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.

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 × 10(8) 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.

Pharmacokinetics of gallium maltolate in Lawsonia intracellularis-infected and uninfected rabbits.

Oral gallium maltolate (GaM) pharmacokinetics (PK) and intestinal tissue (IT) concentrations of elemental gallium ([Ga]) and iron ([Fe]) were investigated in a rabbit model of equine proliferative enteropathy (EPE). New Zealand white does (uninfected controls and EPE-infected, n = 6/group) were given a single oral GaM dose (50 mg/kg). Serial blood samples were collected from 0 to 216 h post-treatment (PT) and IT samples after euthanasia. Serology, qPCR, and immunohistochemistry confirmed, or excluded, EPE. Blood and IT [Ga] and [Fe] were determined using inductively coupled plasma-mass spectrometry. PK parameters were estimated through noncompartmental approaches. For all statistical comparisons on [Ga] and [Fe] α = 5%. The Ga log-linear terminal phase rate constant was lower in EPE rabbits vs. uninfected controls [0.0116 ± 0.004 (SD) vs. 0.0171 ± 0.0028 per hour; P = 0.03]; but half-life (59.4 ± 24.0 vs. 39.4 ± 10.8 h; P = 0.12); Cmax (0.50 ± 0.21 vs. 0.59 ± 0.42 µg/mL; P = 0.45); tmax (1.75 ± 0.41 vs. 0.9 ± 0.37 h; P = 0.20); and oral clearance (6.743 ± 1.887 vs. 7.208 ± 2.565 L/h; P = 0.74) were not. IT's [Ga] and [Fe] were higher (P < 0.0001) in controls. In conclusion, although infection reduces IT [Ga] and [Fe], a 48 h GaM dosing interval is appropriate for multidose studies in EPE rabbits.

In vitro antimicrobial activity against equine Lawsonia intracellularis strains.

BACKGROUND: Lawsonia intracellularis is the aetiologic agent of equine proliferative enteropathy (EPE). This emerging equine disease leads to diarrhoea, severe protein loss and can result in death if left untreated. Timely treatment of EPE is critical for recovery from the disease, and hence, information about antimicrobial susceptibilities of equine L. intracellularis strains to antimicrobials used in horses is needed. However, L. intracellularis is an obligate intracellular bacterium and so must be isolated and maintained in cell cultures. OBJECTIVES: To determine the in vitro antimicrobial activity of 14 antimicrobials against two equine L. intracellularis strains. STUDY DESIGN: In vitro experiments. METHODS: This study was designed to compare the relative in vitro susceptibility of each strain of L. intracellularis to different antimicrobials which included metronidazole, minocycline hydrochloride, erythromycin, cephalothin sodium salt, combination (4:1) of sulfamethazine and trimethoprim, chloramphenicol, rifampicin, penicillin, ampicillin, doxycycline hydrochloride, cefazolin sodium salt, clarithromycin, ceftiofur hydrochloride and enrofloxacin. The minimum inhibitory concentration (MIC) was based on intracellular and extracellular activity that inhibited 99% of L. intracellularis growth in cell culture as compared to the antimicrobial-free control. RESULTS: Rifampicin and clarithromycin were the most active antimicrobials against the two L. intracellularis strains tested, with MICs of ≤0.125 when tested both intracellularly and extracellularly. Doxycycline, minocycline, erythromycin, chloramphenicol and enrofloxacin showed intermediate to high activity, and activity was generally higher when evaluating intracellular activity. Sulfamethazine/trimethoprim showed variable results. Ampicillin, penicillin and metronidazole had low to moderate activity. L. intracellularis was resistant to cefazolin, cephalothin and ceftiofur in in vitro conditions. MAIN LIMITATIONS: Only two equine isolates of L. intracellularis were available for this study due to the difficulty in isolating this obligate intracellular species from intestinal samples. CONCLUSIONS: This is the first report of antimicrobial susceptibility patterns for equine L. intracellularis strains.

Lawsonia intracellularis proliferative enteropathy in a foal.

A weanling foal was diagnosed with proliferative enteropathy caused by Lawsonia intracellularis based on history, clinical findings of depression, anorexia, weight loss, colic, diarrhea, and ventral edema, and a combination of serology and fecal PCR. An epidemiological investigation on the premises revealed that many of the other foals and adult horses were seropositive for L. intracellularis, despite being clinically normal, and identified a dog as a potential carrier and source of infection for the foal. The foal was successfully treated with a combination of azithromycin and rifampin.

Polymerase chain reaction for diagnosis of porcine proliferative enteropathy.

A polymerase chain reaction (PCR) assay for detection of the intracellular bacteria, ileal symbiont intracellularis of porcine proliferative enteropathy is described. The test is based on specific DNA primers and gave positive PCR product from samples of preserved intestinal mucosa and faeces from affected pigs. Mucosa and faeces from normal pigs gave no positive PCR products. The identity of the PCR product was confirmed by DNA-DNA hybridization with a probe, pCLO78, specific for IS intracellularis. Positive results were only observed in animals with active lesions of proliferative enteropathy. PCR is probably the most useful method for diagnosis of proliferative enteropathy that is currently available for live animals.

Southern blot analysis of strain variation in Campylobacter mucosalis.

A panel of three DNA probes were derived at random from a genomic DNA library of Campylobacter mucosalis strain E8384-4. Each probe hybridized specifically to C. mucosalis DNA from bacteria fixed to nylon membranes. The probes did not hybridize to DNA from other Campylobacter species or to other bacteria even at 100-fold higher amounts. Each probe hybridized to all of 24 isolates of C. mucosalis which had been collected over time from different geographic locations. Southern blot analysis of selected C. mucosalis isolates was carried out to determine if the probes would be useful for differentiating among various isolates. It indicated that restriction fragment length polymorphisms (RFLPs) exist at the loci identified by our probes. These differences were used to characterize seven C. mucosalis isolates recovered from pigs in Minnesota. The results suggest that RFLP analysis may be a useful tool for epidemiological studies of C. mucosalis.

Diagnosis of proliferative enteritis in frozen and formalin-fixed, paraffin-embedded tissues from a hamster, horse, deer and ostrich using a Lawsonia intracellularis-specific multiplex PCR assay.

Proliferative enteritis (PE) is an enteric disease that has been reported in a variety of animals. It is caused by an obligate intracellular bacterium identified in swine as Lawsonia intracellularis. The organism can be detected ante-mortem in swine with PE using molecular diagnostic methods. The disease can be diagnosed post-mortem in all species by gross examination of tissues and special histologic staining procedures. In this study we extracted total DNA from frozen or formalin-fixed, paraffin-embedded tissues from cases of pig, hamster, horse, deer and ostrich PE. The samples were subjected to a multiplex PCR reaction using primers specific for a swine isolate of L. intracellularis. Identical sized PCR products were detected in samples from all animals with PE and the specificity of the PCR reaction for L. intracellularis was demonstrated by Southern-blotting and hybridization using specific probes. These results suggest that the intracellular organism of PE in these species are all very closely related to the causative agent of PE in swine, L. intracellularis. In addition, this multiplex PCR assay can be used to detect the organism in frozen or archival tissues, facilitating retrospective diagnosis of PE.

Species-specific DNA probes for Campylobacter species isolated from pigs with proliferative enteritis.

Cloned, chromosomal DNA probes from porcine isolates of Campylobacter hyointestinalis and C. mucosalis were developed for the detection and identification of these putative swine enteric pathogens. High molecular weight chromosomal DNA from each species was used to construct genomic libraries in plasmids. Recombinants were selected which hybridized strongly to the homologous organism, but not to any other species of Campylobacter. Species-specific recombinants were labeled with phosphorus-32 and tested for sensitivity by dot blot hybridization to various dilutions of DNA and bacteria from each swine species, including C. hyointestinalis, C. mucosalis, C. coli and C. jejuni. Specificity was tested by hybridizing these probes against various strains of C. hyointestinalis or C. mucosalis, and against reference strains of all other described Campylobacter species. A C. hyointestinalis-specific probe and a C. mucosalis-specific probe were identified which were capable of detecting 1 ng of DNA or 10(4) cfu by bacterial spot blotting on nylon membranes. These probes hybridized to intestinal mucosal scrapings containing C. hyointestinalis and C. mucosalis obtained from pigs with proliferative enteritis, but not to material from normal pigs. Thus, cloned, chromosomal DNA probes may be useful in the detection and identification of bacteria involved in swine proliferative enteritis.

Multiplex polymerase chain reaction for simultaneous detection of Lawsonia intracellularis, Serpulina hyodysenteriae, and salmonellae in porcine intestinal specimens.

Proliferative enteritis, swine dysentery, and porcine salmonellosis are the most common enteric bacterial diseases affecting pigs in the growing and finishing stages of production. Currently, diagnoses of these diseases by standard cultural techniques of intestinal specimens can be laborious, time consuming, and expensive (swine dysentery, porcine salmonellosis) or impossible (proliferative enteritis). Amplification by polymerase chain reaction (PCR) of DNA sequences specific for each bacterial agent is a highly sensitive and specific method that overcomes the limitations associated with standard detection methods. A multiplex PCR (M-PCR) assay was developed for simultaneous detection and identification of the etiologic agents associated with proliferative enteritis, swine dysentery, and porcine salmonellosis in a single reaction using total DNA obtained directly from intestinal specimens. Purified DNA obtained from pure cultures of each bacterial agent alone or mixed in different combinations and concentrations and total DNA from intestinal specimens were amplified using the Lawsonia intracellularis-, Serpulina hyodysenteriae-, and salmonellae-specific M-PCR assay. Intestinal specimens consisted of feces and mucosal scrapings obtained from field cases of each disease alone or in combinations and feces obtained from pigs challenged with S. hyodysenteriae. The banding pattern of the amplified PCR products, after agarose gel electrophoresis and staining, indicated the presence of individual or combinations of etiologic agents in each specimen. Results from this study indicated that simultaneous amplification of L. intracellularis-, S. hyodysenteriae-, and salmonellae-specific DNA sequences by M-PCR can be used for specific detection and identification of three major enteric bacterial pathogens associated with proliferative enteritis, swine dysentery, and porcine salmonellosis occurring alone or in combinations. Also, the M-PCR assay can be done using DNA obtained directly from intestinal specimens submitted for diagnostic investigation.

Developed and resolving lesions in porcine proliferative enteropathy: possible pathogenetic mechanisms.

Proliferative enteropathy, caused by Lawsonia intracellularis, offers the opportunity to examine bacterial mechanisms that influence epithelial cell proliferation. Ultrastructural features of developed and resolving lesions included the presence of enlarged intestinal crypts containing undifferentiated immature epithelial cells and an absence of goblet cells. Numerous intracytoplasmic bacteria, identified as L. intracellularis, were consistently present within affected cells. In recovering intestinal tissue, additional features were (1) the common presence of pale, swollen, protruding epithelial cells, (2) shrunken, degenerate epithelial cells, (3) apoptotic bodies in both epithelial cells and macrophages, (4) the reappearance of normal goblet cells, and (5) reduced numbers of L. intracellularis within lesions. Bacteria were released from cells via cytoplasmic and cellular protrusions into the intestinal lumen. It is speculated that the presence of the intracytoplasmic bacterium, L. intracellularis, may disrupt normal processes of cell growth, differentiation or apoptosis in the intestinal epithelium.

Equine proliferative enteropathy: a cause of weight loss, colic, diarrhoea and hypoproteinaemia in foals on three breeding farms in Canada.

Proliferative enteropathy (PE) is a transmissible enteric disease caused by Lawsonia intracellularis. An outbreak of equine PE was diagnosed in foals from 3 breeding farms. Most foals had been weaned prior to the appearance of clinical signs, which included depression, rapid and marked weight loss, subcutaneous oedema, diarrhoea and colic. Poor body condition with a rough haircoat and a potbellied appearance were common findings in affected foals. Respiratory tract infection, dermatitis and intestinal parasitism were also found in some foals. Haematological and plasma biochemical abnormalities included hypoproteinaemia, transient leucocytosis, anaemia and increased serum creatinine kinase concentration. Postmortem diagnosis of PE was confirmed on 4 foals based on the presence of characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells of the intestinal mucosa, using silver stains, and by results of PCR analysis and immunohistochemistry. Antemortem diagnosis of equine PE was based on the clinical signs, hypoproteinaemia and the exclusion of common enteric infections. Faecal PCR analysis was positive for the presence of L. intracellularis in 6 of 18 foals tested while the serum of all 7 foals with PE serologically evaluated had antibodies against L. intracellularis. Most foals were treated with erythromycin estolate alone or combined with rifampin for a minimum of 21 days. Additional symptomatic treatments were administered when indicated. All but one foal treated with erythromycin survived the infection. This study indicates that equine PE should be included in the differential diagnosis of outbreaks of rapid weight loss, diarrhoea, colic and hypoproteinaemia in weanling foals.

Porcine proliferative enteritis: serological, microbiological and pathological studies from three field epizootics.

Three outbreaks of porcine proliferative enteritis were evaluated clinically, pathologically, microbiologically and serologically. The disease was characterized by a chronic intermittent diarrhea. Pathological lesions included a thickened, turbid ileum with the microscopic appearance of proliferating ileal crypt epithelial cells. Comma shaped intracytoplasmic organisms were observed in the apical portions of the proliferating crypt epithelial cells with a Warthin-Starry silver stain. Microbiologically, both Campylobacter sputorum subspecies mucosalis and Campylobacter hyointestinalis, were cultured from ileal specimens of seven pigs with lesions of porcine proliferative enteritis. Microagglutination antibody titers were determined on sera from 12 of 14 pigs with porcine proliferative enteritis and on sera from 91 clinically normal swine. Pigs with porcine proliferative enteritis had a low antibody titer to subspecies mucosalis that ranged from 1-3 with a mean of 2.17. A varied C. hyointestinalis titer from 3-7 with mean of 4.83 was determined. Titers to either subspecies mucosalis and C. hyointestinalis were higher in non-porcine proliferative enteritis pigs. The results indicate that the presence of a positive titer to either C. hyointestinalis or subspecies mucosalis in swine is not indicative of clinical disease. The isolation of C. hyointestinalis from diseased ileal specimens (porcine proliferative enteritis) confirms previous reports implicating this agent in the disease.

Campylobacter microagglutination tests of swine with proliferative enteritis.

A microagglutination test was developed to determine campylobacter titers in swine with proliferative enteritis. Formalinized whole cell antigens from 24 Campylobacter isolates, including C hyointestinalis (CHI), C sputorum ss mucosalis (CSM), C jejuni/coli (CJC), C fetus ss fetus (CFF), and C fecalis (CF), were tested with 9 rabbit antisera prepared against each of 3 strains of CHI, CSM, and CJC. The CHI appeared to be antigenically homogeneous. All 6 isolates of CHI agglutinated with homologous antisera at high dilutions and did not react with CSM antisera. Five of 6 isolates of CSM agglutinated with homologous antisera, whereas 1 isolate did not. Seven strains of CJC autoagglutinated in saline solution and various antisera. One of 3 CJC antisera, however, cross-reacted with CHI and CSM antigens at high dilutions. The antigens from 5 strains of CFF and CF did not react with CHI, CSM, and CJC antisera. A survey of sera from 1,052 adult pigs from production herds indicated that the majority had high titers to CHI and CSM (mean, in log2: CHI = 5.57, CSM = 6.05). Similar titers were found in weaned pigs from 3 herds with the disease and 2 of 3 herds without the disease. Pigs with confirmed lesions of proliferative enteritis, however, had low titers (mean in log2: CHI = 2.44, CSM = 3.11). Agglutinating antibodies to CHI and CSM were transmitted from farrowing gilts to neonatal pigs via colostrum. The acquired antibodies decayed to low levels in pigs at 4 weeks of age (mean in log2: CHI = 1.09, CSM = 1.27).

Campylobacter hyointestinalis (new species) isolated from swine with lesions of proliferative ileitis.

Intestines from 48 swine with enteric disease were examined by bacteriologic cultural technique for the presence of various Campylobacter species. Histopathologic techniques were used to determine whether the submitted specimens had lesions of either swine proliferative ileitis or other enteric diseases. Three species of Campylobacter were identified as Campylobacter jejuni/coli, Campylobacter sputorum ss mucosalis, and Campylobacter hyointestinalis (proposed new species) on the basis of biochemical characteristics and response to various inhibitory substances. The C hyointestinalis was isolated from 18 of 27 (67%) swine with proliferative ileitis and from only 1 of 21 (5%) swine with other enteric diseases. The C sputorum ss mucosalis was obtained from 16 of 27 (59%) swine with proliferative ileitis and from 2 of 21 (10%) swine with other enteric disease. The C jejuni/coli was isolated from 2 of 27 (7%) swine with proliferative ileitis and from 8 of 21 (38%) swine with other enteric disease. The new organism, C hyointestinalis, was catalase-positive, hydrogen sulfide positive in triple sugar iron agar, glycine tolerant, intolerant to 3.0% sodium chloride, able to grow at 25 C, sensitive to cephalothin, and resistant to nalidixic acid. On the basis of these characteristics, C hyointestinalis was differentiated from other campylobacters isolated from swine and from other sources.

Immunofluorescent demonstration of Campylobacter hyointestinalis and Campylobacter sputorum subsp mucosalis in swine intestines with lesions of proliferative enteritis.

An indirect fluorescent antibody technique was developed to identify Campylobacter spp in lesions of swine proliferative enteritis (SPE). Rabbit antisera to C hyointestinalis and C sputorum subsp mucosalis were produced. Bacterial smears stained by fluorescent antibody test with homologous antisera differentiated C hyointestinalis from subsp mucosalis. Ileal frozen sections from 29 pigs with histologic lesions of SPE had specific fluorescent staining of C hyointestinalis in all 29 and subsp mucosalis in 24. Bacterial structures of C hyointestinalis were seen in large numbers and were broadly distributed in intestinal luminal exudate, mucosal necrotic tissues, surface epithelium, lamina propria, and proliferative cryptal epithelium. Numerous C hyointestinalis organisms were always present in the apical cytoplasm of proliferative cryptal epithelium. Fluorescent subsp mucosalis bacteria were seen less frequently and were distributed focally in the mucosa. Numerous subsp mucosalis organisms were more common in cellular debris and in necrotic tissues of surface mucosa, and less common in the epithelial cells of proliferative crypts. Ileal sections from 13 pigs without SPE had no fluorescent staining of C hyointestinalis and subsp mucosalis.

Transmission of proliferative enteritis to swine by use of embryonating chicken eggs.

Embryonating eggs were inoculated with filtered porcine ileal mucosa containing intracellular curved rods (ICR) and incubated for 4 to 6 days. Three of 12 pigs given the eggs per os developed microscopic lesions of proliferative enteritis (PE). Nonchallenge-exposed control pigs did not develop lesions of PE. Four of six positive control pigs given ileal mucosa from pigs with PE also developed microscopic lesions of PE. All of the PE lesions were found in pigs necropsied 10 to 29 days after challenge exposure. None of the swine in the study had clinical signs or gross lesions of PE. Campylobacter spp were isolated from pigs with and without exposure to the ileal mucosa from pigs with PE. There was no relationship between Campylobacter spp isolation and development of lesions. Deoxyribonucleic acids extracted from embryonating chicken eggs injected with the equivalent of 0.5 mg of mucosal lesions and incubated for 4 days hybridized to a DNA probe specific for the ICR, whereas DNA extracted from 1.5 mg of mucosal homogenates of the same proliferative tissue did not hybridize with the same probe. Results of these experiments indicated that ICR injected into eggs remained infective for pigs and suggest replication of ICR in the first-passage eggs.

Use of a DNA probe to detect the intracellular organism of proliferative enteritis in swine feces.

A method of extracting bacterial DNA from swine feces was developed and used in a molecular assay for the presence of ileal symbiont (IS) intracellularis, formerly known as the Campylobacter-like organism associated with swine with proliferative enteritis. Hybridization with a digoxigenin-labeled, IS intracellularis-specific probe detected the presence of IS intracellularis at a concentration of 10(7) organisms/g of feces. This method was sufficient to detect IS intracellularis in the feces of swine with experimentally induced and naturally acquired infection. Results of the hybridization were in agreement with those from histologic postmortem examination.

Equine proliferative enteropathy--a review of recent developments.

Equine proliferative enteropathy (EPE) is a disease of foals caused by the obligate intracellular organism Lawsonia intracellularis. This emerging disease affects mainly weanling foals and causes fever, lethargy, peripheral oedema, diarrhoea, colic and weight loss. The diagnosis of EPE may be challenging and relies on the presence of hypoproteinaemia, thickening of segments of the small intestinal wall observed upon abdominal ultrasonography, positive serology and molecular detection of L. intracellularis in faeces. Although the clinical entity, diagnostic approach and treatment of EPE are well established and described, the epidemiology for this disease has remained largely unaddressed. This article focuses on new developments in the field of EPE, including epidemiology, pathophysiology, clinical signs, diagnosis, treatment and prevention.

Evaluation of the field efficacy of an avirulent live Lawsonia intracellularis vaccine in foals.

Equine proliferative enteropathy caused by Lawsonia intracellularis is an emerging disease with as yet unaddressed preventative measures. The hypothesis of this study was that vaccination will prevent clinical and sub-clinical disease. Weanling Thoroughbreds (n=202) from Central Kentucky were randomly assigned into two groups (vaccinated and non-vaccinated). Vaccinated foals received 30 mL of an avirulent, live L. intracellularis vaccine intra-rectally twice, 30 days apart. Foals were monitored for clinical disease, total solids and average weight gain until yearling age. There was an overall decreased disease incidence on the farms involved in the study that did not differ significantly between the groups. This decreased disease prevalence in the study population may be associated with the ongoing vaccine trial on these farms, as disease prevalence in Central Kentucky did not change in 2009 compared to 2008.