Infection dynamics of Lawsonia intracellularis in pig herds

Little information is known about the natural course and within-herd prevalence of porcine proliferative enteropathy caused by Lawsonia intracellularis. The objective of the study was to investigate the within-herd dynamics of naturally acquired L. intracellularis infection in pigs from weaning to slaughter. The study was designed as a longitudinal survey where 100 pigs from five herds were randomly selected at weaning (approximately 4 weeks of age). Every second week until slaughter (10-12 times, i.e. 20-24 weeks) the pigs were weighed and faecal as well as blood samples were collected. Faecal shedding of L. intracellularis was assessed by real time-PCR and sero-conversion by an indirect immunofluorescence antibody test (IFAT). Clinical disease was not reported but infection was present in all herds and the PCR assay indicated infection in 75% of pigs examined. Most L. intracellularis infected pigs were shedding at 10-12 weeks of age (22-29 kg) and shed for 2-6 successive weeks. After 18 weeks of age all shedding had ceased and re-infection at PCR detectable level was not seen. Variable L. intracellularis associated impact on growth rate was observed. Immediately before bacterial shedding and during early infection the average growth rate declined whereas a compensatory impact was observed during later infection and after bacterial shedding had ceased. The performance of the IFAT resembled the bacteriological test almost perfectly. Sero-conversion was first detected at 12-14 weeks of age. Relative to the bacterial shedding, the onset of sero-conversion was a little delayed, in general, most pigs had sero-converted 2 weeks after the first shedding. Once sero-converted, 92% of the pigs remained sero-positive over the entire survey period.     

Seroprevalence of Lawsonia intracellularis in large pig production units

In 11 'farrow-to-finish' outdoor or indoor production units, blood samples from late pregnant gilts were tested by indirect immunofluorescence antibody (IFA) serum assay for Lawsonia intracellularis. The offspring of positively tested gilts were tested at 2, 7, 12, 17, 22 and 27 weeks of age for seroprevalence of Lawsonia intracellularis. All offspring of IFA positive gilts were seronegative at 2 and 7 weeks of age. At 12 weeks of age 81.0% of indoor and 51.0% of outdoor pigs were tested positive. While at 17 weeks of age 82.5% of indoor-raised pigs showed seropositivity, in outdoor units the seropositivity declined to 31.3%. At weeks 22 and 27 indoor-raised pigs still showed marked seropositivity (17.7% and 11.5%) but their outdoor-raised counterparts revealed declining values (7.4% and 0%).     

Prevalence of antibodies to Lawsonia intracellularis in pig herds in Australia

Objective Proliferative enteropathy (PE) of pigs is caused by Lawsonia intracellularis. Clinical severity appears to depend, at least partly, on the infective dose and strain of L. intracellularis. Serological tests are able to detect subclinical disease. The Bioscreen ELISA for detecting L. intracellularis-specific antibodies is widely used to monitor the circulating antibody status of pigs in Australia, but its sensitivity and specificity have not been reported. The aim of the present study was to measure the seroprevalence of antibodies to L. intracellularis in growing pigs in Australia. Methods Test sera were sourced from 1817 serum samples collected from finisher pigs from 63 herds across Australia in 2001, selected from a larger sample of 180 herds to represent the contribution that each herd size makes to the number of pigs produced. The test sera were the most recent collection of pig sera from all states and samples had been stored at −80°C from 2001 until testing was conducted in 2008. Sera were tested using the BioScreen ELISA. Results All herds tested positive for L. intracellularis-specific antibodies. The mean percentage of positive samples within each herd was 84.2% (range 31.3–100%). Conclusions Lawsonia intracellularis is endemic in pig herds in Australia and cost-effective strategies to reduce reliance on antibiotics, such as vaccination and/or all-in/all-out pig flow coupled with cleaning and disinfection of pens, are warranted.     

Patterns of exposure to Lawsonia intracellularis infection on European pig farms

A serological investigation was made of the patterns of exposure of pigs to Lawsonia intracellularis, the causative agent of proliferative enteropathy (ileitis), on farms in France and Spain. Blood samples from groups of adult female pigs in breeding programmes and from postweaning pigs were monitored, the latter every month for five months, by a L. intracellularis-specific immunofluorescence seroassay. Four of 33 farms monitored in France (12 per cent) and three of 29 farms monitored in Spain (10.3 per cent) remained free of clinical signs and seronegative throughout the study. The postweaning pigs on all of the remaining French farms and on 20 of the 26 remaining Spanish farms had a pattern of infection characterised by seroconversion in the grower period, generally between eight and 16 weeks of age. The seroprevalence in these groups ranged from 8 to 20 per cent. On all of these farms at least 15 per cent of the breeding females tested were seropositive, and the farms were under similar management systems, with a continuous flow of pigs or between buildings on one site, so-called 'one site, farrow-to-finish'. On the six remaining Spanish farms, under two management groups, a multiple-site system was used, with the piglets being separated from the adults at weaning and moved to a separate location. On three of these farms, the pattern of infection was characterised by seroconversion later in the finisher period, at between 16 and 20 weeks of age, and none of the breeding females was seropositive. On the three other multiple-site farms the pattern of infection resembled that on the one-site farms. On all of the farms, the seroconversion of groups of pigs was frequently associated with clinical or subclinical signs of ileitis.     

Efficacy of gallium maltolate against Lawsonia intracellularis infection in a rabbit model

Antimicrobial efficacy against Lawsonia intracellularis is difficult to evaluate in vitro, thus, the effects of gallium maltolate's (GaM) were investigated in a rabbit model for equine proliferative enteropathy (EPE). Juvenile (5–6‐week‐old) does were infected with 3.0 × 10⁸ L. intracellularis/rabbit and allocated into three groups (n = 8). One week postinfection, one group was treated with GaM, 50 mg/kg; one, with doxycycline, 5 mg/kg; and one with a sham‐treatment (control). Feces and blood were collected daily and weekly, respectively, to verify presence of L. intracellularis fecal shedding using qPCR, and seroconversion using immunoperoxidase monolayer assay. Rabbits were sacrificed after 1 week of treatment to collect intestinal tissues focusing on EPE‐affected sections. Intestinal lesions were confirmed via immunohistochemistry. No difference was noted between treatments regarding EPE‐lesions in jejunum (P = 0.51), ileum (P = 0.74), and cecum (P = 0.35), or in L. intracellularis fecal shedding (P = 0.64). GaM and doxycycline appear to have similar efficacy against EPE in infected rabbits.     

Preparation and characterization of polyclonal and monoclonal antibodies against Lawsonia intracellularis.

Proliferative enteropathy is an intestinal infectious disease caused by the obligate intracellular bacterium Lawsonia intracellularis. Immunohistochemistry staining has superior sensitivity over hematoxylin and eosin and silver staining for detecting L. intracellularis in histological sections. A L. intracellularis-specific monoclonal antibody (MAb) produced in the UK (IG4 MAb) has been described in the literature. However, no monoclonal or polyclonal antibodies are commercially available. Therefore, the objective of this study was to produce and characterize new polyclonal and monoclonal antibodies against L. intracellularis that are suitable for diagnostic use. The new monoclonal (2001 MAb) and polyclonal antibodies (1999 PAb) were compared with the IG4 MAb using Western blot analysis of outer membrane proteins (OMPs) of 6 L. intracellularis isolates, Bilophila wadsworthia and Brachyspira hyodysenteriae and using immunohistochemistry of known positive and negative histologic samples and pure cultures of L. intracellularis, B. wadsworthia, B. hyodysenteriae, Salmonella choleraesuis, S. typhimurium, and Escherichia coli K88. Immunogold staining using 2001 MAb was performed to show the specificity of the antibody against an L. intracellularis surface protein. Western blot analysis showed that the 2001 MAb targeted an OMP of 77 kD, which made it different from the IG4 MAb that targeted an 18-kD OMP. The immunogold stain demonstrated the specificity of the 2001 MAb to a surface protein of L. intracellularis. The polyclonal antibody (1999 PAb) targeted 5 OMPs (77, 69, 54, 42, and 36 kD). Both the 2001 MAb and 1999 PAb stained known positive, but not negative, histologic samples. Both the 2001 MAb and 1999 PAb reacted with a pure culture of L. intracellularis but not with any other common enteric pathogens. These two new antibodies will be useful for immunodiagnosis of L. intracellularis.     

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

Background

Porcine 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.

Methods

A 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.

Results

Although 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.

Conclusions

The 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.     

Immunohistochemical detection of Lawsonia intracellularis in tissue sections from pigs

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

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.     

Porcine proliferative enteropathy in Estonian pig herds: histopathology and detection of Lawsonia intracellularis by PCR

Lawsonia intracellularis is the causative agent of proliferative enteritis in pigs (PPE). This bacterium is difficult to culture from clinical samples and antemortem demonstration is therefore usually performed by PCR on faecal samples. The aim of this study was to elucidate the frequency of L. intracellularis infection in pig herds in Estonia using PCR, histopathological methods and electronmicroscopical studies. The frequency of demonstration of L. intracellularis was highest in 9-12 weeks old pigs (68.1%). It was more frequent in growing pigs with enteritis on small farms where the system of "all-in all-out" was not practiced and where standards of hygiene were poor. Gross and histopathological studies demonstrated that characteristic macroscopic changes associated with PPE were localised to the distal jejunum and ileum.Thickened longitudinal and circumferential folds occurred in the mucosa of the affected regions of the bowel. Samples from pigs aged 4 to 20 weeks exhibited the most intensive inflammatory changes. The distal part of the jejunum, ileum and the upper third of proximal colon and cecum wall were visibly thickened with reduced luminal diameter. Hyperplasia of lymphoid tissue and, in many cases, pseudomembranous or fibrinous inflammation was found. L. intracellularis was detected in 56 young pigs using histopathological methods. Additionally, in 8 of these pigs intracellular bacteria were demonstrated in ilial epithelial cells by transmission electronmicroscopical (TEM) investigation. On the basis of these TEM investigations it was concluded that L. intracellularis causes disturbances of normal growth, differentiation and apoptosis of the epithelial cells of ileum.     

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

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

胞内劳森菌及其所致的增殖性肠病

本综述介绍的胞内劳森菌 ,是一种弯曲状杆菌 ,无鞭毛 ,抗酸染色阳性 ,能为Warthin Starry或Lavaditti镀银法着色。该菌存在于病畜肠细胞原生质内 ,至今不能人工培养 ,但可在一些肠细胞培养中生长 ,引起猪增殖性肠病 ,还见于仓鼠、马、鹿、雪貂、大鼠、兔、狐、鸵鸟、鸸鹋等动物。可用病理学、血清学、细菌学、PCR等方法诊断 ,防治可用抗生素 ,如金霉素、tylosin、dimetridiazole、carbadax、timulin等治疗 ,至今无疫苗。     

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

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

Evidence of host adaptation in Lawsonia intracellularis infections

Background

Lawsonia intracellularis is the causative agent of proliferative enteropathy, an endemic disease in pigs and an emerging concern in horses. Enterocyte hyperplasia is a common lesion in every case but there are differences regarding clinical and pathological presentations among affected species. We hypothesize that host susceptibility to L. intracellularis infection depends on the species of origin of the bacterial isolate. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using either a porcine or an equine isolate.

Materials and methods

Twelve foals and eighteen pigs were equally divided into three groups and infected with either a porcine or an equine isolate (10⁹L. Intracellularis/challenged animal), and a saline solution (negative control group). The animals were monitored regarding clinical signs, average of daily weight gain, fecal shedding of the bacteria by PCR and humoral serological response.

Results

Foals infected with the equine isolate developed moderate to severe clinical signs and maintained a lower average of weight gain compared to control foals. Fecal quantitative PCR in equine isolate-infected foals revealed higher amounts of bacterial DNA associated with longer duration of shedding compared with porcine isolate-infected foals. All four foals infected with the equine isolate demonstrated higher IgG titers in the serum compared with porcine isolate-infected foals. In the pig trial, diarrhea and seroconversion were only observed in animals infected with the porcine isolate. Pathological changes typical of proliferative enteropathy were observed in the necropsied foal infected with equine isolate and in the two necropsied pigs infected with the porcine isolate.

Conclusions

Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates. These results show that host susceptibility is driven by the origin of the isolated L. intracellularis strain.     

Evaluation of a blocking ELISA for the detection of antibodies against Lawsonia intracellularis in pig sera

Background

Lawsonia intracellularis is a common cause of chronic diarrhoea and poor performance in young growing pigs. Diagnosis of this obligate intracellular bacterium is based on the demonstration of the microbe or microbial DNA in tissue specimens or faecal samples, or the demonstration of L. intracellularis-specific antibodies in sera. The aim of the present study was to evaluate a blocking ELISA in the detection of serum antibodies to L. intracellularis, by comparison to the previously widely used immunofluorescent antibody test (IFAT).

Methods

Sera were collected from 176 pigs aged 8-12 weeks originating from 24 herds with or without problems with diarrhoea and poor performance in young growing pigs. Sera were analyzed by the blocking ELISA and by IFAT. Bayesian modelling techniques were used to account for the absence of a gold standard test and the results of the blocking ELISA was modelled against the IFAT test with a "2 dependent tests, 2 populations, no gold standard" model.

Results

At the finally selected cut-off value of percent inhibition (PI) 35, the diagnostic sensitivity of the blocking ELISA was 72% and the diagnostic specificity was 93%. The positive predictive value was 0.82 and the negative predictive value was 0.89, at the observed prevalence of 33.5%.

Conclusion

The sensitivity and specificity as evaluated by Bayesian statistic techniques differed from that previously reported. Properties of diagnostic tests may well vary between countries, laboratories and among populations of animals. In the absence of a true gold standard, the importance of validating new methods by appropriate statistical methods and with respect to the target population must be emphasized.     

Detection of Lawsonia intracellularis using immunomagnetic beads and ATP bioluminescence

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

Isolation of Lawsonia intracellularis specific single-chain Fv antibody fragments from phage display library

Single-chain antibodies (scFv) exhibiting specific binding to Lawsonia intracellularis were isolated from a phagemid library expressing scFvs molecules on the surface of filamentous bacteriophages. For scFv selection whole bacterial cells were used and individual clones were tested in ELISA test. The total of seven unique clones with different fingerprint profiles was isolated. All clones were able to bind specifically in immunofluorescence assay. This is the first report of species specific recombinant antibodies against L. intracellularis.     

Transmission of Lawsonia intracellularis to weanling foals using feces from experimentally infected rabbits

The aim of this study was to investigate whether feces from rabbits experimentally infected with Lawsonia intracellularis were infectious to foals. Two rabbits were infected with L. intracellularis, while two rabbits served as controls. Eight foals received daily feces from either the infected or the control rabbits. All rabbits and foals were monitored daily for clinical signs for the entire study period (21 days for rabbits, 42 days for foals). Feces and blood were collected for the PCR detection of L. intracellularis and serologic analysis, respectively. None of the infected rabbits or foals developed clinical signs compatible with proliferative enteropathy. All infected rabbits and foals shed L. intracellularis in their feces and all seroconverted. The results support the role of rabbits as asymptomatic amplifiers of L. intracellularis and their role as sources of infection for susceptible foals.