wildlife, one vaccine strain, and 17 public strains. The VNTR patterns were highly diverse: a total of 130 samples identified 99 distinct patterns, and the 98 horses were classified into 71 different patterns. A phylogenetic tree based on the MLVA showed three clusters: porcine, equine, and miscellaneous cluster. The equine cluster contained 46 horse samples, of which 42 (91.3%) were collected from two sampling areas. The MLVA could discriminate horse samples from pig, but the horse samples in the miscellaneous cluster could not be distinguished from wildlife samples. As for clinical data of the horses, the VNTR patterns were unrelated to horse age, clinical signs, and clinical outcomes. This study shows that VNTR patterns had no clear connection with equine clinical status, but the MLVA could be useful to investigate its epidemiological relationships, and interspecific transmission of L. intracellularis between horse and wildlife cannot be ruled out.

Comparison of feces versus rectal swabs for the molecular detection of Lawsonia intracellularis in foals with equine proliferative enteropathy.

The purpose of the current study was to compare the molecular detection rate of Lawsonia intracellularis between feces and rectal swabs collected from 42 foals with suspected equine proliferative enteropathy (EPE). Fecal samples and rectal swabs were processed for DNA purification by using an automated extraction system. The purified DNA was then analyzed by real-time polymerase chain reaction (PCR) for the presence of the aspartate ammonia lyase (aspA) gene of L. intracellularis. Absolute quantitation was calculated by using a standard curve for L. intracellularis and expressed as copy numbers of the aspA gene of L. intracellularis per microliter of purified DNA. The combined PCR detection rate for L. intracellularis was 90%, with 38 foals testing PCR positive in feces (33 samples), rectal swabs (32), or both (27). Six foals tested PCR positive only in feces, whereas 5 tested positive only in rectal swabs. Feces yielded a significantly higher aspA gene copy number of L. intracellularis than rectal swabs. Feces and rectal swabs tested PCR negative from 4 foals. In conclusion, the results showed that feces yielded similar numbers of PCR-positive results, with a higher L. intracellularis aspA gene load than rectal swabs. By analyzing dual samples, the PCR detection rate for L. intracellularis increased from 76% and 79% for rectal swabs and feces, respectively, to 90%. Rectal swabs should be considered as an alternative sample type for EPE-suspected patients with decreased or no fecal output.

Metagenomic sequencing of clinical samples reveals a single widespread clone of Lawsonia intracellularis responsible for porcine proliferative enteropathy.

Lawsonia intracellularis is a Gram-negative obligate intracellular bacterium that is the aetiological agent of proliferative enteropathy (PE), a common intestinal disease of major economic importance in pigs and other animal species. To date, progress in understanding the biology of L. intracellularis for improved disease control has been hampered by the inability to culture the organism in vitro. In particular, our understanding of the genomic diversity and population structure of clinical L. intercellularis is very limited. Here, we utilized a metagenomic shotgun approach to directly sequence and assemble 21 L. intracellularis genomes from faecal and ileum samples of infected pigs and horses across three continents. Phylogenetic analysis revealed a genetically monomorphic clonal lineage responsible for infections in pigs, with distinct subtypes associated with infections in horses. The genome was highly conserved, with 94 % of genes shared by all isolates and a very small accessory genome made up of only 84 genes across all sequenced strains. In part, the accessory genome was represented by regions with a high density of SNPs, indicative of recombination events importing novel gene alleles. In summary, our analysis provides the first view of the population structure for L. intracellularis, revealing a single major lineage associated with disease of pigs. The limited diversity and broad geographical distribution suggest the recent emergence and clonal expansion of an important livestock pathogen.

Temporal detection of Lawsonia intracellularis using serology and real-time PCR in Thoroughbred horses residing on a farm endemic for equine proliferative enteropathy.

The goals of this study were to evaluate titers of antibodies against Lawsonia intracellularis in 68 resident broodmares from a farm known to be endemic for equine proliferative enteropathy (EPE) and to evaluate maternal antibodies, occurrence of seroconversion and fecal shedding in their foals. Serum samples collected from mares at delivery and from foals pre- and post-colostrum ingestion and monthly thereafter were tested for the presence of L. intracellularis antibodies by immunoperoxidase monolayer assay (IPMA). Further, feces collected from mares at delivery and foals post-partum and monthly thereafter were assayed for L. intracellularis using real-time PCR. Thirty-seven mares (54.4%) had detectable antibody titers (> or =60) against L. intracellularis by IPMA at the time of foaling. Passive transfer of colostral antibodies against L. intracellularis was documented in 37 foals (54.4%) and the colostral antibodies remained detectable in the serum of foals for 1-3 months. Overall, 22 foals (33.3%) showed evidence of natural exposure to L. intracellularis throughout the study period, however, none of the study foals developed signs compatible with EPE. The serological results showed that mares residing on a farm known to be endemic for EPE are routinely exposed to L. intracellularis and that antibodies against L. intracellularis are passively transferred to foals.

Quantification of Lawsonia intracellularis in porcine faeces by real-time PCR.

AIM: To develop and to validate a method for the quantification of Lawsonia intracellularis in porcine faeces by real-time PCR. METHODS AND RESULTS: A real-time PCR including a calibrator based on plasmid DNA for quantification by means of DeltaDeltaCt method was evaluated. The parameters specificity, detection limit, quantification limit, linearity, range, repeatability, precision and recovery were validated. The detection limit of the agent was 1 copy per reaction, and quantification was reliable between 10(1) and 10(7) copies per microl reaction volume. The linearity calculated by logistic regression revealed a slope of -3.329 reflecting an efficiency of 99.7% for the assay. Moreover, it was shown that storage of samples and repetition of tests including DNA isolation by same or other investigators did not influence the outcome. CONCLUSION: The quantification method described herein revealed consistent results for the quantitation of L. intracellularis in porcine faeces samples. SIGNIFICANCE AND IMPACT OF THE STUDY: In contrast to common PCR in combination with gel electrophoresis, this validated quantification method based on real-time PCR enhances a reliable quantification and is even more sensitive.

LI1035, a putative effector secreted by Lawsonia intracellularis, targets the MAPK pathway and regulates actin organizationin yeast and mammalian cells.

Lawsonia intracellularis is an obligate intracellular Gram-negative bacterium that has been identified as the etiological agent of the contagious disease proliferative enteropathy (PE) in a wide range of animals, mainly pigs. The genome sequence of L. intracellularis indicates that this bacterium possess a type III secretion system (T3SS), which may assist the bacterium during cell invasion and host innate immune system evasion and could be a mechanism for inducing cellular proliferation. However, the effectors secreted by the T3SS (T3Es) of L. intracellularis have not been reported. T3Es often target conserved eukaryotic cellular processes, and yeast is an established and robust model system in which to reveal their function. By screening the growth inhibition of an ordered array of Saccharomyces cerevisiae strains expressing the hypothetical genes of L. intracellularis, LI1035 was identified as the first putative effector that inhibits yeast growth. The LI1035-induced growth inhibition was rescued in two of the 14 mitogen-activated protein kinase (MAPK) yeast haploid deletion strains, suggesting that LI1035 interacts with the components of the MAPK pathway in yeast. Phosphorylation assays confirmed that LI1035 inhibits MAPK signaling cascades in yeast and mammalian cells. Actin staining assays revealed that LI1035 regulates actin organization in yeast and mammalian cells. Taken together, these results indicate that LI1035 alters MAPK pathway activity and regulates actin organization in the host. These findings may contribute to the understanding the pathogenesis of L. intracellularis and support the use of yeast as a heterologous system for the functional analysis of pathogen-specific gene products in the laboratory.

Lawsonia intracellularis contains a gene encoding a functional rickettsia-like ATP/ADP translocase for host exploitation.

ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [NTT1(Li)]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of NTT1(Li) were determined by measuring the uptake of radioactively labeled substrates by E. coli. NTT1(Li) transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 (+/- 36.5) microM for ATP and 275.2 (+/- 28.1) microM for ADP, identifying this protein as a functional ATP/ADP translocase. NTT1(Li) is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors.

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 89bp and 90bp 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.

A real-time loop-mediated isothermal amplification method for rapid detection of Lawsonia intracellularis in porcine fecal samples.

Porcine proliferative enteritis is a common diarrheal disease characterized by thickening of the intestinal mucosa in swine due to enterocyte proliferation, which is caused by Lawsonia intracellularis. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay was developed to detect L. intracellularis based on the conserved region of the 16S ribosomal RNA gene. The optimal reaction conditions of the real-time LAMP was 65 °C for 60 min. The LAMP products could be detected by both real-time turbidity and direct visual inspection. The assay was specific for L. intracellularis, as no cross-reaction was observed with other pathogens. The detection limit of the real-time LAMP assay was 1.4 × 10-1pg of L. intracellularis DNA, which was the same as that of real-time PCR and approximately 100 times more sensitive than that of conventional PCR. Of the 136 clinical samples, L. intracellularis DNA was identified in 60 samples by real-time LAMP, which was the same as real-time PCR and higher than conventional PCR (36.8%, 50/136). The specific, sensitive and rapid real-time LAMP assay developed in this study could be a useful alternative tool in point-of-care (POC) diagnosis of L. intracellularis infection.

In situ hybridization for Lawsonia intracellularis--specific 16s rRNA sequence in paraffin-embedded tissue using a digoxigenin-labeled oligonucleotide probe.

An in situ hybridization (ISH) procedure with a digoxigenin-labeled oligonucleotide probe for detection of Lawsonia intracellularis in paraffin-embedded tissue is described. This technique recognized 71% of PCR-positive cases and was thus superior to Warthin-Starry silver stain, which only detected 41%. The presented ISH is of comparable sensitivity to previously published immunohistochemical assays and is recommended for laboratories wishing to diagnose L. intracellularis infections in tissue sections but without access to antibodies.

Identification of Lawsonia intracellularis putative hemolysin protein A and characterization of its immunoreactivity.

Despite the recent global increase in fatal endemic outbreaks of proliferative enteropathy (PE) caused by the obligate intracellular bacterium Lawsonia intracelluralis (LI) in the swine industry, development of effective prevention strategies or immunodiagnostic tests has been delayed due to the difficulty of cultivating this pathogen in vitro. Although several genetic analyses have been performed at the level of gene transcription after the complete genome sequence of LI was made available, the mechanism of LI infection and virulence genes remain unidentified. In the present study, we assessed the antigenic features of the LI0004 protein, which we putatively defined as Lawsonia hemolysin A (LhlyA), by employing bioinformatics tools and in vivo and in vitro protein-based molecular assays. The amino acid sequence of LhlyA showed approximately 60% homology to the hemolysin-like proteins of Bilophila wadsworthia and Desulfovibrio piger. Presence of computationally predicted linear antigenic B-cell epitopes on the LhlyA protein was demonstrated by immunoblotting; a band with a molecular mass corresponding to the predicted size of the protein was strongly recognized by sera collected from artificially infected mice. Further, in an in vivo cytotoxicity assay, no splenomegaly was observed in mice inoculated with purified LhlyA. Collectively, the data presented here suggest that the LhlyA protein is a highly immuno-reactive antigen of L. intracellullaris and can potentially be used to develop effective protection strategies against PE.

Human inflammatory bowel disease does not associate with Lawsonia intracellularis infection.

BACKGROUND: There is increasing evidence that bacterial infection of the intestinal mucosa may contribute to the pathogenesis of inflammatory bowel diseases (IBD). In pigs, an obligate intracellular bacterium, Lawsonia intracellularis (LI), was shown to cause proliferative enteropathy (PE) of which some forms display histological and clinical similarities to human IBD. Since LI-similar Desulfovibrio spp. may infect human cells, we hypothesized that LI might be associated with the development of human IBD. RESULTS: In human intestinal tissue samples, PCR using LLG, 50SL27, LSA and strictly LI-specific 16SII primers, yielded either no amplicons or products with weak homology to human genomic sequences. Sequencing of these amplicons revealed no specificity for LI. However, amplification of DNA with less specific 16SI primers resulted in products bearing homology to certain Streptococcus species. These 16SI-amplified products were present in healthy and diseased specimens, without obvious prevalence. CONCLUSION: LI is not associated with the pathogenesis of UC or CD. Whether an immunologic response to commensal bacteria such as streptococci may contribute to the chronic inflammatory condition in IBD, remained to be determined.

PREVALENCE OF ANTIBODY TO AND DNA OF LAWSONIA INTRACELLULARIS IN SAMPLES FROM WILD ANIMALS IN KOREA.

We evaluated the prevalence of Lawsonia intracellularis infection in three wild animal species in Korea; the Korean water deer ( Hydropotes inermis ), Siberian roe deer ( Capreolus pygargus ), and raccoon dogs ( Nyctereutes procyonoides ). We collected 136 sera and 109 fecal samples from individuals in 10 Wildlife Rescue and Conservation Centers. Serum samples were tested for anti- L. intracellularis antibodies using a blocking enzyme-linked immunosorbent assay (bELISA), and fecal samples were subjected to a real-time PCR assay for L. intracellularis . Thirty-five (25.7%) sera and 36 (33.0%) fecal samples were positive. We found a higher proportion of positive sera (64.7%, χ2=15.439, P<0.01) and feces (58.8%, χ2=6.126, P<0.05) in raccoon dogs (χ2=11.855, P<0.01) than in the other species (20% positive sera and 29% positive feces in Korean water deer; 20% positive sera and 25% positive feces in Siberian roe deer). Our data indicate infection by L. intracellularis in Korean water deer, Siberian roe deer, and raccoon dogs throughout the country. It is imperative to know whether these infected animal species are natural hosts for L. intracellularis in addition to domestic pigs ( Sus scrofa domesticus).

Proliferative enteropathy: a global enteric disease of pigs caused by Lawsonia intracellularis.

Proliferative enteropathy (PE; ileitis) is a common intestinal disease affecting susceptible pigs raised under various management systems around the world. Major developments in the understanding of PE and its causative agent, Lawsonia intracellularis, have occurred that have led to advances in the detection of this disease and methods to control and prevent it. Diagnostic tools that have improved overall detection and early onset of PE in pigs include various serological and molecular-based assays. Histological tests such as immunohistochemistry continue to be the gold standard in confirming Lawsonia-specific lesions in pigs post mortem. Despite extreme difficulties in isolating L. intracellularis, innovations in the cultivation and the development of pure culture challenge models, have opened doors to better characterization of the pathogenesis of PE through in vivo and in vitro L. intracellularis-host interactions. Advancements in molecular research such as the genetic sequencing of the entire Lawsonia genome have provided ways to identify various immunogens, metabolic pathways and methods for understanding the epidemiology of this organism. The determinations of immunological responsiveness in pigs to virulent and attenuated isolates of L. intracellularis and identification of various immunogens have led to progress in vaccine development.

A Lawsonia intracellularis transmission study using a pure culture inoculated seeder-pig sentinel model.

Transmission of Lawsonia intracellularis from experimentally inoculated pigs to naive swine was demonstrated in this study. The study was conducted using conventional pigs divided into three groups as follows: principles inoculated with L. intracellularis, sentinels, and controls. The pigs were inoculated and paired on 13 and 9 days post-inoculation with a sentinel pig for 7 days. Fecal samples and serum samples were collected throughout the study for polymerase chain reaction (PCR) and antibody testing by indirect fluorescent antibody techniques. After co-mingling, the inoculated group was necropsied; sentinel and control pigs were necropsied 7-14 days later. The intestinal tracts were evaluated grossly and microscopically for lesions. PCR was performed on intestinal mucosal scrapings and feces. Warthin-Starry and fluorescent antibody staining procedures were conducted to confirm colonization with L. intracellularis. Gross and microscopic lesions typical of porcine proliferative enteropathy (PPE) were observed in both the inoculated and sentinel groups. Transmission was demonstrated from inoculated principle pigs to sentinel pigs. PCR results detected cyclical shedding of L. intracellularis in the feces. Seroconversion occurred in pigs that were exposed to L. intracellularis. From this study, it was demonstrated that transmission of L. intracellularis can occur easily in an environment with experimentally infected pigs and that PCR can be a useful tool to monitor fecal shedding of the organism.

Routine diagnostics of Lawsonia intracellularis performed by PCR, serological and post mortem examination, with special emphasis on sample preparation methods for PCR.

The aim of this study was to find suitable and reliable tools for demonstrating Lawsonia intracellularis in routine clinical diagnosis. Firstly, a method to prepare tissue samples before a polymerase chain reaction (PCR) was evaluated in pigs submitted for necropsy. Secondly, seven different faecal preparation methods and four different DNA polymerases were tested in single or nested PCR, with co-amplification of a mimic molecule. Thirdly, in selected pigs submitted for necropsy, tissue and faecal samples were examined histopathologically and by PCR, and blood samples were analysed serologically. Detection of L. intracellularis in tissue preparations by PCR showed good specificity and correlated to lesions found at necropsy. The sensitivity in spiked tissue samples was 10(1)-10(2) mimic molecules per tube. In faecal samples, nested PCR on boiled lysate gave the best result with a sensitivity of 10(2)-10(3) mimic molecules per reaction tube. However, because of the time-consuming procedure and the increased risk for contamination, a commercially available kit was preferred for routine diagnoses, despite a somewhat lower detection rate in subclinically infected pigs. In a few cases, the serological results differed from those obtained by PCR and by necropsy but the reason for this is not clear. This study indicates that the best method for diagnosis of acute enteritis in growers is PCR on faecal or tissue samples. To determine the presence of the bacteria in a herd, serology or repeated faecal sampling for PCR from target animals, or both, should be used.