High Seroprevalence of Lawsonia intracellularis in Thoroughbred Farms in Southern Brazil.

The aim of the present study was to carry out a serological survey to identify the seroprevalence of Lawsonia intracellularis in six Thoroughbred farms in the Southern region of the state of Rio Grande do Sul, Brazil. During 2019 and 2020, blood samples from 686 Thoroughbred horses were obtained from six different breeding farms. Horses were divided into groups according to age: (1) broodmares (>5 years), (2) two-year-old foals, (3) yearlings, and (4) 0-6 months-old foals. Blood samples were collected by venipuncture of the external jugular vein. The detection of antibodies (IgG) against L. intracellularis was performed by Immunoperoxidase Monolayer Assay. The detection of specific antibodies (IgG) against L. intracellularis in the evaluated population was 51%. The highest detection (86.8%) of IgG was in the broodmares category, while the lowest (5.2%) was in foals of 0-6 months of age. Regarding the farms, the Farm 1 had the highest (67.4%) prevalence of seropositivity against L. intracellularis, while Farm 4 had the lowest (30.6%). There was no record of clinical manifestation of Equine Proliferative Enteropathy in the sampled animals. The results of this study show the high seroprevalence of L. intracellularis in Thoroughbred farms in the Southern of Rio Grande do Sul, suggesting a large and continuous exposure to the agent.

Productivity parameters, antimicrobial consumption, and prevalence of enteric pathogens before and after intramuscular vaccination against Lawsonia intracellularis in naturally infected Danish weaner and finisher pig herds.

In Danish pig production, gastro-intestinal diseases account for most of the antimicrobials (AM) used in growing pigs. Diarrhoea is most frequently caused by Lawsonia intracellularis (LI), Brachyspira pilosicoli (BP), E coli fimbria type F4 (F4) and E. coli fimbria type F18 (F18). With a new LI vaccine available from 2019, it was relevant to investigate the effect of this vaccine in a Danish field study including both weaner and finisher sites. The aim was to evaluate the efficacy of Porcilis® Lawsonia Vet. in naturally LI-infected pig herds by comparing of productivity parameters, AM consumption and dynamics of enteric pathogens over two 6-months periods before and after LI vaccination. Further, faecal sock samples were collected from each site before and after vaccination and analysed by qPCR for excretion levels of LI, BP, F18 and F4. In total, 28 weaner and 41 finisher sites were included in the study. Vaccination reduced Feed Conversion Ratio by 0.12 Feed Unit/kg (p = 0.029) and 0.08 Feed Unit/kg (p = 0.005) in weaners and finishers, respectively. Increased Average Daily Weight Gain of 45.6 gr./day (p < 0.001) was found in the finishers. Mortality risk fell by 8.8% in weaners (RR = 0.912; p < 0.001). AM prescriptions for oral group treatments were reduced by 38.8% active compound/kg pig produced (p = 0.005) or 33.3% Weighted Animal Daily Doses per 100 animals per day in finishers (p = 0.004). LI prevalence was reduced in weaners and finishers (both p < 0.001) and BP prevalence was reduced in finishers (p = 0.043). Mean excretion levels of LI and BP decreased at weaner sites (-1.32 and -1.02 log(10) copies/gr faeces, respectively; both p < 0.001) and at finisher sites (-1.04 and -1.16 log(10) copies/gr faeces, respectively; both p < 0.001). Prevalence and excretion levels of F18 and F4 were unaffected by LI vaccination. In conclusion, vaccination against LI using Porcilis® Lawsonia Vet. improved productivity parameters, cut AM consumption, and reduced prevalence and excretion levels of LI and BP in naturally LI-infected pig herds.

Development and validation of a flow cytometry antibody test for Lawsonia intracellularis.

Lawsonia intracellularis is the etiologic agent of porcine proliferative enteropathy (PPE), an inflammatory bowel disease with a major economic impact on the pig industry. The serological diagnosis of PPE can be performed using Blocking or Indirect ELISA, Immunoperoxidase Monolayer Assay (IPMA) and Indirect Fluorescence Antibody Test (IFAT). Here, we designed a most sophisticated immunological method for the detection of porcine anti-L. intracellularis IgGs, named Flow Cytometry Antibody Test - FCAT. This assay uses whole, live-attenuated L. intracellularis bacteria derived from a commercial vaccine. For the assay, we set up the optimal antigen concentration (106 bacterium/assay), primary antibody dilution (1:100), time of incubation (20 min), antigen stability (15 days), precision (coefficient of variation - CV < 10%), reproducibility (CV ≤ 13%) and Receiver Operating Characteristic (ROC). When using a cut-off of >15.15% for FCAT, we determined that it showed a sensitivity of 98.8% and specificity of 100%. The rate of agreement with IPMA was 84.09% with a kappa index of 0.66. FCAT was used to screen 1,000 sera from non-vaccinated pigs housed in 22 different farms and we found that 730 pigs (73%) from 16 farms (72.7%) had L. intracellularis IgG. This high prevalence confirms that L. intracellularis is endemic on Brazilian pig farms. Finally, we determined that FCAT is an easy to perform diagnostic assay and we would highly recommend it for: i) seroepidemiological studies; ii) evaluation of infection dynamics; and iii) characterization of the humoral response profile induced by vaccines.

Effect of intramuscular vaccination against Lawsonia intracellularis on production parameters, diarrhea occurrence, antimicrobial treatment, bacterial shedding, and lean meat percentage in two Danish naturally infected finisher pig herds.

Lawsonia intracellularis (LI) is an economically important enteric pathogen in pigs with a worldwide endemic prevalence. The objective of this study was to evaluate the effect of an intramuscularly administrated LI vaccine (Porcilis®Lawsonia Vet.) in Danish finisher pigs (30-115 kg) measured on key production figures, antimicrobial (AB) treatments, occurrence of diarrhea and LI shedding. The study was a group-randomized block-trial with parallel groups in two herds, Herd 1 and Herd 2, experiencing a natural subclinical-clinical LI infection in early finisher period. Vaccination occurred at weaning, but the study focused on the first eight weeks in the finisher period. Further, slaughterhouse data were included. In total, 52 and 50 finisher pens comprising 2184 and 2254 finisher pigs were included in each of two herds, respectively. LI vaccination significantly reduced feed conversion ratio (FCR) by 0.05 and 0.09 FU/kg (p = 0.007 and p < 0.001) alongside a significantly increased average daily weight gain (ADWG) by 31 and 43 gr/day (p = 0.001 and p < 0.001) in each of the herds, respectively. In the vaccinated group, less variation was found in ADWG compared to the control group (p < 0.001 in both herds) as an expression of a more uniform growth, which was further confirmed by less variation in lean meat percent in the vaccinated group in one herd (p = 0.007). No significant difference between groups were found in mortality and pigs excluded due to welfare reasons. AB flock treatment against diarrhea was significantly reduced in Herd 1 with all pens treated in the control group compared to 30.8 % in the vaccinated group (p < 0.001). In Herd 2, the difference was non-significant with 68.0 % in the control group compared to 50.0 % in the vaccination group (p = 0.252). Low levels of individual treatments against diarrhea were seen in both herds (≤ 5.0 %) but still significantly reduced in vaccinated pigs compared to control pigs (p < 0.050 in both herds). Mean diarrheic blot counts were significantly reduced in vaccinated pens compared to control pens (p < 0.001 in both herds). In vaccinated pigs, shedding of LI was reduced in both prevalence (p < 0.001 in both herds), excretion level in positive samples (p < 0.001 in both herds) and, in one herd, also in duration (p = 0.003) when compared to control pigs. In conclusion, pigs vaccinated with Porcilis®Lawsonia Vet against LI in both of two high-health and high-productive finisher herds had, compared to non-vaccinated pigs, significantly improved key production figures, and reduced AB treatment, occurrence of diarrhea, LI shedding, and growth variation.

Infection of juvenile falcons (Falco spp.) with intestinal Lawsonia intracellularis.

Intestinal infection of many host species with Lawsonia intracellularis are widely reported. Analyses of infections among carnivorous falcons have not previously been reported. Fifty juvenile captive falcons (Falco spp.) with or without Lawsonia infection were investigated in the United Arab Emirates, including clinical laboratory methods. Fresh intestinal biopsy samples were analysed by microbiological techniques for Lawsonia and other bacteria and by standard parasitological and pathological methods. Lawsonia intracellularis infection was diagnosed by microbiological examination and qPCR in 10 of 50 juvenile falcons at case examination. Seven of these 10 falcons were of normal clinical appearance, and the other three had other contributing factors to ill-thrift. A range of other conditions were noted in 40 case control falcons. This first report of Lawsonia infection in falcons suggests that the agent may have a limited contribution to clinical disease in these birds, including ill-thrift syndromes. This lack of clinical disease association mimics that noted among Lawsonia infections recorded in other avian families.

Isolation and In Vitro cultivation of Lawsonia intracellularis from China.

Lawsonia intracellularis is an obligate intracellular bacterium that cannot be cultured by conventional bacteriological methods. Pigs infected with L. intracellularis suffer from decreased daily weight gain and poor feed conversion ratio. China is a large producer of pigs, but epidemiological investigation data of L. intracellularis has not been obtained in recent years. Additionally, there is no information about a L. intracellularis strain being successfully isolated and established in cell culture in China, and the above shortcomings limit understanding of the pathogenesis of L. intracellularis and alternative prevention and control methods. The aims of this study were to estimate the seroprevalence of L. intracellularis antibodies in eight major pig-producing provinces in China during 2019-2020, to isolate L. intracellularis from infected intestines and then to establish an infection model of L. intracellularis in mice. Our results showed that of the 3586 serum samples, 2837 (79.1%, 95% CI: 77.7%, 80.4%) were seropositive for the L. intracellularis antibody. Subsequently, the L. intracellularis strain LJS19051 from China was successfully isolated and established in cell culture. Furthermore, L. intracellularis DNA and antibodies could be detected in the feces and serum samples of infected mice, respectively. Moreover, infected crypts showed typical proliferative enteropathies (PE) lesions and L. intracellularis antigen was detected in infected mice by immunofluorescence at 28 days post inoculation. The results indicated that the new L. intracellularis strain LJS19051 was obtained and could successfully proliferate in ICR mice.

Evaluation of host and bacterial gene modulation during Lawsonia intracellularis infection in immunocompetent C57BL/6 mouse model.

BACKGROUND: Proliferative enteritis caused by Lawsonia intracellularis undermines the economic stability of the swine industry worldwide. The development of cost-effective animal models to study the pathophysiology of the disease will help develop strategies to counter this bacterium. OBJECTIVES: This study focused on establishing a model of gastrointestinal (GI) infection of L. intracellularis in C57BL/6 mice to evaluate the disease progression and lesions of proliferative enteropathy (PE) in murine GI tissue. METHODS: We assessed the murine mucosal and cell-mediated immune responses generated in response to inoculation with L. intracellularis. RESULTS: The mice developed characteristic lesions of the disease and shed L. intracellularis in the feces following oral inoculation with 5 × l07 bacteria. An increase in L. intracellularis 16s rRNA and groEL copies in the intestine of infected mice indicated intestinal dissemination of the bacteria. The C57BL/6 mice appeared capable of modulating humoral and cell-mediated immune responses to L. intracellularis infection. Notably, the expression of genes for the vitamin B12 receptor and for secreted and membrane-bound mucins were downregulated in L. intracellularis -infected mice. Furthermore, L. intracellularis colonization of the mouse intestine was confirmed by the immunohistochemistry and western blot analyses. CONCLUSIONS: This is the first study demonstrating the contributions of bacterial chaperonin and host nutrient genes to PE using an immunocompetent mouse model. This mouse infection model may serve as a platform from which to study L. intracellularis infection and develop potential vaccination and therapeutic strategies to treat PE.

Equine Proliferative Enteropathy in Weanling Foals on A German Breeding Farm: Clinical Course, Treatment and Long-Term Outcome.

The goal of the current report was to describe the clinical signs, therapy and outcome of foals with suspected equine proliferative enteropathy (EPE) due to an infection with Lawsonia intracellularis. Forty foals, born on the same breeding farm, were diagnosed with suspected clinical EPE between September 2019 and January 2020. Data of these cases were analyzed retrospectively regarding the course of the disease, treatment, outcome and long-term prognosis. All horses, including randomly selected control horses, were reassessed about nine months after the suspicion of EPE. The horses affected were between 5 and 10 months of age. Fever was the most common clinical sign. Hypoproteinemia was shown consistently in all cases. Seroconversion was detected in all horses affected, while fecal shedding of Lawsonia intracellularis via qualitative real-time polymerase chain reaction was only found in 21 cases. Treatment was based on tetracyclines and the administration of equine plasma IV. A total of 39 of 40 foals survived EPE. No long-term effects in terms of poor body condition or abnormal blood values were observed. If diagnosed and treated early, EPE can generally be described as a disease with a good prognosis and no long-term effects in Warmblood horses.

Characterisation of autophagy disruption in the ileum of pigs infected with Lawsonia intracellularis.

Lawsonia intracellularis is the aetiological agent of proliferative enteropathy, an enteric disease endemic in swine. Survival in its intracellular niche of the ileum epithelial lining requires the capacity to subvert, repress or exploit the host immune response to create an environment conducive to bacterial propagation. To better understand how L. intracellularis survives in its intracellular niche, we have performed an investigation into the dynamic relationship between infection and the host autophagy response by immunohistochemistry in experimentally infected porcine ileum samples.Beclin1, a protein required early in the autophagy pathway was observed to be distributed with a basal to apical concentration gradient in the crypts of healthy piglets, whilst infected piglets were observed to have no gradient of distribution and an increase in the presence of Beclin1 in crypts with histological characteristics of L. intracellularis residence. Detecting microtubule-associated protein light chain 3 (LC3) is used as a method for monitoring autophagy progression as it associates with mature autophagosomes. For LC3 there was no notable change in signal intensity between crypts with characteristic L. intracellularis infection and healthy crypts of uninfected pigs. Finally, as p62 is degraded with the internal substrate of an autophagosome it was used to measure autophagic flux. There was no observed reduction or redistribution of p62.These preliminary results of the autophagy response in the ileum suggest that L. intracellularis affects autophagy. This disruption to host ileum homeostasis may provide a mechanism that assists in bacterial propagation and contributes to pathogenesis.

Evaluation of immune efficacy of Omp2 protein against Lawsonia intracellularis in mice.

Porcine proliferative enteropathy (PPE) is caused by the obligate intracellular bacterium Lawsonia intracellularis. Infection results in an enteric disease characterised by decreased growth performance of pigs, and presents a major economic burden for swine industries worldwide. Since vaccination is an effective technique for controlling PPE, novel effective vaccine platforms are need to be developed. In this study, five proteins of L. intracellularis were screened through animal experiments and the highly immunoprotective Omp2 protein was identified. Then, the immune efficacy of Omp2 was further evaluated based on humoral and cell mediated immune (CMI) responses, faecal bacterial shedding, histopathological lesions, immune barrier function of intestinal mucosa as well as digestive and absorptive capacity following challenge of mice with L. intracellularis. Mice immunised with Omp2 had reduced faecal shedding, fewer histopathological lesions and reduced bacteria colonisation of the ileum. Additionally, Omp2 immunised mice showed stronger serum IgG and IFN-γ levels, up-regulated Occludin and zonula occludens-1 (ZO-1) mRNA levels, as well as increased numbers of intestinal intraepithelial lymphocytes (IELs) and levels of sIgA. On the contrary, the activities of LPS, α-AMS and AKP were significantly increased. Our investigation indicated that immunization with Omp2 reduced the severity of clinical signs and provided efficacious immunoprotection for target animals against L. intracellularis infection in mouse model.

Salmonella delivered Lawsonia intracellularis novel epitope-fusion vaccines enhance immunogenicity and confers protection against Lawsonia intracellularis in mice.

Attenuated Salmonella-mediated vaccine constructs were designed by employing selected discontinuous immunodominant epitopes of LatA, FliC, and PAL antigens of Lawsonia intracellularis to create vaccines against porcine proliferative enteropathy (PPE). Whole protein sequences were subjected to in silico prediction of dominant epitopes, the stability of fusions, and hydropathicity and to ensure that the fused epitopes were feasible for expression in a Salmonella system. Two fusion constructs, one comprising LatA epitopes and the other FliC-PAL-FliC epitopes, were built into a prokaryotic constitutive expression system and transformed into the auxotrophic Salmonella host strain JOL1800. Epitope selection eliminated the majority of less immunodominant regions of target proteins and resulted in an efficient secretion platform that induced significant protective responses. Overall, our results demonstrated that the Salmonella-mediated LI- multi-epitope vaccines elicited significant humoral and cellular immune responses. Additionally, the challenge study suggested that the vaccinated mice were protected against experimental Lawsonia intracellularis infection. Based on the outcomes of the study, Salmonella-mediated LI- multi-epitope vaccines have the potential to prevent PPE.

Lawsonia intracellularis infected enterocytes lack sucrase-isomaltase which contributes to reduced pig digestive capacity.

Lawsonia intracellularis is endemic to swine herds worldwide, however much is still unknown regarding its impact on intestinal function. Thus, this study aimed to characterize the impact of L. intracellularis on digestive function, and how vaccination mitigates these impacts. Thirty-six L. intracellularis negative barrows were assigned to treatment groups (n = 12/trt): (1) nonvaccinated, L. intracellularis negative (NC); (2) nonvaccinated, L intracellularis challenged (PC); and (3) L. intracellularis challenged, vaccinated (Enterisol® Ileitis, Boehringer Ingelheim) 7 weeks pre-challenge (VAC). On days post-inoculation (dpi) 0 PC and VAC pigs were inoculated with L. intracellularis. From dpi 19-21 fecal samples were collected for apparent total tract digestibility (ATTD) and at dpi 21, pigs were euthanized for sample collection. Post-inoculation, ADG was reduced in PC pigs compared with NC (41%, P < 0.001) and VAC (25%, P < 0.001) pigs. Ileal gross lesion severity was greater in PC pigs compared with NC (P = 0.003) and VAC (P = 0.018) pigs. Dry matter, organic matter, nitrogen, and energy ATTD were reduced in PC pigs compared with NC pigs (P ≤ 0.001 for all). RNAscope in situ hybridization revealed abolition of sucrase-isomaltase transcript in the ileum of PC pigs compared with NC and VAC pigs (P < 0.01). Conversely, abundance of stem cell signaling markers Wnt3, Hes1, and p27Kip1 were increased in PC pigs compared with NC pigs (P ≤ 0.085). Taken together, these data demonstrate that reduced digestibility during L. intracellularis challenge is partially driven by abolition of digestive machinery in lesioned tissue. Further, vaccination mitigated several of these effects, likely from lower bacterial burden and reduced disease severity.

PREVALENCE OF LAWSONIA INTRACELLULARIS INFECTION IN NONHUMAN PRIMATES AND PEST RODENTS IN A ZOOLOGICAL COLLECTION.

In 2016 and 2017, Lawsonia intracellularis was isolated from several pileated gibbons (Hylobates pileatus) presenting with diarrhea in Mulhouse Zoo (eastern France). To this day, infection with this bacterium has rarely been described in nonhuman primates (NHP) in captivity or in the wild and there are no data about the prevalence or transmission of the disease. This study focuses on finding the prevalence of this infection amongst Mulhouse Zoo's NHP collection and trying to identify a source of contamination responsible for this epizooty. Forty-eight real-time PCR were conducted on feces from all NHP species in the zoo and on small mammals trapped in the NHP housing structures. No NHP was experiencing symptoms at the time of the study, however test results showed that Lawsonia intracellularis can be found in 61.76% (21/34) of the group total (n = 34) and the prevalence even increases to 92.3% (12/13) in the Lemuriform infraorder (n = 13). In small mammals (n = 14), prevalence of the bacterium is 57.17% (8/14) including 77.78% in rodents (7/9). The results of this study show that several NHP species are healthy carriers and some species of small mammals can be considered as a potential source of contamination. Because of the difficulty encountered trying to isolate the bacterium, it is plausible that infections caused by Lawsonia intracellularis have been underdiagnosed to this day, and that it could be an emerging disease in Europe. Therefore, using real-time PCR to search for this bacterium seems essential in case of diarrhea occurring in nonhuman primates. Moreover, even though further studies on contamination sources need to be conducted, the issue of the presence of rodents in NHP housing structures has to be taken very seriously and tackled with the utmost care.

Evaluation of immunogenicity and protection mediated by Lawsonia intracellularis subunit vaccines.

Lawsonia intracellularis is an economically important bacterium that causes ileitis in pigs. Current vaccines for L. intracellularis do not allow for differentiation between infected and vaccinated animals (DIVA), which is beneficial for disease tracking and surveillance. Previously, we identified five putative surface L. intracellularis proteins that were targeted by antibodies from pigs infected with L. intracellularis which could serve as antigens in a subunit vaccine. We conducted two trials to determine whether these antigens were immunogenic and provided protection against infectious challenge and whether truncated glycoprotein D could be used as a DIVA antigen. For Trial 1, 5 week-old piglets were administered intramuscular monovalent vaccines comprised of a recombinant (r) flagella subunit protein (rFliC,) and DIVA antigen (truncated glycoprotein D (TgD), a herpes virus antigen) both formulated with a combination adjuvant consisting of polyinosinic:polycytidylic acid(poly I:C), host defense peptide 1002 and polyphosphazene, referred to as Triple Adjuvant (TriAdj). Relative to control animals, animals vaccinated with rFliC and rTgD had significantly elevated antigen-specific humoral immunity in sera suggesting that rFliC and TgD are immunogenic. Control animals had negligible anti-TgD titres suggesting that TgD may be a suitable DIVA antigen for pigs. For Trial 2, piglets were immunized with a trivalent vaccine (FOG vaccine consisting of rFLiC, rOppA protein (a ABC Type dipeptide transport system) and rGroEL (a stress response protein)) and a divalent vaccine (CM vaccine consisting of rClpP (an ATP-dependent Clp protease proteolytic subunit) and rMetK (a S-adenosyl methionine synthase)) formulated with Emulsigen®. Relative to the control pigs, pigs immunized with the FOG vaccine produced robust and significantly higher serum IgG antibodies against rFliC and rGroEL, and significantly higher anti-FliC and anti-GroEL IgA antibodies in jejunal (GroEL only) and ileal intestinal mucosa. Pigs immunized with CM vaccine produced significantly higher serum antibodies against rClpP and rMetK and significantly higher anti-rClpP IgA antibodies in the ileum relative to the control pigs. Quantitative polymerase chain reaction (qPCR) analysis showed that 18 days after challenge with infectious L. intracellularis, challenged/control pigs and pigs that received the CM vaccine, but not the pigs vaccinated with the FOG vaccine, shed significantly more bacteria in feces than the unchallenged controls pigs. These data suggest that the FOG vaccinated pigs showed limited protection. While promising, more work is needed to enhance the efficiency of the intramuscular vaccine to show significant disease protection.

Review of methods for the detection of Lawsonia intracellularis infection in pigs.

Lawsonia intracellularis is an obligate intracellular bacterium associated with enteric disease in pigs. Clinical signs include weight loss, diarrhea, and, in some cases, sudden death. The hallmark lesion is the thickening of the intestinal mucosa caused by increased epithelial cell replication, known as proliferative enteropathy. The immune response to L. intracellularis is not well defined, and detection of the infection, especially in the early stages, is still a significant challenge. We review here the main approaches used to identify this important but poorly understood pathogen. Detection of L. intracellularis infection as the cause of clinical disease is confounded by the high prevalence of the pathogen in many countries and that several other pathogens can produce similar clinical signs. A single L. intracellularis-specific ELISA and several amplification assays are available commercially to aid detection and surveillance, although histopathology remains the primary way to reach a conclusive diagnosis. There are major gaps in our understanding of L. intracellularis pathogenesis, especially how the host responds to infection and the factors that drive infection toward different clinical outcomes. Knowledge of pathogenesis will increase the predictive value of antemortem tests to guide appropriate interventions, including identification and treatment of subclinically affected pigs in the early stages of disease, given that this important manifestation reduces pig productivity and contributes to the economic burden of L. intracellularis worldwide.

Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BACKGROUND: Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate. RESULTS: Batch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers. CONCLUSIONS: Considering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.

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.

High Seroprevalence Against Lawsonia intracellularis Among Adult Horses in Belgium.

Lawsonia intracellularis (LI) is an obligate intracellular gram-negative rod causing equine proliferative enteropathy (EPE). Occasional cases of EPE have been reported in foals living in Belgium, but the seroprevalence of equine LI in this country is unknown. The target population included clinically healthy adult horses, whose blood samples were collected and analyzed for specific IgG antibodies against LI using a blocking enzyme-linked immunosorbent assay test. The results were expressed as percentage of inhibition (PI). Samples that had a PI <20% were judged as negative, those between 20 and 30% as inconclusive, and those >30% were considered positive. A total of 356 blood samples were analyzed with 352 horses (98.8%) testing positive, 2 horses (0.6%) testing negative, and 2 horses (0.6%) showing inconclusive results. The large percentage of seropositive samples obtained in this study confirms a widespread exposure of Belgian horses to LI.

Investigation of the Usefulness of Serum Amyloid A in Supporting the Diagnosis of Equine Proliferative Enteropathy.

The objective of this study was to determine if serum amyloid A (SAA), a major acute-phase protein, could help support the diagnosis of equine proliferative enteropathy (EPE) caused by Lawsonia intracellularis infection in foals. Archived serum samples from 101 foals with enteric signs and hypoproteinemia were available for SAA testing. Based on immunodiagnostics for L. intracellularis, the foals were divided into EPE-suspect (67) and non-EPE-suspect cases (34). Serum amyloid A values ranged from 0 to 2,761 µg/mL (median 466 µg/mL) and from 0 to 2,555 µg/mL (median 192 µg/mL) for the EPE-suspect and the non-EPE-suspect cases, respectively. Although SAA can be measured patient-side and help determine the severity of the underlying inflammatory condition, SAA was unable to consistently support the diagnosis of EPE in hypoproteinemic foals with enteric signs.