The bacterial strains, plasmids, and primer pairs used in this study are listed in Table 1 . We used the ST mutant strain JOL912 (∆asd ∆lon ∆cpxR) to deliver the LI vaccine antigens. The asd auxotrophy of the strain enables antibiotic-free stable maintenance of the strain, which is grown by supplementing Luria–Bertani medium with diaminopimelic acid (50 µg/mL). The live-attenuated LI strain (Enterisol Ileitis; Boehringer Ingelheim Vetmedica, USA) was purchased and dissolved in sterile phosphate-buffered saline (PBS, pH = 7.2). The final concentration of the strain was adjusted to 106.9 times the median tissue culture infectious dose (TCID50) per 100 µL volume for the challenge experiment.
Enterisol ileitis
Manufactured by Boehringer Ingelheim
Sourced in Germany, United States
Enterisol Ileitis is a veterinary diagnostic lab equipment used for the detection of Lawsonia intracellularis, the causative agent of porcine proliferative enteropathy, also known as ileitis. The product utilizes an enzyme-linked immunosorbent assay (ELISA) method to identify the presence of specific antibodies against Lawsonia intracellularis in serum or plasma samples collected from pigs.
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10 protocols using enterisol ileitis
1
Attenuated Salmonella Vaccine Delivery
2
Evaluation of Ileitis Vaccine Response
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The vaccine challenge was performed using the vaccine against L. intracellularis with an intestinal tropism (Enterisol ® Ileitis; Boehringer Ingelheim Vetmedica GmbH). This vaccine is a live attenuated vaccine composed of L. intracellularis as active substance from 10 4•9 to 10 6•1 Tissue Culture Infective Dose 50 . At PND 33, 117 weaned pigs were vaccinated (n 31 CTRL/CTRL, n 30 CTRL/scFOS, n 28 scFOS/CTRL and n 28 scFOS/scFOS) with a ten-times dose (10×) of vaccine in 2-ml sterile water by oral drenching. The 10× dose of vaccine was used to induce detectable levels of L. intracellularis-specific Ig. Indeed, the administration of a 1× dose of vaccine induced moderate or even undetectable levels of specific Ig in the serum and ileal mucosa (24) . In each litter, one or two pigs assigned to either a standard post-weaning diet or a scFOS-supplemented diet were not vaccinated (four pigs per dietary group). This reference group was used to detect differences between the effect of dietary treatments and the effect of vaccination on serum and intestinal parameters. Non-vaccinated pigs were housed separately (in another room) from the vaccinated ones. Vaccine response was evaluated 3 weeks after oral immunisation (PND 54-56). No other vaccine was administered during the experiment.
3
Oral Live Attenuated Ileitis Vaccine
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Pigs in the VAC-LAW group were vaccinated with a commercial L. intracellularis vaccine (Enterisol® Ileitis, Boehringer Ingelheim, serial number 3040187B) at 3 weeks of age. This particular vaccine is a one dose product, contains a live bacterial culture, and is given orally. Prior to vaccination the vaccine was freshly reconstituted according to the manufacturer’s instructions and administered to each VAC-LAW pig by slowly dripping 2 mL of the vaccine with a syringe into the mouth.
4
Serological Assay for L. intracellularis Vaccine Strain
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The in vitro cultivation of L. intracellularis is extremely difficult, which limits the production of this bacterium to few laboratories in the world. Since our objective was to develop a serological assay that could be easily performed in any laboratory, we used the vaccine strain of L. intracellularis from Enterisol® Ileitis (Boehringer Ingelheim), a live attenuated vaccine, as a source of antigen. This licensed vaccine is distributed worldwide, and all new batches of live antigen are qualified by the manufacturer, an ideal situation to guarantee antigen quality and ensure repeatability between antigens batches and laboratories that will perform this diagnosis.
5
Gilt Management and Estrus Stimulation
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At the initial receiving GDU, gilts were housed (providing approximately 0.84-m2 floor space per gilt) in groups of 25 on fully slatted floors. Upon arrival, gilts were acclimated by inoculation with porcine epidemic diarrhea virus, as well as vaccinating for ileitis (EnterisolIleitis, Boehringer Ingelheim) and erysipelas (Ery Vac, ARKO Laboratories). Once transported to the sow farm-specific GDU, all gilts were inoculated with a site-specific porcine reproductive and respiratory syndrome virus strain. At the sow farm, gilts were again allocated approximately 0.84-m2 floor space per head. At approximately 20 wk of age, gilts were again subjected to selection criteria unrelated to reproductive performance. At approximately 20 to 26 wk of age, gilts entered the designated sow farm with boar exposure beginning immediately using mature boars (>18 mo of age) which were rotated daily and not used more than once per week. Boar exposure (approximately 10 min of contact per pen) for puberty stimulation and heat detection was done via fence-line contact. Gilts not demonstrating behavioral estrus by 36 wk of age were culled, and their culling reason noted as failure to display estrus.
6
Cultivation of Intracellular Bacteria
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The L. intracellularis strain (a commercial live attenuated L. intracellularis vaccine, Enterisol®ileitis) was purchased from Boehringer Ingelheim Vetmedica, Germany, and Escherichia coli (CAU0751), Salmonella choleraesuis (CVCC2139), and Salmonella typhimurium (CVCC2220) were purchased from the China Veterinary Culture Collection Center (CVCC). Desulfovibrio (ATCC 27774) and Bilophila wadsworthia (ATCC 49260) were purchased from the American Type Culture Collection (ATCC). Brachyspira hyodysenteriae is courtesy of the Shanghai Animal Disease Prevention and Control Center. The pGex-6p-1 (+) vector was purchased from Novagen, USA. The myeloma cell line SP2/0 (stored in our laboratory) and rat small intestine cells (IEC-18; ATCC CRL 1589) were cultured in DMEM/high glucose (Gibco, USA) in a humidified 5% CO2 atmosphere at 37°C. All the cell culture media were supplemented with 10% fetal bovine serum (FBS, Gibco, USA). L. intracellularis was grown as described (5 (link)). In brief, 30% confluent IEC-18 monolayers were infected with L. intracellularis and incubated in an atmosphere of 83.2% nitrogen (N2), 8.8% carbon dioxide (CO2), and 8% oxygen (O2) for 7 days at 37°C.
7
Salmonella-based Vaccine Delivery Protocol
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The bacterial strains, plasmids and primer pairs used in this study are listed in Table 1 . The JOL912 ST mutant strain was used to deliver the LI-specific vaccine antigens. An asd+ plasmid (pJHL65) was used for cloning and expression of LI antigens in the JOL912 strain. All bacterial strains were incubated at 37°C in Luria-Bertani broth. The live attenuated LI strain (Enterisol Ileitis; Boehringer Ingelheim Vetmedica, Inc., USA) was purchased, and dissolved in sterile phosphate buffered saline (PBS; pH = 7.2). Final concentration of the strain was adjusted to 106.9 50% tissue culture infectious dose (TCID50) per 100 µL volume for the challenge experiment.
8
Murine Ileitis: Pathogenesis and Immune Responses
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The test group mice were experimentally infected with about 5 × 107L. intracellularis (Enterisol Ileitis; Boehringer Ingelheim, Germany) administered by gavage. The control group received phosphate-buffered saline (PBS). For 5 consecutive weeks, the mice were weighed and their feces were collected. Blood samples were collected once weekly and stored at −20°C. Additionally, the ileum and spleen (n = 5/group) were collected for further analyses. The ileal tissue was processed for quantification of the L. intracellularis 16S rRNA gene. Further, the expression of genes encoding for mucin, pro- and anti-inflammatory cytokines, the vitamin B12 transporter and Lawsonia chaperonin GroEL. The spleen was processed for profiling of the T-cell population.
9
Effect of L. intracellularis Vaccination in Piglets
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Four weekly weaning groups were followed in the study. One group consisted of approximately 500 piglets, of which half were vaccinated with L. intracellularis vaccine at the age of 3 weeks about 7 days before weaning (V = vaccine group). The other half of non-vaccinated piglets served as controls (C = control). The litters were chosen randomly and all piglets in a litter were either vaccinated or served as controls. One vaccine group piglet at a time was lifted from the ground and administered 2 mL of L. intracellularis vaccine (Enterisol Ileitis®, Boehringer Ingelheim Vetmedica GmbH) directly into the mouth with a drench and injected intramuscularly 1 mL of circovirus vaccine (Ingelvac CircoFLEX®, Boehringer Ingelheim Vetmedica GmbH) behind its ear. The control group piglets were lifted similarly but only received the circovirus vaccine. Vaccinated piglets were marked with an individually numbered red ear tag in their right ear. Similarly, control piglets received a numbered yellow ear tag in their left ear.
10
Immunoreactivity Analysis of LFliC Protein
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Western blot analysis was performed to evaluate immunoreactivity of the LFliC protein in vitro using anti-LI hyperimmune serum obtained from the mice immunized with an L. intracellularis modified-live vaccine (Enterisol® Ileitis, Boehringer Ingelheim, Ingelheim am Rhein, Germany). The mice were inoculated with 5 × 106.9 50% tissue culture infectious doses (TCID50) via an oral route twice at 2-week intervals. The purified protein (10 μg/μL) was subjected to 15% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with phosphate-buffered saline (PBS) buffer containing 3% bovine serum albumin (BSA) for 1 h, followed by incubation with the anti-LI mouse hyperimmune serum at a 1:300 dilution for 2 h. The antigen–antibody interaction was detected with a 1:5000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotechnology, Birmingham, AL, USA; diluted 1:5000).
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