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Bovine Tuberculosis Protocols Database


CULTURE
EXTRACTION & IDENTIFICATION
MOLECULAR CHARACTERIZATION
INTERFERON GAMMA DETECTION
POTENCY TESTS

Bovine Tuberculosis Protocols Database

As defined in the specific responsibilities and tasks for the EU-RL for Bovine Tuberculosis in the Commission Regulation (EC) No 737/2008, the EU-RL has to facilitate the harmonization of techniques throughout the Community, in particular specifying standard test methodologies.


With this objective in mind, the EU-RL sent to all Member States questionnaires regarding a) stain and bacteriological culture; b) chemical test, DNA extraction and identification of mycobacteria; d) molecular characterization; and e) interferon gamma detection. The main aim was to compile all the information available and to define the state of the art of the different methodologies used in the National Reference Laboratories in Europe. In a second stage, methodologies will be revised by the EU-RL and actions (proficiency tests, determine analytical sensitivity, etc.) will be taken towards harmonization of the protocols


Initially a manual review of protocols was programmed to be edited but since the main objective of the EU-RL is to produce useful material to the rest of the NRLs all the information has been included in an online database (housed in the EU-RL for Bovine Tuberculosis) with restricted access to the NRLs.


In this protocol database all the information regarding the different methodologies are included in different searches by technique, reagent, country, etc. that will allow the NRL to find out the information. Moreover, information regarding accreditation by the European Standard EN ISO/IEC 17025 and internal quality controls are also included.


This database should be updated periodically in case any NRLs modify/change a protocol. Therefore if you need to modify any of the information included regarding your NRL send an e-mail to Dr. Beatriz Romero.


Stain and bacteriological culture


This database includes the information related to the methodology for the acid fast bacillus (AFB) stain and bacteriological culture.


Most of the laboratories currently use the AFB stain (mainly Ziehl-Neelsen) for the detection bacilli from tissue samples and/or from solid or liquid media.


Regarding the bacteriological culture the main differences between the NRLs are the decontamination step (decontaminant and time of exposure) and the solid media (type and incubation period). In the case of decontamination of samples for the isolation of mycobacteria the decontaminants used are: hexadecil pyridinium chloride, HPC (0.35% or 0.75%); sodium hydroxide, NaOH (2% or 4%); oxalic acid, H2C2O4 (4.5% or 5%); hydrogen chloride, HCl (1M or 2.87M); sulphuric acid, H2SO4 (4% or 5%); n-acetyl-L-cysteine-sodium hydroxide, NALC-NaOH (1%). In relation to the culture media most laboratories use solid commercial media (mainly Lowenstein Jensen and Lowenstein Jensen with sodium pyruvate) and the incubation time ranges from 54 to 90 days. The main liquid automatized system for the recovery of the MTB complex bacteria is the BACTEC MGIT system. Moreover, information regarding preservation of the strain is also included in this sections being the liquid medium Middlebrook 7H9 with glycerol the one used by most NRLs.


Culture protocols (restricted to NRLs).


Chemical test, DNA extraction and identification of mycobacteria


Information regarding chemical test used for the identification of mycobacteria is included in the database, although nowadays only a few Member States still use this methodology.


Regarding DNA extraction, the NRLs that perform the DNA extraction by heat inactivation or by extraction kits are described in the database.


In relation to the diagnostic techniques used for the identification of mycobacteria all MSs use protocols based on the polymerase chain reaction (conventional or real time PCR, multiplex or single). There is a wide variety of protocols, some of them based on commercialized kits or in-house/published PCRs. The main targets for the identification of the Mycobacterium tuberculosis complex (MTBC) members are the insertion sequence IS6110 and the mpb70 gene. For visualization of DNA in the agarose gel several stain markers are currently used in the NRLs (ethidium bromide, SyBR Safe, Gel Red or Gold View). If the laboratory includes sequence analysis for the identification of mycobacteria is also specified as well as the NRLs that have a DNA collection.


Extraction & Identification protocols (restricted to NRLs).


Molecular characterization


The molecular characterization is the methodology used for the differentiation of the strains. Among the MTBC three techniques are mainly used: Restriction fragment length polymorphism (RFLP) directed to the detection of the insertion sequence IS6110, Direct Variable Repeat - Spacer Oligonucleotide Typing (DVR-spoligotyping) and Mycobacterial Interspersed Repetitive Units- Variable Number of Tandem Repeats (MIRU-VNTR) typing.


RFLP-IS6110 is not commonly used for the characterization of M. bovis isolates due to the low number of insertion sequences and therefore the discrimination of the strains is very low.


The spoligotyping technique is a routine technique for molecular characterization and the protocol is easily standardized. The main difference between NRLs is the source of the spoligotyping membrane (commercial, NRL in-house membrane or EU-RL in-house membrane). All the NRLs performing this protocol assign the same coding number to each spoligotyping profile based on the database mbovis.org.


The MIRU-VNTR protocol for the molecular characterization of the members is also a widely used technique. The main differences are based in the technique used to determine the number of repeats per locus (gel or capillary electrophoresis) and the selection of the MIRU-VNTR markers that is quite variable among the MSs. However most laboratories always include the panel of 6 loci defined by VENoMYC European Coordination Action (ETR-A, ETR-B, ETR-D, QUB11a, QUB11b and QUB3232).


Molecular characterization protocols (restricted to NRLs).


Interferon gamma detection


The interferon gamma detection for the diagnosis of bovine tuberculosis is applied routinely in those countries that are not officially TB-free and in some certain situations in the other countries.


The parameters that are included in this database are the time of blood stimulation (8, 24 or 30 hours post-collections), well- plate format (24 or 96), the final concentration of PPDs (10-30 ng/ l), the use of additional antigens or mitogens and the interpretation cut-off.


IFN Gamma detection protocols (restricted to NRLs).