Controlling Gumboro Disease, is what I am doing enough?- Part 2

Infectious  Bursal  Disease  (IBD)  represents  one  of  the  main  health  threats  for  the  poultry  industry,  mainly due to the severe immunosuppression induced by the virus (IBDV) in affected birds.  IBD requires a quick diagnosis for immediate reaction in the case of an outbreak.  Thus, molecular tools such as PCR and sequencing have become indispensable for controlling the disease. For this reason, HIPRA offers a molecular diagnosis programme for IBD, known as “GUMBOCHECK”.

GUMBOCHECK

GUMBOCHECK is a diagnostic pack, which includes an FTA card, a form to annotate the data of  sampled animals and a short guide that shows how to carry out a good sampling to obtain a positive result.

The main objective of GUMBOCHECK is to evaluate the colonization of the bursa of Fabricius by the vaccine virus, for an evaluation of the efficacy of vaccination. However, it can also be used as a diagnostic tool in the case of IBD field infection.

To achieve this, it is advisable to carry out this evaluation at different times in the life of the birds.  It is recommend to be done at 21, 28 and 35 days of age. Nevertheless, when colonization of the bursa by HIPRA 1052 (GUMBOHATCH® strain) is confirmed, the subsequent sampling points may be skipped.

GUMBOCHECK STEPS

1. SAMPLING

The sampling of IBD is performed with an FTA card. What is an FTA card and how does it work?

•  An FTA card is a piece of paper with a cellulose matrix designed to capture cells inside the biological samples.

• After this capture, the membranes of the cells break (bacterial walls or the viral capsids), which facilitate the stability of the genetic material at room temperature for a long time.

• Due to the rupture of these membranes, biological samples inoculated in FTA cards are considered as non-infectious substances, since the pathogens are inactivated.

• FTA cards facilitate the shipment and storage of biological samples.

• Once the FTA card arrives at the laboratory, the genetic material is easily extracted to perform the PCR.

There are some critical points that we should take into account when using FTA® cards:

• There are different types of FTA® cards: not all of them work properly or give the same results.  For this reason, HIPRA just recommends using FTA cards provided in the GUMBOCHECK packs.

• Do not to use expired FTA® cards: expired FTA® cards can give false negative results, so it is important to look at the expiration date on the label.

• Proper inoculation of the sample: samples should not be inoculated outside the delimited area in order to guarantee the correct inactivation of the pathogens.

• Correct number of animals inoculated: GUMBOCHECK FTA® cards have four areas where samples are applied. Each area on the card has a capacity of two birds, meaning a total capacity of eight birds for each FTA® card/sampling day.

• Enough time: all the reactions need some time after the inoculation to break the membranes and preserve the genetic material (at least 1 hour at room temperature).

2. PCR

PCR is a molecular diagnostic technique with the aim of amplifying the genome of a pathogen present in the sample. In the case of IBD virus, the amplified gene is VP2, since it is the most immunogenic protein, located in the surface of the virus.

How does the PCR work?

• This technique generates millions of copies of a specific gene in order to be able to detect a very small amount of the virus. It allows having an exponential amplification of the initial amount of virus present in the sample.

Diagnostic reports show a parameter known as Ct value, which is the number of cycles that a sample needs to be detected as positive. For example, if the Ct value shown is 32, it will mean that the sample needs 32 cycles of the PCR to be detected as positive.



*Ct value is inversely proportional to the amount of genetic material present in the sample

3. VIRUS TYPING

Once positive samples are detected with PCR, the typing of the virus is performed in order to know the kind of virus present in the sample, or rather, to know if it is a vaccine strain or a field strain. This typing is done by sequencing.

IBD virus can be classified regarding antigenicity but also regarding pathogenicity. Moreover, this virus is constantly evolving because of mutations or combination of events. For this reason in 2017 Jackwood  proposed that all the viruses circulating worldwide can be classified into seven different genogroups, being genogroups 1, 2 and 3 the more prevalent.

• Genogroup 1 represent all the classified as classical strains.

• Genogroup 2 represent all the viruses known as variant strains.

• Genogroup 3 represent the very virulent strains.

• All the other viruses that do not belong into any of this three genogroups are classified into groups from 4 to 7 depending on molecular characteristics of the of the virus.

GUMBOCHECK programme offers two different reports:

1. A table with the percentage of nucleotide similarity compared with referenced strains in each genogroup, in order to know which kind of virus is present in the sample and in what genogroup belongs to.

2. A phylogenetic tree, a graphical way to see the genetic distance between all the different strains.

• Phylogenetic trees are composed by different branches or clusters. Each of the branches belongs to one specific genogroup, so the first step will be to see in which branch is located the sampled strain.

• After knowing the genogroup of the sampled strain, the similarity of the different viruses can be compared by looking at the horizontal distance of the tree. Therefore, the longer is the horizontal line, the bigger will be the genetic distance between two specific strains.

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