Microbiota of vaccinated and non-vaccinated clinically inconspicuous and conspicuous piglets under natural Lawsonia intracellularis infection.

Lawsonia (L.) intracellularis is a widespread, economically important bacterium causing the porcine proliferative enteropathy (PPE). In this study, we evaluated intestinal microbiota of naturally exposed L. intracellularis-positive pigs under standardized conditions. To obtain three independent repetitions, 27 L. intracellularis-infected pigs (19.0 ± 1.50 kg body weight) from one farm were divided into three groups at an age of 7 to 8 weeks (nine pigs/group). Pigs were either vaccinated against L. intracellularis via oral drenching on their 21st day of life (attenuated live vaccine) or non-vaccinated and selected according to clinical findings (pigs without deviating fecal consistency or with moderate to soft fecal consistency). Comparison of the clinically inconspicuous piglets that differed regarding their vaccination status showed fewer significant differences in fecal microbiota composition. The vaccination led to an overall enrichment of bacterial species belonging to the order Clostridiales, while species of the genus Collinsella and Prevotella were decreased. Several bacterial species belonging to the order Bacteroidales, mainly of the family Prevotellacecae, often closely matching Prevotella copri differed significantly between non-vaccinated clinically inconspicuous and conspicuous piglets. Whether those bacterial species play a role in mitigating the severity of an L. intracellularis infection remains to be defined.

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.

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.

Correlation of Lawsonia intracellularis positivity in quantitative PCR and herd factors in European pig herds.

BACKGROUND: Lawsonia intracellularis is causing diarrhea, poor growth and sudden death in pigs. It can be found in most pig populations leading to large economic losses worldwide. Many potential risk factors for the occurrence of disease or seropositivity have been described. The current study therefore focused on herd characteristics in European countries associated with direct detection of the pathogen determined by quantitative polymerase chain reaction. RESULTS: A median number of less than 30 nursery pigs per pen was correlated to less positive nursery pigs (p < 0.01) and generally less samples positive per herd (p < 0.05) as well as a lower median of genome equivalents determined per herd (p < 0.05). Routine use of zinc oxide at/ around weaning, which was mentioned by 41.0% of all farmers, was correlated to higher number of positive nursery pigs (p < 0.01) as well as higher median genome equivalents determined per herd (p < 0.05). Slatted flooring of more than 78.0% of the surface in nursery units was correlated to lower number of positive animals (p < 0.05) and a lower median of genome equivalents per herd (p < 0.05). A weight of more than 7.8 kg at weaning was correlated to a higher number of positive growing pigs (p < 0.05) as well as general higher number of positive samples/ herd (p < 0.01). CONCLUSIONS: Weaning and subsequent accommodation of nursery pigs seem to be of particular importance in prevention of infection with Lawsonia intracellularis and the spread of the pathogen within the herd.

Use of oral fluids to detect anti Lawsonia intracellularis antibodies in experimentally infected pigs / Utilização de fluidos orais para detecção de anticorpos anti Lawsonia intracellularis em suínos experimentalmente infectados

Several pathogens and antibodies derived from serum or produced in tissues associated with the oral cavity are present in the oral fluid (OF). Considering the applicability of this alternative sample, recent studies in veterinary medicine have tested OF as a replacement for serum in diagnostic assays. The aim of this study was to standardize the immunoperoxidase monolayer assay (IPMA) to detect anti-Lawsonia intracellularis immunoglobulin A (IgA) and immunoglobulin G (IgG) in OF samples from experimentally infected pigs. Sixty-two pigs were divided into two groups: control (T1, n=30) and inoculated with L. intracellularis (T2, n=32). Blood, OF and fecal samples were collected at 0, 7, 14, 21, 28 and 42 days post-inoculation (dpi). Some adaptations of the standard technique for serum were made to IPMA for the detection of IgA and IgG in OF. The IPMA showed high specificity and sensitivity for serum samples and high specificity and moderate sensitivity for the detection of IgA and IgG in OF. There was high agreement between the results of serum IgG and OF IgA and IgG. Based on our results, oral fluid samples may be used for the evaluation and determination of anti-L. intracellularis antibodies in pigs, but not for individual diagnosis of swine proliferative enteropathy.(AU)

Vários patógenos e anticorpos derivados do soro ou produzidos em tecidos associados a cavidade oral estão presentes no fluido oral (FO). Considerando a aplicabilidade dessa amostra alternativa, estudos recentes em medicina veterinária têm testado o FO como substituto do soro para testes diagnósticos. O objetivo desse estudo foi padronizar a imunoperoxidase em monocamada de célula (IPMC) para a detecção de imunoglobulina A e imunoglobulina G anti-Lawsonia intracellularis em amostras de FO de suínos experimentalmente infectados. Um total de 62 suínos foram divididos em dois grupos: controle (T1, n=30) e inoculados com L. intracellularis (T2, n=32). Sangue, FO e amostras de fezes foram coletados aos 0, 7,14, 21, 28 e 42 dias após a inoculação (dpi). Algumas adaptações da técnica foram realizadas na técnica padrão da IPMC para a detecção de IgA e IgG. A IPMC demostrou alta especificidade e sensibilidade para amostras de soro e alta especificidade de moderada sensibilidade para a detecção de IgA e IgG em FO. Houve alta concordância entre resultados de detecção de IgG em soro com a IgA e IgG em amostras de FO. Baseado em nossos resultados, amostras de fluido oral podem ser usadas em avaliações e detecção de anticorpos anti-L. intracellularis em suínos, porém não de forma individual.(AU)

Efficacy of a novel intradermal Lawsonia intracellularis vaccine in pigs against experimental infection and under field conditions.

BACKGROUND: The efficacy of a novel inactivated intradermal Lawsonia intracellularis vaccine, Porcilis® Lawsonia ID, was evaluated in two experimental vaccination-challenge studies and under field conditions on a farm with a history of recurrent acute ileitis. In addition, the efficacy of the vaccine was compared to that of a commercially available live attenuated vaccine. The novel inactivated vaccine consists of a freeze-dried antigen fraction that is dissolved just prior to use in either the adjuvant or in Porcilis® PCV ID; an existing intradermal vaccine against porcine Circovirus type 2. In the two experimental vaccination-challenge studies, groups of 25 piglets were vaccinated once at 3 weeks of age or left unvaccinated as challenge control. Vaccines tested were Porcilis® Lawsonia ID as standalone (study 1) or in associated mixed use with Porcilis® PCV ID (study 2) and an orally administered commercially available live vaccine (study 1). The pigs were challenged with virulent L. intracellularis at 4 weeks (study 1) or 21 weeks (study 2) after vaccination. Post-challenge, the pigs were evaluated for clinical signs, average daily weight gain, shedding and macroscopic as well as microscopic immuno-histological ileum lesion scores. In the field study, the mortality and key performance parameters were evaluated over a period of 8 months. RESULTS: The results of the two experimental vaccination-challenge studies showed that Porcilis® Lawsonia ID as single vaccine or in associated mixed use with Porcilis® PCV ID, induced statistically significant protection against experimental L. intracellularis infection, 4 weeks or 21 weeks after vaccination. This was demonstrated by lower clinical scores, improved weight gain, reduction of L. intracellularis shedding and reduction of macroscopic as well as microscopic ileum lesion scores when compared to the controls. The protection induced was superior to that of the commercially available live vaccine. In the field study Porcilis® Lawsonia ID was highly efficacious in reducing L. intracellularis associated mortality and improving key production parameters. CONCLUSION: The results support that this new intradermal vaccine is efficacious against L. intracellularis and may be used in associated mixed use with Porcilis® PCV ID.

Efficacy of a novel inactivated Lawsonia intracellularis vaccine in pigs against experimental infection and under field conditions.

The efficacy of a novel inactivated Lawsonia intracellularis vaccine, Porcilis® Lawsonia, was compared to that of a commercially available live attenuated vaccine in three experimental vaccination-challenge studies in pigs. The efficacy of the new vaccine was further tested under field conditions on a farm with a history of acute ileitis. The novel inactivated vaccine consists of a freeze-dried antigen fraction that is dissolved just prior to use in either the adjuvant or in Porcilis® PCV M Hyo; an existing combination vaccine against porcine circovirus type 2 and Mycoplasma hyopneumoniae. The three experimental vaccination-challenge trials had a similar design and for each trial 75 piglets were used, randomly allotted to three groups of 25 piglets. The pigs were vaccinated at 4 or 5 weeks of age with either Porcilis® Lawsonia in adjuvant or in associated mixed use with Porcilis® PCV M Hyo (group 1), with the live vaccine (group 2), or left as unvaccinated controls (group 3). The pigs were challenged with virulent Lawsonia intracellularis 3, 4 or 17 weeks after vaccination. Post-challenge the pigs were evaluated for clinical signs, average daily weight gain, shedding and macroscopic as well as microscopic immuno-histological ileum lesion scores. In the field study, the mortality and key performance parameters were evaluated over a period of 8 months. The results of all three experimental vaccination-challenge trials showed that Porcilis® Lawsonia induced statistically significant protection against experimental Lawsonia intracellularis infection. This was demonstrated by lower clinical scores, improved weight gain, reduction of Lawsonia intracellularis shedding and reduction of macroscopic as well as microscopic ileum lesion scores when compared to the controls. The protection induced was superior to that of the commercially available live vaccine. In the field study, Porcilis® Lawsonia proved to be highly efficacious; reducing Lawsonia associated mortality to zero and improving key production parameters.

Oral fluid for detection of exposure to Lawsonia intracellularis in naturally infected pigs.

To demonstrate the utility of oral fluid (OF) for indirect diagnostic detection of Lawsonia intracellularis (Li), 15 pig farms were studied. Serum and fecal samples were collected from 20 animals from five different age groups on each farm. OF samples were collected from animals in two pens of the same age groups. Serum and OF samples were analyzed in an immunoperoxidase in monolayer assay (IPMA) for the detection of anti-Li immunoglobulin G (IgG) and A (IgA). Compatible results were found between PCR and IgG in OF in four of the five ages evaluated. Simultaneous detection of IgG in serum and OF was mainly observed on farms showing clinical signs suggestive of porcine proliferative enteropathy (PPE). These findings demonstrate the potential usefulness of OF in detecting anti-Li antibodies as a diagnostic tool that can be used to monitor PPE in herds with clinical signs compatible with the disease.

Tylosin injectable for the treatment of porcine proliferative enteropathy in experimentally inoculated pigs / Tilosina injetável no tratamento da enteropatia proliferativa suína em leitões experimentalmente inoculados

Porcine proliferative enteropathy (PPE) is one of the most common enteric diseases in growing and finishing pigs. PPE is characterized by reduced growth performance, accompanied or not by diarrhea. PPE is highly prevalent in several countries of the Americas, Europe and Asia, causing high economic losses in swine herds. The most common form of PPE control in pigs is antibiotic therapy. The objective of this study was to evaluate a new product based on tylosin injectable (Eurofarma Laboratórios S.A.) to control PPE in experimentally inoculated animals. Sixty 5-week-old pigs with mean weight of 9.5kg were divided into two experimental groups of 30 animals: medication and control. All pigs were challenged with Lawsonia intracellularis, the etiologic agent of PPE, on day zero. Fecal score, body condition score, and behavior were daily evaluated. Pigs were weighted on days -2, 13 and 21 of the experiment. Pigs in the Medication Group received tylosin injectable 13 days after inoculation, in three doses with a 12-hour interval between them. Pigs in the Control Group received injectable saline solution following the same protocol. In the Control Group, 23pigs presented with diarrhea before day 13. After day 13, the number of diarrheic animals in this group was reduced to 17. In the Medication Group, 26 pigs presented with diarrhea in the initial period, and in the period after medication, only 11 animals had diarrhea. The score of gross intestinal PPE lesions in the Medication Group was lower than that in the Control Group (p=0.031). The Medication Group also showed lower score for Lawsonia intracellularis antigen-labeling by immunohistochemistry compared with that of the Control Group (p=0.032), showing lower level of infection. These results demonstrate that tylosin injectable (Eurofarma Laboratórios S.A.), administrated in three doses (1mL/20kg) every 12 hours, was effective for the control of PPE in experimentally inoculated pigs.(AU)

Enteropatia proliferativa suína (EPS), causada pela bactéria Lawsonia intracellularis, é uma das doenças entéricas mais comuns em suínos de recria e terminação. A EPS caracteriza-se por redução no desempenho dos animais, acompanhada ou não por diarreia. É uma doença altamente prevalente em diversos países da América, Europa e Ásia, provocando elevados prejuízos econômicos nos rebanhos suínos. A forma de controle da EPS mais adotada em rebanhos suínos é a antibioticoterapia. O objetivo deste estudo foi avaliar um novo produto à base de tilosina (Eurofarma Laboratórios S.A.) na forma injetável para controlar a EPS em animais experimentalmente inoculados. Foram utilizados 60 leitões, de cinco semanas de idade, com peso médio de 9,5kg, divididos em dois grupos experimentais (n=30), medicados e não medicados. Todos os leitões foram desafiados com Lawsonia intracellularis no dia zero. Avaliações clínicas de escore fecal, escore corporal e comportamento foram realizadas diariamente além da pesagem individual dos animais realizada nos dias -2, 13 e 21 do experimento. Os leitões do grupo medicado receberam tilosina injetável 13 dias após a inoculação em três doses com intervalo de 12 horas cada. Já os leitões do grupo não medicado receberam solução salina injetável com o mesmo protocolo. O grupo não medicado apresentou 23 animais com diarreia antes do dia 13 e 17 após este período. No grupo medicado, 26 animais apresentaram diarreia previamente à medicação e apenas 11 após a medicação a partir do dia 13. Os leitões medicados apresentaram extensão de lesão macroscópica, caracterizada por espessamento de mucosa intestinal, menor em comparação com o grupo não medicado (p=0,031). A imunomarcação para Lawsonia intracellularis foi menor no grupo medicado (p<0,032), mostrando redução no grau de infecção por L. intracellularis nos animais medicados. Estes resultados demonstram que a tilosina injetável (Eurofarma Laboratórios S.A.) (1mL/20kg) em três doses, a cada 12 horas, foi eficaz no tratamento da enteropatia proliferativa suína em animais experimentalmente inoculados.(AU)

Serum folate, cobalamin, homocysteine and methylmalonic acid concentrations in pigs with acute, chronic or subclinical Lawsonia intracellularis infection.

Lawsonia intracellularis is the causative agent of porcine proliferative enteropathy. The clinical presentation can be acute (i.e. proliferative hemorrhagic enteropathy, PHE), chronic (i.e. porcine intestinal adenomatosis, PIA) or subclinical. In humans with chronic enteropathies, low serum folate (vitamin B(9)) and cobalamin (vitamin B(12)) concentrations have been associated with increased serum concentrations of homocysteine and methylmalonic acid (MMA), which reflect the availability of both vitamins at the cellular level. The aim of this study was to evaluate serum folate, cobalamin, homocysteine and MMA concentrations in serum samples from pigs with PHE, PIA or subclinical L. intracellularis infection, and in negative controls. Serum folate, cobalamin, homocysteine and MMA concentrations differed significantly among pigs in the PHE, PIA, subclinical and negative control groups. Serum folate concentrations in the PHE and PIA groups were lower than in the subclinical and negative control groups, while serum cobalamin concentrations were lower in the PIA group than in other groups. Serum concentrations of homocysteine were higher in the PHE, PIA and subclinical groups than in the negative control group. Serum concentrations of MMA were higher in the subclinical and PIA groups than in the control group. These data suggest that pigs infected with L. intracellularis have altered serum cobalamin, folate, homocysteine and MMA concentrations.

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.

Risk factors associated with Lawsonia intracellularis in English pig farms.

Porcine proliferative enteropathy (PPE) caused by the bacterium Lawsonia intracellularis causes considerable economic loss to the pig industry. The objective of this study was to evaluate the seroprevalence of L. intracellularis exposure in different age groups of pigs (growers to finishers) within English farms and to identify potential risk factors. Samples were obtained in a cross-sectional study of 147 farms between 2008 and 2009. Twelve samples (six growers and six finishers) from each farm were tested for L. intracellularis by antibody ELISA. At animal level there was a significant positive linear trend between seroprevalence and age in weeks (r(2)=2.65, P<0.001), with seroprevalence lowest (24.73%) at 11 weeks and highest (93.33%) at 24 weeks. At farm level, seroprevalence was significantly lower in growers than finishers (56.80% vs. 94.26%, P<0.001). Farms reporting minor Salmonella problems and those that brought boars onto the farm had higher odds of testing positive in growers (OR 5.69 and 4.31, respectively. On the other hand, farms where producers considered temperature as an important stress factor (OR=0.3) and which had more than two sites on which pigs are kept (OR=0.16) were less likely to test positive in growers. The current study confirmed the high prevalence of L. intracellularis in English pig farms. The potential risk factors and further information of the disease impact on the farm productivity will aid the development of appropriate control strategies through better understanding of the disease.

Antimicrobianos para o controle da enteropatia proliferativa suína / Antimicrobial use for the control of porcine proliferative enteropathy

A enteropatia proliferativa suína (EPS), causada pela bactéria Lawsonia intracellularis, causa perdas econômicas importantes mundialmente, devido à diarréia e baixa taxa de crescimento de suínos na recria (forma crônica) e à morte súbita de animais de terminação e reposição (forma aguda). Programas de controle têm sido focalizados na medicação com antibióticos na ração. Essencialmente, a eficiência de um antimicrobiano pode ser testada in vitro ou in vivo. Testes in vivo podem ser desenvolvidos utilizando-se animais natural ou experimentalmente infectados. No caso de infecção experimental, o inóculo pode ser preparado com culturas puras de L. intracellularis ou com homogenado de mucosa intestinal de leitões enfermos. O uso de antimicrobianos tem-se mostrado eficiente em reduzir os sinais clínicos da EPS, resultando em melhor performance dos animais tratados em relação aos não-medicados e na redução da eliminação fecal da bactéria e da extensão e severidade das lesões macroscópicas. A maioria dos fármacos eficientes que são discutidas no texto são dos grupos dos macrolídeos, das tetraciclinas, lincosamidas e pleuromutilinas. Todos esses fármacos agem bloqueando a síntese protéica de bactérias.

Porcine proliferative enteropathy (PPE), caused by the bacterium Lawsonia intracellularis, causes serious economic losses worldwide due to diarrhea and poor growth rate medication in young growing pigs (chronic disease form) and sudden death in finisher and replacement pigs (acute hemorrhagic form). Typical control programs have focused on antibiotics. Essentially, the effectiveness of an antimicrobial can be tested in vitro or in vivo. In vivo test can be developed with natural or experimentally infected pigs. In tests that the animals are experimentally challenged, the inoculation is done with pure culture of L. intracellularis or intestinal mucosal homogenate of pig with PPE. Antimicrobial use have been shown to be effective in reducing the clinical signs of PPE and to result in better performance in treated pigs than in untreated animals. In addition, it decreases fecal shedding and the severity of gross lesions. The most efficient antimicrobial groups of drugs discussed in this manuscript are macrolides, tetracyclines, lincosamides and pleuromutilins. All of them act by blocking bacterial protein synthesis.