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Lawsonia intracellularis cultivation, anti-lawsonia intracelluaris vaccines and diagnostic agents

A Lawsonia intracellulare and vaccine technology, applied in the direction of bacterial antigen components, bacteria, biological testing, etc., can solve the problems of unsuitability for large-scale cultivation, laborious cultivation methods, understanding of disease treatment and effective control obstacles, etc.

Inactive Publication Date: 2004-08-11
NOBL LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These existing culture methods are laborious and not suitable for large-scale cultivation
[0007] Current understanding of L. intracellulare infection and the treatment and effective control of the disease are hampered by the harsh growth conditions required to culture L. intracellulare in vitro

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Isolation of Lawsonia intracellulare from the gut of American pigs with porcine proliferative enteropathy

[0076] Materials and methods

[0077] Selection of Inoculation Samples

[0078] Obtaining sample N24912 in a herd on a farm in Iowa, 15 of 300 5-month-old finisher pigs were observed to have consistently bloody stools despite treatment with penicillin. After autopsy of the pig, the intestine (ileum) was found to have a thick mucosa. Histopathological examination with silver staining revealed the presence of curved, intracellular bacterial and crypt intestinal epithelial cell proliferation, confirming the diagnosis of PPE. Sample N72994 was obtained from a 1.5-year-old second-parity SPF sow on a farm in Minnesota. The herd size was 70-80 sows and the antibiotic treatment used was unknown. Autopsy revealed thickened and somewhat hemorrhagic ileal mucosa. Giminez staining of the mucosa revealed the presence of numerous Curlybacteria. Sample N1014 94 was obtaine...

Embodiment 2

[0100] Growth of Lawsonia intracellulare in suspension cultures of HEp-2 cells

[0101] Prepare intestinal homogenate for inoculation:

[0102] Intestinal tissue homogenates were prepared by scraping the mucosa from 6.0-8.0 cm of the ileum in the intestinal samples of Example 1. Trypsin (1%) was added to the scraped mucosa and samples were briefly homogenized and then incubated at 37°C for 35 minutes. Another 10 ml of SPG / 10% FBS was added and the samples were ground in a tissue grinder. Another 10 ml of SPG / 10% FBS was added. The homogenate was filtered through Whatman 113V filter paper followed by 5.0, 1.0 and 0.65 micron filter membranes. The filtrate was divided into 1.0 ml portions and stored at -70°C.

[0103] Infection of cell cultures

[0104] Method A:

[0105] Tissue cells at 1×10 7 Cells were seeded in 100 ml spinner flasks containing 50 ml DMEM / 10% FBS. Cultures were incubated for 24 hours before addition of vancomycin and amphotericin B. A vial of frozen ...

Embodiment 3

[0118] Growth of Lawsonia intracellulare in suspension cultures of McCoys cells

[0119] Prepare intestinal homogenate for inoculation:

[0120] According to the method described in Example 2, intestinal tissue homogenate was prepared. The Lawsonia intracellulare sample cultivated with the following example method was deposited in the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Maryland U.S.A 20852) on May 19, 1995 according to the Budapest Treaty, and the deposit number is 55672.

[0121] Infection of cell cultures

[0122] McCoys cells at 1.25×10 5 Cells were seeded in two 25cm tubes containing DMEM / 10%FBS 2 In a regular bottle, incubate for 18-24 hours. Cells were 30% confluent upon seeding. Inoculum was diluted in DMEM / 5% FBS. When the inoculum was from intestinal homogenate, the medium also contained vancomycin (100 μg / ml) and amphotericin B (2.0 μg / ml). The culture was placed in an air tank, evacuated, and refilled with hydrogen and c...

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PUM

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Abstract

A method for large scale cultivation and attenuation of L. intracellularis bacteria by inoculating cells with L. intracellularis bacteria to infect the cells, incubating the infected cells in a reduced oxygen concentration and maintaining the infected cells in suspension. Anti-L. intracellularis vaccines are prepared from cultures grown in suspension. Diagnostic agents are also disclosed.

Description

[0001] This divisional application is a divisional application of the Chinese patent application CN 96114542.0 entitled "Cultivation of Lawsonia intracellulare, Vaccine against the Bacteria and Diagnostic Reagent". technical field [0002] The present invention relates to vaccines against Lawsonia intracellularis and methods of preventing and diagnosing LI infections. The products and methods of the present invention are partly a result of our discovery of improved methods for the large-scale cultivation of L. intracellulare. Background technique [0003] Lawsonia intracellulare is the causative agent of porcine proliferative enteropathy (PPE), which affects almost all animals, including humans, rabbits, ferrets, hamsters, foxes, horses, and a variety of other animals such as Ostrich and rhea etc. [0004] Lawsonia intracellulare is a particularly important cause of losses in pig herds. Estimates of the incidence and infection rates of PPE in the United States are that up ...

Claims

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Application Information

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IPC IPC(8): A61K35/74A61K39/02A61P31/00C12N1/20C12N1/36C12Q1/04G01N33/53
Inventor J·P·克尼特尔M·B·鲁夫
Owner NOBL LAB
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