Survival of Lawsonia intracellularis in porcine peripheral blood monocyte-derived macrophages.

Lawsonia intracellularis, an obligately intracellular enteric bacterium, infects intestinal epithelial cells, but may also be found within macrophages in the intestinal lamina propria of affected pigs. Macrophages play an important role in host defense against infectious agents, but the role of this cell in L. intracellularis infection is not well understood. The aim of this study was to evaluate the permissibility of macrophages to L. intracellularis infection in vitro. Pure culture of L. intracellularis was added to swine peripheral blood monocyte-derived macrophages. Viability of intracytoplasmic L. intracellularis was evaluated at different time points by transmission electron microscopy (TEM). Potential replication of L. intracellularis in macrophages was also evaluated by qPCR. By TEM, phagocytosis L. intracellularis within of phagolysosomes were observed 1-hour post-infection (hpi) and bacterial structures in binary fission at 48 hpi. The number of intracellular bacteria was determined at 1, 4, 24, 48, and 72 hpi by qPCR in infected macrophages and compared to the number of intracellular bacteria from culture in McCoy cells. In both cell lines, the amount of L. intracellularis was decreased at 4 hpiand increased at 24 hpi. The number of intracellular bacteria continued to increase in McCoy cells over time. This is the first study showing interaction, survival and propagation of L. intracellularis in macrophages. These findings are critical to establish an experimental model for future studies of the pathogenesis of porcine proliferative enteropathy and the potential persistence of L. intracellularis in macrophages during chronic infections.

Use of flow cytometry and PCR analysis to detect 5-carboxyfluoroscein-stained obligate intracellular bacteria Lawsonia intracellularis invasion of McCoy cells.

In this study, we describe a method to quantify invasion of obligate intracellular bacteria, Lawsonia intracellularis, inside McCoy cells. In immunological research, the cell-permeable fluorescent dye 5'-carboxyfluoroscein succidyl ester (CFSE) is commonly used to quantify eukaryotic cellular proliferation. Instead of using it in this traditional way, we stained L. intracellularis with CFSE dye prior to infection of McCoy cells. Flow cytometry was performed to quantify the percentage of eukaryotic cells which had taken up or were associated with fluorescent bacteria. As obligate intracellular bacteria, they cannot replicate outside of eukaryotic cells and thus qPCR analysis was used to quantify bacterial growth. Indirectly, PCR analysis confirmed invasion rather than adherence to the McCoy cell surface. Fluorescent activated cell sorting (FACS) was used to sort the CFSE(+) (i.e. infected) McCoy cells from the CFSE(-) (i.e. non-infected) McCoy cells and confocal microscopy was used to confirm bacterial invasion and cytosolic localization of CFSE-L. intracellularis. To show that this approach could be used in conjunction with functional assays, we investigated the effect that serum antibodies had on CFSE-bacterial invasion and growth. Instead of blocking invasion, rabbit hyperimmune serum augmented invasion of the bacteria inside McCoy cells and qPCR analysis confirmed bacterial growth over the course of 5days. We conclude that CFSE-labeling of bacteria and qPCR can be used to track and quantify bacterial invasion and may be a valuable tool for studying the invasive properties of bacteria, especially if commercial antibodies are not available. This approach may be adapted for use in other obligate intracellular bacteria and intracellular pathogens.

The critical threshold of Lawsonia intracellularis in pig faeces that causes reduced average daily weight gains in experimentally challenged pigs.

Serology indicates that Lawsonia intracellularis infection is widespread in many countries, with most pigs seroconverting before 22 weeks of age. However, the majority of animals appear to be sub-clinically affected, demonstrated by the low reported prevalence of diarrhoea. Production losses caused by sub-clinical proliferative enteropathy (PE) are more difficult to diagnose, indicating the need for a quantitative L. intracellularis assay that correlates well with disease severity. In previous studies, increasing numbers of L. intracellularis in pig faeces, quantified with a real time polymerase chain reaction (qPCR), showed a strong negative correlation with average daily gain (ADG). In this study, the association between faecal L. intracellularis numbers and PE severity was examined in two L. intracellularis experimental challenge trials (n1=32 and n2=95). The number of L. intracellularis shed in individual faeces was determined by qPCR on days 0, 7, 14, 17 and 21 days post challenge, and average daily gain was recorded over the same period. The severity of histopathological lesions of PE was scored at 21 days post challenge. L. intracellularis numbers correlated well with histopathology severity and faecal consistency scores (r=0.72 and 0.68, respectively), and negatively with ADG (r=-0.44). Large reductions in ADG (131 g/day) occurred when the number of L. intracellularis shed by experimentally challenged pigs increased from 10(7) to 10(8)L. intracellularis, although smaller ADG reductions were also observed (15 g/day) when the number of L. intracellularis increased from 10(6) to 10(7)L. intracellularis.

An alternative method for cultivation of Lawsonia intracellularis.

An alternative method for the cultivation of Lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, was developed using an Original Space Bag inflated with a mixture of gas containing 10% hydrogen, 10% carbon dioxide, and 80% nitrogen. The flexibility of this protocol allows the testing of various environmental conditions for static cultivation of this bacterium and the development of diagnostic techniques.

Analysis of bacterial load and prevalence of mixed infections with Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli in German pigs with diarrhoea.

Lawsonia (L.) intracellularis, Brachyspira (B.) hyodysenteriae and B. pilosicoli are important pathogens in domestic pig production world-wide, responsible for porcine intestinal adenomatosis, swine dysentery, and porcine intestinal spirochetosis, respectively. Conventional PCR is the major diagnostic tool in the detection of the three pathogens, but the sole detection of bacterial DNA might lead to misinterpretations of results with respect to their clinical relevance, especially with mixed infections. Thus, the present study targeted the detection and quantification of the three pathogens in samples from herds with a case history of diarrhoea. Herds and samples were selected by the practitioners on a voluntary basis. Results were based on 1176 individual samples from 95 herds from Southern Germany. The pathogens were detected simultaneously by multiplex real-time PCR. The overall prevalence for L. intracellularis, B. hyodysenteriae and B. pilosicoli was 12.6%, 8.4% and 3.2% in faecal samples and 48.4%, 24.2% and 31.6% in herds, respectively. Sixty one percent, 82.6%, and 73.4% of herds positive for L. intracellularis, B. hyodysenteriae, and B. pilosicoli, respectively, had mixed infections. Median log values of DNA equivalents/g of faeces for L. intracellularis, B. hyodysenteriae and B. pilosicoli were 3.3, 5.9 and 3.2, with maxima of 8.3, 8.0 and 6.3, respectively. Within herd prevalence of B. hyodysenteriae and B. pilosicoli as well as the load of B. hyodysenteriae were significantly associated with the severity of diarrhoea.

In vitro assessment of the effectiveness of powder disinfectant (Stalosan F) against Lawsonia intracellularis using two different assays.

The objective of this study was to determine the in vitro efficacy of Stalosan F, a mixed chemical and heavy metal disinfectant, against two strains of Lawsonia intracellularis using both a modified tissue culture and a direct count method. For testing as a powder, 1g, 0.5g, or 0.25g of Stalosan F was applied to bacterial solutions spread into sterile dishes. For use as an aqueous suspension, Stalosan F was prepared to final concentrations of 1%, 4%, 8%, 16%, and 32%. In both applications, L. intracellularis was exposed to Stalosan F for 0.5h, 1h, 2h, and 4h. The results showed that both strains were similar in their susceptibilities to Stalosan F. The modified tissue culture assay showed no detectable L. intracellularis in cell culture after exposure to all levels of Stalosan F powder for 0.5h. Furthermore, the number of viable bacteria was markedly reduced in the aqueous concentration of 4% and no L. intracellularis was detected at concentrations of > or =8% for 0.5h. Using the direct count method, detection of live bacteria was less than 1% after exposure to the powder for 0.5h. After exposure to the aqueous form, the number of viable bacteria killed was over 99% in concentrations of > or =16% compared to controls. Our results indicate that Stalosan F in both powder and suspension forms is able to inactivate over 99% of L. intracellularis after 30min of exposure. Furthermore, both laboratory methods can be used to determine the effect of disinfectants on L. intracellularis viability.

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.

Re-challenge of pigs following recovery from proliferative enteropathy.

An experimental challenge model was developed to demonstrate Lawsonia intracellularis colonization and reproduction of proliferative enteropathy (PE) in naïve weaner pigs. Groups of pigs were orally dosed with between 10(10) and 10(5)L. intracellularis extracted from haemorrhagic PE affected mucosa. Pigs were monitored for clinical signs and intestinal lesions of PE and evidence of bacterial colonization by serology and faecal polymerase chain reaction (PCR). One group of challenged pigs were necropsied after 21 days to confirm the reproduction of PE. L. intracellularis colonization and seroconversion was delayed in pigs dosed with lower numbers of L. intracellularis. When faecal shedding of L. intracellularis ceased to be detected in all of the challenged pigs, they were re-dosed orally with approximately 10(10)L. intracellularis and monitored for evidence of re-colonization and clinical disease. This study demonstrated that pigs previously challenged with L. intracellularis were protected from re-colonization and clinical disease on subsequent exposure 10 weeks later, regardless of the initial dose of L. intracellularis.

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.

Measurement of the viability of Lawsonia intracellularis.

The objective of this study was to develop and test both a flow cytometry method (FCM) and a direct count method (DCM) that both use fluorescent stains to determine the viability of Lawsonia intracellularis (LI), an obligate intracellular bacterium and the cause of proliferative enteropathy (PE) in pigs and other animal species. Live LI were passaged in cell culture and harvested from infected McCoy cells. Dead LI were prepared by exposing live LI to 70% isopropyl alcohol for 30 min. Seven samples with dead:live ratios of 0:100 (live control), 10:90, 30:70, 50:50, 70:30, 90:10, and 100:0 (dead control) were prepared for testing by both the FCM and the DCM. For the FCM, TO-PRO-3 iodine was applied to the samples, and viable LI were counted. For the DCM, the samples were stained with LIVE/DEAD BacLight, which contains SYTO 9 and propidium iodine, then filtered through 0.2-microm Nuclepore black polycarbonate filters, viewed, and counted with the use of an epifluorescence microscope. Data were evaluated by estimating 95% limits of agreement and the concordance correlation coefficient (CCC). The limits of agreement between the FCM and the DCM versus the standard ratio of added LI showed mean differences not equal to zero, suggesting that systematic bias was introduced. The CCC showed almost perfect agreement (r = 0.9898). With a specific fluorescent probe, the FCM is useful and as good as the DCM for determining LI viability.

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.

The influence of diet on Lawsonia intracellularis colonization in pigs upon experimental challenge.

The objective of this investigation was to study if different feeding strategies influence experimental infections of pigs with Lawsonia intracellularis, the causative agent of proliferative enteropathy. In three sequential trials, a total of 144 weaned pigs were fed five different diets all made from a standard diet based on wheat and barley as carbohydrate source and soybean as protein source. The five diets were: a standard diet (fine ground and pelleted), the standard diet fed as fermented liquid feed, the standard diet added 1.8% formic acid, the standard diet added 2.4% lactic acid and a diet similar to the standard diet (made from the same ingredients), but fed coarse ground. Twenty-four pigs on each diet were orally inoculated with L. intracellularis and growth performance and faecal excretion of bacteria were monitored. Twenty-four pigs fed the standard diet were included as not experimentally infected controls. Pigs in the first two trials were sacrificed 4 weeks post-inoculation, whereas animals in the third trial were sacrificed after 5 weeks. Pigs in all experimentally infected groups excreted L. intracellularis. The fermented liquid diet delayed the excretion of L. intracellularis and furthermore, pigs fed the standard diet supplemented with lactic acid had limited pathological lesions when the intestines were examined 4 weeks after inoculation. The growth performance was reduced in pigs experimentally challenged with L. intracellularis, however the prevalence and severity of diarrhea was limited.

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.

Prevalence of porcine proliferative enteropathy and its control with tylosin in Korea.

Porcine proliferative enteropathy(PPE) is an enteric disease been caused by Lawsonia intracellularis. It has become one of the critical problems in the pig industry. To investigate the prevalence of PPE in Korea, serum samples of 828 pigs from 65 herds were tested using indirect immunofluorescence antibody technique(IFA). The infection rate in individual pigs varied from 44 to 69%, whereas 100% in pig farms. The infection frequency was 57, 44.9, and 59.4% according to age respectively. Administration of tylosin in feed at a concentration of 110 ppm for 14 days reduced the infection rate of the farms. These data indicated that the high prevalence of PPE may be controlled by tylosin.