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.

The effects of zinc amino acid complex supplementation on the porcine host response to Lawsonia intracellularis infection.

Lawsonia intracellularis is among the most important enteric pathogens of swine and antibiotic alternatives are needed to help mitigate the negative effects of infection. Zinc is an essential trace mineral known to be crucial for maintaining intestinal barrier function and proper immune response. In this study, we investigated the porcine host response to L. intracellularis infection when supplemented with a zinc-amino acid complex, a form of zinc that can lead to greater bioavailability when compared to traditional inorganic forms of zinc. Our results show that a zinc-amino acid complex supplementation with a final concentration of 125 ppm of zinc in feed significantly (p < 0.05) decreased the number of animals with lesions and severity of lesions caused by L. intracellularis. Animals supplemented with the zinc-amino acid complex also exhibited a significantly (p < 0.05) earlier onset of seroconversion as well as an increased number of T cells in infected and non-infected intestinal tissue. This study demonstrated that this zinc-amino acid complex aids the host in responding to L. intracellularis infection and may be a new approach to help minimize negative effects of disease.

Relationships among Fecal, Air, Oral, and Tracheal Microbial Communities in Pigs in a Respiratory Infection Disease Model.

The association of the lower respiratory tract microbiome in pigs with that of other tissues and environment is still unclear. This study aimed to describe the microbiome of tracheal and oral fluids, air, and feces in the late stage of Mycoplasma hyopneumoniae infection in pigs, and assess the association between the tracheal microbiome and those from air, feces, and oral fluids. Tracheal fluids (n = 73), feces (n = 71), oropharyngeal fluids (n = 8), and air (n = 12) were collected in seeder pigs (inoculated with M. hyopneumoniae) and contact pigs (113 days post exposure to seeder pigs). After DNA extraction, the V4 region from 16S rRNA gene was sequenced and reads were processed using Divisive Amplicon Denoising Algorithm (DADA2). Clostridium and Streptococcus were among the top five genera identified in all sample types. Mycoplasma hyopneumoniae in tracheal fluids was associated with a reduction of diversity and increment of M. hyorhinis, Glaesserella parasuis, and Pasteurella multocida in tracheal fluids, as well as a reduction of Ruminiclostridium, Barnesiella, and Lactobacillus in feces. Air contributed in a greater proportion to bacteria in the trachea compared with feces and oral fluids. In conclusion, evidence suggests the existence of complex interactions between bacterial communities from distant and distinct niches.

Oral Vaccination Reduces the Effects of Lawsonia intracellularis Challenge on the Swine Small and Large Intestine Microbiome.

Porcine proliferative enteropathy remains one of the most prevalent diseases in swine herds worldwide. This disease is caused by Lawsonia intracellularis, an intracellular bacterial pathogen that primarily colonizes the ileum. In this study, we evaluated changes to the microbiome of the ileal mucosa, ileal digesta, cecal digesta, and feces subsequent to challenge with L. intracellularis and to an oral live vaccine against L. intracellularis. Given that gut homogenates have been used since 1931 to study this disease, we also characterized the microbial composition of a gut homogenate from swine infected with L. intracellularis that was used as challenge material. The L. intracellularis challenge led to a dysbiosis of the microbiome of both the small and large intestine marked by an increase of pathobionts including Collinsella, Campylobacter, Chlamydia, and Fusobacterium. This microbiome response could play a role in favoring L. intracellularis colonization and disease as well as potentially predisposing to other diseases. Vaccination altered both small and large intestine microbiome community structure and led to a significant 3.03 log10 reduction in the amount of L. intracellularis shed by the challenged pigs. Vaccination also led to a significant decrease in the abundance of Collinsella, Fusobacterium, and Campylobacter among other microbial changes compared with non-vaccinated and challenged animals. These results indicate that L. intracellularis infection is associated with broad changes to microbiome composition in both the large and small intestine, many of which can be mitigated by vaccination.

The effects of Lawsonia intracellularis, Salmonella enterica serovar Typhimurium and co-infection on IL-8 and TNFα expression in IPEC-J2 cells.

Lawsonia intracellularis is among the most important enteric pathogens of swine and has been shown to be a risk factor for increased Salmonella enterica shedding. S. enterica serovar Typhimurium, in addition to being a significant pathogen of swine, also remains one of the most common causes of foodborne illness worldwide. Inflammation and the expression of IL8 and TNFα are an important process in the establishment of S. Typhimurium infection. Yet the effect of L. intracellularis on the expression of these cytokines by enterocytes, the niche both pathogens occupy during infection, is poorly understood. In this study we compared cytokine gene expression between singly and dually infected IPEC-J2 cells, a non-transformed porcine enterocyte cell line. Our results show that L. intracellularis leads to increased expression of IL8 and TNFα and has an additive effect on their expression in co-infection. The increase in expression of inflammatory cytokines may be one mechanism by which L. intracellularis favors S. Typhimurium infection.

A Cell Proliferation and Inflammatory Signature Is Induced by Lawsonia intracellularis Infection in Swine.

Lawsonia intracellularis causes porcine proliferative enteropathy. This is an enteric disease characterized by thickening of the wall of the ileum that leads to decreased growth of animals and diarrhea. In this study, we investigated the host response to L. intracellularis infection by performing transcriptomic and pathway analysis of intestinal tissue samples from groups of infected and noninfected animals at 14, 21, and 28 days postchallenge. At the peak of infection, when animals developed the most severe lesions, infected animals had higher levels of several gene transcripts involved in cellular proliferation and inflammation, including matrix metalloproteinase-7 (MMP7), transglutaminase-2 (TGM2), and oncostatin M (OSM). Histomorphology also revealed general features of intestinal inflammation. This study identified important pathways associated with the host response in developing and resolving lesions due to L. intracellularis infection.IMPORTANCELawsonia intracellularis is among the most important enteric pathogens of swine, and it can also infect other mammalian species. Much is still unknown regarding its pathogenesis and the host response, especially at the site of infection. In this study, we uncovered several novel genes and pathways associated with infection. Differentially expressed transcripts, in addition to histological changes in infected tissue, revealed striking similarities between L. intracellularis infection and cellular proliferation mechanisms described in some cancers and inflammatory diseases of the gastrointestinal tract. This research sheds important light into the pathogenesis of L. intracellularis and the host response associated with the lesions caused by infection.

Vaccination Against Lawsonia intracellularis Decreases Shedding of Salmonella enterica serovar Typhimurium in Co-Infected Pigs and Alters the Gut Microbiome.

Salmonella enterica serovar Typhimurium continues to be a major cause of foodborne illness worldwide and pork can serve as a source of infection. Co-infection of S. enterica with Lawsonia intracellularis, a common intestinal pathogen of swine, has been found as risk factor for increased S. enterica shedding. The objective of this study was to investigate if vaccination against L. intracellularis could lead to decreased S. Typhimurium shedding. To test this hypothesis, pigs were challenged with either S. Typhimurium or S. Typhimurium and L. intracellularis, with and without L. intracellularis vaccination (n = 9 per group). A non-challenged group served as a negative control. Vaccination decreased the shedding of S. Typhimurium in co-infected animals by 2.12 log10 organisms per gram of feces at 7 days post infection. Analysis of the microbiome showed that vaccination led to changes in the abundance of Clostridium species, including Clostridium butyricum, in addition to other compositional changes that may explain the protection mediated against S. Typhimurium. These results indicate that vaccination against L. intracellularis in co-infected herds may provide a new tool to increase food safety by helping to prevent S. enterica without the need for antibiotics.

Fecal Hyodeoxycholic Acid Is Correlated With Tylosin-Induced Microbiome Changes in Growing Pigs.

The changes in the gut microbiome play an important role in the promoting effects of antibiotics, such as tylosin, to the health, and productivity of farm animals. Microbial metabolites are expected to be key mediators between antibiotics-induced microbiome changes and growth-promoting effects. The objective of this study was to extend the identification of tylosin-responsive microbes to the identification of tylosin-responsive metabolites in growing pigs. The feeding trial was conducted on a commercial farm using two pens of pigs fed diets with and without tylosin (40 mg/kg of diet). Fecal samples were collected from 10 pigs per pen at weeks 10, 13, 16, 19, and 22 of age, and subsequently analyzed using liquid chromatography-mass spectrometry (LC-MS) analysis. The multivariate model of LC-MS data showed that time-dependent changes occurred in the fecal metabolome of both control and tylosin-treated pigs. More importantly, the metabolomic profiles were similar between the tylosin treatment and control groups in weeks 10 and 22, but diverged during weeks 13-19. Subsequent analyses of the fecal metabolites contributing to the separation of two groups of pigs showed that hyodeoxycholic acid (HDCA), together with tylosin and its metabolites in feces, was greatly increased during weeks 13-19 (P < 0.05) in the group of pigs fed tylosin. The integration of current metabolomics data and the microbiome data from a previous study revealed the consistency between HDCA and a specific genus of microbes in the Clostridia family. Further studies are required to determine the causative relations between tylosin-elicited changes in HDCA and the microbiome as well as the role of HDCA in the growth promoting effects of tylosin.

Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) to identify individual proteins within the complexes. Identity of individual proteins within complexes was further confirmed by MS upon excision of spots from 2D SDS-PAGE gels. Among the seven putative membrane complexes observed, major membrane protein (MAP2121c), a key MAP antigen involved in invasion of epithelial cells, was found to form a complex with cysteine desulfurase (MAP2120c). Other complexes found included those involved in energy metabolism (succinate dehydrogenase complex) as well as a complex formed by Cfp29, a characterized T cell antigen of Mycobacterium tuberculosis. To determine antigenicity of proteins, Western blot was performed on replicate 2D SDS-PAGE gels with sera from noninfected control cows (n=9) and naturally infected cows in the subclinical (n=10) and clinical (n=13) stages of infection. Clinical animals recognized MAP2121c in greater proportion than subclinical and control cows, whereas cysteine desulfurase recognition was not differentiated by infection status. To further characterize antigenicity, recombinant proteins were expressed for 10 of the proteins identified and evaluated in an interferon-gamma (IFN-γ) release assay as well as immunoblots. This study reveals the presence of protein complexes in the cell envelope of MAP, suggesting protein interactions in the envelope of this pathogen. Furthermore the identification of antigenic proteins with potential as diagnostic targets was characterized.

ZAP-70, CTLA-4 and proximal T cell receptor signaling in cows infected with Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis is a chronic intestinal disease of ruminant animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). A hallmark of paratuberculosis is a transition from a cell-mediated Th1 type response to a humoral Th2 response with the progression of disease from a subclinical to clinical state. The objective of this study was to investigate the expression of two crucial molecules in T cell function, ZAP-70 (zeta-chain-associated protein of 70 kDa) and CTLA-4 (cytotoxic T-lymphocyte antigen-4), in cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMCs) isolated from control non-infected cows (n=5), and cows in subclinical (n=6) and clinical stages of paratuberculosis (n=6) were cultured alone (medium only), and with concanavalin A, and a whole cell sonicate of MAP for 24, 72 and 144 h to measure the dynamic changes of ZAP-70 and CTLA-4 expression on CD4, CD8, and gamma delta (γδ) T cells. Flow cytometry was also performed to measure ZAP-70 phosphorylation to examine proximal T cell receptor signaling in animals of different disease status. The surface expression of CTLA-4 was increased in animals in subclinical stage of infection while levels of ZAP-70 were decreased in CD4+ T cells of both subclinical and clinical animals, indicating a change in T cell phenotype with disease state. Interestingly, proximal T cell receptor signaling was not altered in infected animals. This study demonstrated changes in crucial signaling molecules in animals infected with MAP, thereby elucidating T cell alterations associated with disease progression.

Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction.

Culture of Mycobacterium avium subsp. paratuberculosis (MAP) from feces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct fecal polymerase chain reaction (PCR) is becoming more widely used, demonstrating similar sensitivity and specificity to culture. To ensure efficient and reproducible PCR results from a difficult sample matrix such as feces, there are many obstacles that a DNA extraction method must overcome, including the presence of inhibitors and the thick waxy cell wall of MAP. In the current study, 6 commercial DNA extraction kits were evaluated using fecal samples from naturally infected cattle shedding various amounts of MAP. Upon extraction, DNA purity and yield were measured, and real-time PCR was performed for detection of the insertion sequence (IS)900 and ISMAP02 targets. The kits evaluated showed significant differences in the purity and yield of DNA obtained. The best results were observed with kits E and A, having identified 94% (16/17) and 76% (13/17) of the positive samples by IS900 PCR, respectively. Both of these kits utilized bead beating in a lysis solution for cell disruption, followed by spin column technology (kit E) or magnetic bead-based technology (kit A) for nucleic acid isolation and purification. Two kits (A and F) demonstrated improved performance when used in conjunction with the respective manufacturer's PCR test. The present study demonstrates the importance of choosing the correct methodology for the most accurate diagnosis of paratuberculosis through fecal PCR.

Perfil fenotípico e susceptibilidade antimicrobiana de Streptococcus equi isolados de equinos da região Sul do Brasil / Phenotypic profile and antimicrobial susceptibility of Streptococcus equi isolated from horses in southern Brazil

As características fenotípicas [morfológicas, bioquímicas, susceptibilidade aos antimicrobianos, índice de resistência múltipla aos antimicrobianos (IRMA), concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) da benzilpenicilina] de 38 isolados de Streptococcus equi oriundos de amostras clínicas de animais com adenite equina foram alvo deste estudo. A fenotipia demonstrou três padrões de colônias, três biotipos de fermentação de carboidratos e variação de 0 a 0,4 no IRMA. Todos os isolados de S. equi demonstraram sensibilidade à penicilina, tanto pelo método de disco difusão quanto pelo método de microdiluição. A CIM e CBM média de benzilpenicilina foi de 0,0095μg/mL e 0,0267μg/mL para S. equi subesp. equi e de 0,0128μg/mL e 0,0380μg/mL para S. equi subesp. zooepidemicus. Os valores de CIM e CBM diferiram entre as subespécies (p<0,05). O diâmetro do halo de inibição de penicilina demonstrou relação com a CIM (ì=0,03638 - 0,00072x) para S. equi subesp. equi. Também foi demonstrada relação entre o diâmetro do halo de inibição de penicilina com a CBM para S. equi subesp. equi (ì=0,10931- 0,00223x). Entretanto para as amostras de S. equi subesp. zooepidemicus esta relação somente foi verificada para a CBM (ì=0,1322 - 0,00271x). A CIM de benzilpenicilina frente às amostras isoladas da região Central, Planalto e Sul do estado do Rio Grande do Sul foram estatisticamente semelhantes, mas diferiram do isolado do estado do Paraná, sugerindo o caráter atípico desta cepa. Todos os isolados de S. equi são sensíveis à penicilina e sulfazotrim, confirmando a eleição destes antimicrobianos para o tratamento das infecções por este agente na clínica veterinária. Os resultados obtidos não dispensam a utilização prudente dos antimicrobianos.

Phenotypic characteristics [morphology, biochemical fermentation, antimicrobial susceptibility, index of multiple resistances to antimicrobials (IMRA), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of benzilpenicillin] of 38 Streptococcus equi isolates from clinical samples of horses with strangles were the aim of this study. The phenotypic analyses demonstrated three colony patterns, three carbohydrate fermentation biotypes and IMRA variation from 0 to 0.4. All the isolates of S. equi demonstrated sensitivity to penicillin, both by the disc diffusion method and microdilution method. The average MIC and MBC for benzilpenincillin were of 0.0095μg/mL and 0.0267μg/mL for S. equi subsp. equi and of 0.0128μg/mL and 0.0380μg/mL for S. equi subsp. zooepidemicus. The values of MIC and MBC differed between the subspecies (p<0.05). The diameter of penicillin inhibition halo demonstrated a relation with the MIC (ì=0.03638 - 0.00072x) for Streptococcus equi subsp. equi. A relation between the diameter of the inhibition halo of penincillin was also observed with the MBC for S. equi subsp. equi (ì=0.10931 - 0.00223x). However for the samples of S. equi subsp. zooepidemicus this relation was only verified with the MBC (ì=0.1322 - 0.00271x). The MIC of benzilpenicillin of the samples isolated from the Central, Planalto and South regions of Rio Grande do Sul were statistically similar, although different from the Paraná state sample, suggesting the atypical character of this strain. All the S. equi isolates are sensitive to penicillin and sulfazotrim, confirming these as antibiotics of choice for the treatment of infections caused by this agent in the clinical veterinary practice. The results obtained do not discard the prudent use of antimicrobials.

Surto de ceratoconjuntivite infecciosa bovina e hemoncose causando mortalidade em bezerros / Outbreak of infectious bovine keratoconjunctivitis and haemonchosis causing moratality in calves

É descrito um surto de ceratoconjuntivite infecciosa bovina (CIB) e hemoncose intercorrente afetando um grupo de 116 bezerros de 1-2 anos de idade na região central do Rio Grande do Sul. Vinte bezerros foram afetados e nove morreram. Os sinais clínicos incluíam perda de peso, marcada palidez da mucosa oral, lacrimejamento, opacidade focal da córnea e avermelhamento da conjuntiva. Sinais mais avançados incluíam lacrimejamento copioso, opacidade da córnea, ceratocone, ceratomalacia e cegueira. O diagnóstico de CIB foi baseado nos achados epidemiológicos e clínico-patológicos e confirmado pela cultura de uma cepa hemolítica de Moraxella bovis dos olhos de dois terneiros afetados. O alto coeficiente de letalidade (45 por cento) neste surto foi atribuído a grave hemoncose intercorrente.

An outbreak of infectious bovine keratoconjunctivitis (IBK) with concomitant haemonchosis is described affecting a group of 116 yearling calves in southern Brazil. Twenty calves were affected and nine died. Clinical signs included wasting, marked oral mucosal pallor, serous ocular discharge, focal central corneal opacity, and reddening of the conjunctiva. More advanced stages displayed copious serous ocular discharge, corneal opacity, keratoconus, keratomalacia, and blindness. The diagnosis of IBK was based on the epidemiological, clinical, and pathological findings and confirmed by bacteriologic culture of a hemolytic strain of Moraxella bovis from the eye globe of two affected calves. The high lethality rate (45 percent) in this outbreak was caused by concomitant and severe haemonchosis.