Synergic Effect of Brachyspira hyodysenteriae and Lawsonia intracellularis Coinfection: Anatomopathological and Microbiome Evaluation.

Brachyspira hyodysenteriae and Lawsonia intracellularis coinfection has been observed in the diagnostic routine; however, no studies have evaluated their interaction. This study aimed to characterize lesions and possible synergisms in experimentally infected pigs. Four groups of piglets, coinfection (CO), B. hyodysenteriae (BRA), L. intracellularis (LAW), and negative control (NEG), were used. Clinical signals were evaluated, and fecal samples were collected for qPCR. At 21 days post infection (dpi), all animals were euthanized. Gross lesions, bacterial isolation, histopathology, immunohistochemistry, and fecal microbiome analyses were performed. Diarrhea started at 12 dpi, affecting 11/12 pigs in the CO group and 5/11 pigs in the BRA group. Histopathological lesions were significantly more severe in the CO than the other groups. B. hyodysenteriae was isolated from 11/12 pigs in CO and 5/11 BRA groups. Pigs started shedding L. intracellularis at 3 dpi, and all inoculated pigs tested positive on day 21. A total of 10/12 CO and 7/11 BRA animals tested positive for B. hyodysenteriae by qPCR. A relatively low abundance of microbiota was observed in the CO group. Clinical signs and macroscopic and microscopic lesions were significantly more severe in the CO group compared to the other groups. The presence of L. intracellularis in the CO group increased the severity of swine dysentery.

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.

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.

A review on the evolution of methods for intestinal in vitro organ culture and its application in veterinary science.

Different techniques have been reported in studies of intestinal in vitro organ culture (IVOC). A robust compilation of all available methods is lacking in the literature, making it difficult to choose a method that corresponds to the study's demands. In this review, readers can assess the most available methods, allowing them to evaluate which is more suitable for their purposes and requirements. A simplified view of culturing intestinal explants is presented, highlighting the approachability of IVOC. Relevant findings from diverse veterinarian studies, where explants played a major role, as well as the technique used in each, are described to illustrate its applications. Finally, the strengths and limitations of the innovative intestinal IVOC methods are discussed. This review provides a collection of methods for intestinal explant culture and their possible applications in veterinary research. In this way, it aims to broaden access to IVOC techniques and aid decision-making regarding the best suited for a study's purposes.

Evaluation of the role of clathrin and bacterial viability in the endocytosis of Lawsonia intracellularis.

Lawsonia intracellularis is an obligate intracellular bacterium and causative agent of proliferative enteropathy. The pathogenesis of L. intracellularis is not completely understood, including the endocytic mechanisms to access the host cell cytoplasm. In this study, we evaluated the mechanisms involved in endocytosis of L. intracellularis in vitro using intestinal porcine epithelial cells (IPEC-J2). Confocal microscopy was used to co-localize L. intracellularis and clathrin. Clathrin gene knockdown was then applied to verify whether L. intracellularis endocytosis is clathrin-dependent. Finally, internalization of viable and non-viable (bacteria were inactivated by heat) L. intracellularis organisms were assessed to study the role of the host cell during bacterial endocytosis. L. intracellularis organisms were observed co-localized with clathrin by confocal microscopy but the amount of L. intracellularis internalized in cells, with and without clathrin knockdown, did not differ statistically. The internalization of non-viable L. intracellularis showed a decrease in the internalization in cells with less clathrin synthesis (P<0.05). The present study is the first to elucidate the involvement of clathrin in the endocytosis of L. intracellularis. Clathrin-mediated endocytosis was shown to be an important, but not required, process for L. intracellularis internalization in porcine intestinal epithelial cells. Independence of bacterial viability for host cell internalization was also confirmed.

A dietary spray-dried plasma feeding programme improves growth performance and reduces faecal bacterial shedding of nursery pigs challenged with enterotoxigenic Escherichia coli K88.

This study aimed to assess the effects of different spray-dried plasma (SDP) feeding programmes to pigs on performance, intestinal histomorphology and faecal bacterial shedding after an Escherichia coli K88 challenge. A total of 96 piglets (5.77 ± 0.01 kg) were weaned at 21 days of age (Day 0) and challenged with 3 ml of 1 × 1010 CFU of E. coli K88 in total 3.0 × 1010 CFU/animal on Days 0, 2 and 4. Pigs were fed nursery diets containing 0.0%, 3.0%, 6.0% or 9.0% SDP from weaning to 35 days of age; 0.0%, 1.5%, 3.0% or 4.5% SDP from 36 to 49 days; and the same control diet (without SDP), for the last 10 days of the experiment (50-59 days of age). Performance was measured from 35 to 59 days of age and faecal bacterial shedding and intestinal histomorphometry were evaluated at Days 28 and 49 of age respectively. From 21 to 35 days of age, there was a linear effect for body weight (BW) and average daily gain (ADG), a trend of linear effect for average daily feed intake (ADFI) and a quadratic effect for feed:gain ratio (FG). From 21 to 49 days, the 9.0:4.5% and 6.0:3.0% SDP feeding programmes improved BW, ADG and FG when compared to the other treatments. At 59 days of age, BW and ADG were increased by the two highest SDP feeding programmes. The 9.0:4.5% SDP feeding programme increased ADFI from 21 to 59 days of age, with 6.0:3.0% being intermediate and the other two treatments being lowest. The CFU counts of E. coli/g of faeces decreased linearly with increasing addition of SDP. These results indicate that an extended inclusion of increased SDP levels in post-weaning diets can improve growth potential and decrease bacterial shedding induced by E. coli K88.

Extracts Prepared from Feed Supplements Containing Wood Lignans Improve Intestinal Health by Strengthening Barrier Integrity and Reducing Inflammation.

Lignans are known to exhibit a broad spectrum of biological activities, indicating their potential as constituents of feed supplements. This study investigated two extracts derived from the feed supplements 'ROI' and 'Protect'-which contain the wood lignans magnolol and honokiol ('ROI'), or soluble tannins additional to the aforementioned lignans ('Protect')-and their impact on selected parameters of intestinal functionality. The antioxidant and anti-inflammatory properties of the extracts were determined by measuring their effects on reactive oxygen species (ROS) and pro-inflammatory cytokine production in vitro. The impact on intestinal barrier integrity was evaluated in Caco-2 cells and Drosophila melanogaster by examining leaky gut formation. Furthermore, a feeding trial using infected piglets was conducted to study the impact on the levels of superoxide dismutase, glutathione and lipid peroxidation. The Protect extract lowered ROS production in Caco-2 cells and reversed the stress-induced weakening of barrier integrity. The ROI extract inhibited the expression or secretion of interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα). Moreover, the ROI extract decreased leaky gut formation and mortality rates in Drosophila melanogaster. Dietary supplementation with Protect improved the antioxidant status and barrier integrity of the intestines of infected piglets. In conclusion, wood lignan-enriched feed supplements are valuable tools that support intestinal health by exerting antioxidant, anti-inflammatory and barrier-strengthening effects.

Experimental Infection of Pigs with a ST 245 Brachyspira hyodysenteriae Isolated from an Asymptomatic Pig in a Herd with No History of Swine Dysentery.

Swine dysentery (SD) is characterized by a severe mucohemorrhagic colitis caused by infection with Brachyspira species. In infected herds the disease causes considerable financial loss due to mortality, slow growth rates, poor feed conversion, and costs of treatment. B. hyodysenteriae is the most common etiological agent of SD and infection is usually associated with disease. However, isolated reports have described low pathogenic strains of B. hyodysenteriae. The aim of this study was to describe an experimental infection trial using a subclinical B. hyodysenteriae isolated from an animal without clinical signs and from a disease-free herd, to evaluate the pathogenicity and clinical pathological characteristics compared to a highly clinical isolate. Forty-eight 5-week-old pigs were divided into three groups: control, clinical and the subclinical isolates. The first detection/isolation of B. hyodysenteriae in samples of the animals challenged with a known clinical B. hyodysenteriae strain (clinical group) occurred 5th day post inoculation. Considering the whole period of the study, 11/16 animals from this group were qPCR positive in fecal samples, and diarrhea was observed in 10/16 pigs. In the subclinical isolate group, one animal had diarrhea. There were SD large intestine lesions in 3 animals at necropsy and positive B. hyodysenteriae isolation in 7/15 samples of the subclinical group. In the control group, no diarrhea, gross/microscopic lesions, or qPCR positivity were observed. Clinical signs, bacterial isolation, macroscopic and histologic lesions were significantly difference among groups, demonstrating low pathogenicity of the subclinical isolate in susceptible pigs.

Laboratory diagnosis of Clostridioides (Clostridium) difficile infection in domestic animals: A short review.

Despite the known importance of Clostridioides (Clostridium) difficile infection (CDI) in animals, there are no published guidelines for the diagnosis of CDI. The performance of the available commercial methods, all standardized for human stool samples, can vary according to the animal species. Thus, the aim of the present study was to review the literature on the detection of C. difficile in pigs, horses, and dogs. The detection of toxins A and B using enzyme immunoassays seems to have low performance in piglet and dog samples, while it shows high sensitivity for the diagnosis of CDI in foals. On the other hand, tests for the detection of glutamate dehydrogenase (GDH) have a high sensitivity towards detection of C. difficile in animal samples, suggesting that it can be an adequate screening method. A few studies have evaluated real-time PCR or nucleic acid amplification tests in animal samples and, so far, these methods have also shown a low performance for the detection of C. difficile in animals. Although the intestinal lesions caused by CDI can vary among animal species, histopathology can be a useful auxiliary tool for postmortem diagnosis in animals.

Diversity and potential genetic relationships amongst Brazilian Brachyspira hyodysenteriae isolates from cases of swine dysentery.

The aim of this study was to evaluate genetic diversity, distribution, evolution and population structure of Brazilian Brachyspira hyodysenteriae strains isolated from swine. Multilocus Sequence Typing (MLST) analysis using seven housekeeping genes was applied to 46 isolates obtained from outbreaks of swine dysentery that occurred between 2011 and 2015 in the states of Minas Gerais, São Paulo, Mato Grosso, Rio Grande do Sul and Santa Catarina. Historical isolates from Rio Grande do Sul obtained in 1998 were also included in the study. An independent international profile of the global sequences deposited in the B. hyodysenteriae database was used for comparisons with the Brazilian strains. All isolates from 2011 to 2015 were classified into nine sequence type (STs) and divided into four clonal complexes. These findings indicated genetic relationships among the B. hyodysenteriae from different Brazilian states, among historical strains isolated in 1998 and from recent outbreaks, and relatedness with global isolates. Seven STs were unique and, to date, only reported in Brazil.

Re-Emergence of Salmonellosis in Hog Farms: Outbreak and Bacteriological Characterization.

Clinical salmonellosis has been increasing significantly in Brazil in recent years. A total of 130 outbreaks distributed among 10 swine-producing states were investigated. One representative Salmonella isolate from each outbreak was characterized through serotyping, antimicrobial resistance profiles, PFGE, and WGS. From 130 outbreaks: 50 were enteric, 48 were septicemic, 17 cases were characterized as hepato-biliary invasive, 13 as nodal and two were not classified. The most prevalent serovars were a monophasic variant of S. typhimurium (55/130), Choleraesuis (46/130), and Typhimurium (14/130). Most of the strains (86.92%) demonstrated a high rate of multi-drug resistance. The identification of a major Choleraesuis clonal group in several Brazilian states sharing the same resistance genes suggested that these strains were closely related. Six strains from this clonal group were sequenced, revealing the same ST-145 and 11 to 47 different SNPs. The detected plasmid type showed multiple marker genes as RepA_1_pKPC-CAV1321, the first to be reported in Salmonella. All AMR genes detected in the genomes were likely present on plasmids, and their phenotype was related to genotypic resistance genes. These findings reveal that salmonellosis is endemic in the most important pig-producing states in Brazil, emphasizing the need to make data available to aid in reducing its occurrence.

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)

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

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

Metagenomic sequencing of clinical samples reveals a single widespread clone of Lawsonia intracellularis responsible for porcine proliferative enteropathy.

Lawsonia intracellularis is a Gram-negative obligate intracellular bacterium that is the aetiological agent of proliferative enteropathy (PE), a common intestinal disease of major economic importance in pigs and other animal species. To date, progress in understanding the biology of L. intracellularis for improved disease control has been hampered by the inability to culture the organism in vitro. In particular, our understanding of the genomic diversity and population structure of clinical L. intercellularis is very limited. Here, we utilized a metagenomic shotgun approach to directly sequence and assemble 21 L. intracellularis genomes from faecal and ileum samples of infected pigs and horses across three continents. Phylogenetic analysis revealed a genetically monomorphic clonal lineage responsible for infections in pigs, with distinct subtypes associated with infections in horses. The genome was highly conserved, with 94 % of genes shared by all isolates and a very small accessory genome made up of only 84 genes across all sequenced strains. In part, the accessory genome was represented by regions with a high density of SNPs, indicative of recombination events importing novel gene alleles. In summary, our analysis provides the first view of the population structure for L. intracellularis, revealing a single major lineage associated with disease of pigs. The limited diversity and broad geographical distribution suggest the recent emergence and clonal expansion of an important livestock pathogen.

Correction to: Genetic variation of Mycoplasma hyopneumoniae from Brazilian field samples.

Following publication of the original article [1], we have been notified that two of the author names were incomplete or incorrect.

Genetic variation of Mycoplasma hyopneumoniae from Brazilian field samples.

BACKGROUND: Porcine enzootic pneumonia is a worldwide problem in swine production. The infected host demonstrates a respiratory disease whose etiologic agent is Mycoplasma hyopneumoniae (Mhp). A total of 266 lung samples with Mycoplasma-like lesions were collected from two slaughterhouses. We analyzed the genetic profile of Mhp field samples using 16 genes that encode proteins involved in the mechanisms of bacterial pathogenesis and/or the immune responses of the host. Bioinformatic analyses were performed to classify the Mhp field samples based on their similarity according to the presence of the studied genes. RESULTS: Our results showed variations in the frequency of the 16 studied genes among different Mhp field samples. It was also noted that samples from the same farm were genetically different from each other and samples from different regions could be genetically similar, which is evidence of the presence of different genetic profiles among the Mhp field strains that circulate in Brazilian swine herds. CONCLUSION: This work demonstrated the genetic diversity of several Mhp field strains based on 16 selected genes related to virulence and/or immune response in Brazil. Our findings demonstrate the difference between Mhp field strains could influence the virulence, and we hypothesize that the most frequent genes in Mhp field strains could possibly be used as vaccine candidates. Based on our results, we suspect that Mhp genetic variability may be associated with the frequency of genes among the field strains and we have demonstrated that some Mhp field samples could not have many important genes described in the literature.

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)

Isolation and in vitro antimicrobial susceptibility of porcine Lawsonia intracellularis from Brazil and Thailand.

BACKGROUND: Lawsonia intracellularis is an obligate intracellular bacterium which cannot be cultured by conventional bacteriological methods. Furthermore, L. intracellularis needs enriched medium and a unique atmosphere for isolation, cultivation and propagation. Because of this,there are only a few isolates of L. intracellularis available and few studies in vitro demonstrating the susceptibility of this bacterium to antimicrobial agents. The objectives of this study were to isolate South American and Southeast Asia strains of L.intracellularis and to determine the in vitro antimicrobial activity against these isolates. Tested antimicrobials included: chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin(against both Brazilian and Thailand strains) and additionally, amoxicillin, zinc-bacitracin, carbadox, enrofloxacin, gentamicin, sulfamethazine, trimethoprim, spectinomycin and a combination (1:1) of spectinomycin and lincomycin were also tested against the Thai isolates. The minimum inhibitory concentration (MIC) was determined by the antimicrobial activity that inhibited 99% of L. intracellularis growth in a cell culture as compared to the control (antimicrobial-free). RESULTS: Two strains from Brazil and three strains from Thailand were successfully isolated and established in cell culture. Each antimicrobial was evaluated for intracellular and extracellular activity. Pleuromutilin group (valnemulin and tiamulin) and carbadox were the most active against L. intracellularis strains tested. Tylosin showed intermediate activity, chlortetracycline had variable results between low and intermediate activity, as well as spectinomycin, spectinomycin and lincomycin, amoxicillin, sulfamethazine and enrofloxacin. L. intracellularis was resistant to lincomycin, gentamicin, trimethoprim, colistin and bacitracin in in vitro conditions. CONCLUSIONS: This is the first report of isolation of L. intracellularis strains from South America and Southeast Asia and characterization of the antimicrobial susceptibility patterns of these new strains.

Phenotypic characterization of swine peripheral blood monocyte-derived macrophages and ex vivo infection with Salmonella enterica serovar Typhimurium.

Macrophages are critical mediators of the inflammatory process, playing a relevant role in the pathogenesis of Salmonella Typhimurium. The protocols for isolation, culture, and differentiation of monocytes into macrophages and their interaction with Salmonella are well established in humans and murine models, but little information is available in swine. The aims of this study were to establish an efficient protocol for macrophage culture and to evaluate the interaction of the invA mutant strain and the wild type (WT) Salmonella Typhimurium with porcine macrophages. Peripheral blood monocyte-derived macrophages from pigs were obtained, separated by density-gradient centrifugation, and cultured in Teflon vials for 10 days. After the differentiation period, cultures consisted of 92.4% CD14+ cells. In addition, these cells showed phagocytic ability, demonstrated by the presence of the same amount of WT and invA mutant Salmonella Typhimurium 1 h after interaction with macrophages. The early cytotoxic effect was Salmonella pathogenicity island (SPI)-[1]dependent, in which log-phase WT strains were more efficient (p < 0.01) than the invA mutant strain at inducing the death of macrophages.