Methods for culture and identification of mycrobacterium avium subspecies in crohn's disease

Abstract

Method and media for diagnosing Crohn's disease are provided. A method of diagnosing Crohn's disease in patients includes: obtaining a sample from an individual; culturing the sample to determine the presence or absence of Mycobacterium avium subspecies hominissuis (MAH) in the sample; and diagnosing the individual with Crohn's disease based on the determining the presence of MAH in the sample.

What is claimed: 
     
         1 . A method of diagnosing Crohn's disease in patients, comprising:
 obtaining a sample from an individual;   determining the presence or absence of  Mycobacterium avium  subspecies  hominissuis  (MAH) in the sample; and   diagnosing the individual with Crohn's disease based on the determining the presence of MAH in the sample.   
     
     
         2 . The method of  claim 1 , wherein the obtaining the sample comprises obtaining whole blood from the individual, and further comprising preparing the sample prior to the determining. 
     
     
         3 . The method of  claim 2 , wherein the preparing the sample comprises:
 removing plasma proteins from the sample; and   lysing red blood cells of the sample.   
     
     
         4 . The method of  claim 3 , further comprising culturing the prepared sample using three different media. 
     
     
         5 . The method of  claim 4 , wherein the determining comprising using PCR direct sequencing to detect the presence of both IS1245 and 16S rDNA in an isolate of the cultured sample. 
     
     
         6 . The method of  claim 1 , further comprising culturing the sample using a liquid medium comprising:
 Middlebrook 7H9;   Yeast extract;   Glycerol;   Sucrose;   Tween 80;   Mycobactin J;   Oleic acid; and   NAD.   
     
     
         7 . The method of  claim 6 , wherein the liquid medium is composed of:
 Middlebrook 7H9 0.47% volume/volume;   Yeast extract 0.1% volume/volume;   Glycerol 0.5% volume/volume;   Sucrose 0.2% volume/volume;   Tween 80 0.05% volume/volume;   Mycobactin J 2 μg/ml weight/volume;   Oleic acid 0.1% volume/volume; and   NAD 20 μg/ml weight/volume.   
     
     
         8 . The method of  claim 1 , further comprising culturing the sample using a solid medium comprising:
 Middlebrook 7H10   Yeast extract;   Glycerol;   Sucrose;   Tween 80;   Mycobactin J;   Oleic acid; and   NAD.   
     
     
         9 . The method of  claim 8 , wherein the solid medium is composed of:
 Middlebrook 7H10 1.9% volume/volume;   Yeast extract 0.1% volume/volume;   Glycerol 3% volume/volume;   Sucrose 20% volume/volume;   Tween 80 0.05% volume/volume;   Mycobactin J 2 μg/ml weight/volume;   Oleic acid 0.1% volume/volume; and   NAD 20 μg/ml weight/volume.   
     
     
         10 . The method of  claim 8 , wherein the solid medium is composed of:
 Middlebrook 7H10 1.9% volume/volume;   Yeast extract 0.1% volume/volume;   Glycerol 0.5% volume/volume;   Sucrose 0.2% volume/volume;   Tween 80 0.05% volume/volume;   Mycobactin J 2 μg/ml weight/volume;   Oleic acid 0.1% volume/volume; and   NAD 20 μg/ml weight/volume.   
     
     
         11 . The method of  claim 1 , further comprising treating the individual for Crohn's disease based on the diagnosing. 
     
     
         12 . The method of  claim 1 , further comprising performing serology testing of plasma of the individual. 
     
     
         13 . The method of  claim 12 , wherein the serology testing is used to identify a specific antibody against  Mycobacterium avium  subspecies  paratuberculosis  (MAP). 
     
     
         14 . The method of  claim 12 , wherein the serology testing is performed using a 12-well glass-slide for antibody titration. 
     
     
         15 . The method of  claim 1 , wherein the determining comprises:
 transfer 4 ml blood of the sample from a vacutainer tube to a sterile 15-ml centrifuge tube, and spin the blood in a centrifuge at 6000 g for 10 minutes at room temperature;   remove plasma from the centrifuge tube using a sterile pipette and store plasma in a 2-ml microfuge tube at −20° C. for antibody titration later;   add 10-ml red blood cell lysis buffer to the cells in the centrifuge tube at room temperature, and resuspend the cells by turning the capped centrifuge tube up and down several times;   spin the centrifuge tube at 6000 g for 10 minutes in a centrifuge, and discard the supernatant to a biohazard container;   add 4-ml liquid media to the centrifuge tube, and resuspend the cell pellet by turning the capped centrifuge tube up and down;   remove two aliquots of 100 μl the resuspended cells in liquid media, and plant them in one solid induction plate/slant and one solid maintenance plate/slant;   incubate all the culture tube and plates/slants at 37° C. for 2 weeks without additional CO2, wherein the liquid culture tube, solid plates/slants are examined visually every week, and wherein the culture media are kept at 37° C. for minimally 12 weeks;   after the incubating, an aliquot of 100 μl liquid culture is removed by a sterile pipette, and the culture is transferred to a microfuge tube, the cells are collected by centrifugation at 12000 g for 5 minutes, and discard the supernatant;   resuspend the cells with 500 μl acetone, such that the cells are fully resuspended;   collect cells by centrifugation at 12000 g for 5 minutes, discard the supernatant;   resuspend the cells in 200 μl sterile TE buffer by vortexing the tube, and heat the resuspended cells at 95° C. for 15 minutes;   chill the tubes on ice, and use the content directly for PCR analysis; and   if there are colonies on the solid plates/slants, pick a single colony by a sterile toothpick, and resuspended in 200 μl TE buffer, the bacteria in TE buffer is heated at 95° C. for 15 minute and it is directly used for PCR analysis.   
     
     
         16 . A composition usable as a culture medium, comprising:
 one of Middlebrook 7H9 and Middlebrook 7H10;   Yeast extract;   Glycerol;   Sucrose;   Tween 80;   Mycobactin J;   Oleic acid; and   NAD.   
     
     
         17 . The composition of  claim 16 , wherein the composition is a liquid comprising:
 Middlebrook 7H9 0.47% volume/volume;   Yeast extract 0.1% volume/volume;   Glycerol 0.5% volume/volume;   Sucrose 0.2% volume/volume;   Tween 80 0.05% volume/volume;   Mycobactin J 2 μg/ml weight/volume;   Oleic acid 0.1% volume/volume; and   NAD 20 μg/ml weight/volume.   
     
     
         18 . The composition of  claim 16 , wherein the composition is a solid comprising:
 Middlebrook 7H10 1.9% volume/volume;   Yeast extract 0.1% volume/volume;   Glycerol 3% volume/volume;   Sucrose 20% volume/volume;   Tween 80 0.05% volume/volume;   Mycobactin J 2 μg/ml weight/volume;   Oleic acid 0.1% volume/volume; and   NAD 20 μg/ml weight/volume.   
     
     
         19 . The composition of  claim 16 , wherein the composition is a solid comprising:
 Middlebrook 7H10 1.9% volume/volume;   Yeast extract 0.1% volume/volume;   Glycerol 0.5% volume/volume;   Sucrose 0.2% volume/volume;   Tween 80 0.05% volume/volume;   Mycobactin J 2 μg/ml weight/volume;   Oleic acid 0.1% volume/volume; and   NAD 20 μg/ml weight/volume.   
     
     
         20 . A method of serology testing, comprising:
 serology testing plasma of an individual to identify a specific antibody against  Mycobacterium avium  subspecies  paratuberculosis  (MAP), wherein the serology testing is performed using a 12-well glass-slide for antibody titration.