Immunosuppression and viral persistent in the

host with reference to IBDV

Dr Udi Ashash, Abic Biological Laboratories Teva Ltd

Commercial rearing of chickens depends on many factors; among them is the control of infectious diseases. Management efforts of disease control (e.g. Vaccination) can be negatively influenced by immunosuppression.

Definition of Immunosuppression: "A state of temporary or permanent dysfunction of the immune response resulting from insults to the immune system and leading to increased susceptibility to disease and often a suboptimal antibody response and/or suboptimal innate and cell-mediated immune responses." ( Dohms and Saif  1984,  Schat 2008).

"Immunosuppression" is a term widely used to describe different situations and often cause confusion so it is important to understand the differences between the term "immunosuppression" and "Immunodeficiency" (ID).

"Immunodeficiency" will result in total failure of the immune system to react to an immune stimulus (e.g. vaccination) so ID is always a big problem.

On the other hand, if chickens with certain levels of "immunosuppression", can still gain immunity and be protected from diseases, then this "Immunosuppression" is not a problem at all.

The immune system operates as an integrated system, not a set of components working independently. All three compartments (Innate, Cell-mediated and Humoral responses) interact in multiple ways to generate the desired immunity.

It is relatively easy to measure the function of the humoral response (Antibodies) and directly correlate it with protection, but most of our methods for measuring the cell-mediated and innate immunity in chickens are only indirectly correlated with protection and as a result we have much greater knowledge of the effects of an immunosuppressant on the antibody response, and small knowledge of the effects on innate or cell-mediated immunity.

Due to that, the best way to measure the overall immunosuppression level is to do a comparative challenge trial and then asses the overall protective response.

Effects of immunosuppression: Most studies have focused on resistance to clinical disease and relatively few studies have considered the sub-clinical aspect of immunosuppression.

Studies have shown that immunosuppression can increase disease likelihood and severity (Kim et al., 2003, 2004) and also reduces the efficacy of vaccination. vvIBDV infection reduces the efficacy of vaccination against Marek's disease virus (Jen et al., 1980). Potentially, immunosuppression can exacerbate the effects of attenuated vaccines and increase the susceptibility of the infected birds to the vaccine strain e.g. CAV infection leads to development of clinical ND disease after vaccination with the La-Sota strain (Deboer et al., 1994). Interaction between different potential immunosuppressive viruses can cause increasing susceptibility 

To each other (Davidson et al 2008)

Sub-clinical effects of immunosuppressive agents are generally either ignored or over emphases. Effects can extend from interfering with control of other diseases, to effects on productivity

Causes of Immunosuppression: immune competence can be affected by ontological, toxicological, environmental, nutritional and infectious factors.

Toxins: Most studies (done on mycotoxins) show that at high doses it causes depletion of lymphoid cells and immunosuppressive effects at lower doses.   Aflatoxins suppress antibody responses to viruses and can depress the cell-mediated immune response (Giambrone et a l., 1978) and innate immune mechanisms Ochratoxin A  have a greater effect on T lymphocytes than on humoral responses but can affect antibody responses and phagocytic cell function (Chang et al., 1980; Dwivedi et al.,1984). T2 Toxin cause lymphoid depletion and increase susceptibility to salmonellosis in DOC .

Environment condition: The physical size of the lymphoid organs, the antibody response and the phagocytic activity of macrophages can be adversely affected by heat stress (Bartlett et al., 2003). Differences in housing systems can influence antibody responses, but not phagocytic activity of macrophages. And bursa size is lower in chickens reared at high stock densities compared to those reared at lower densities. (Muniz et al 2006)

Nutrition: Development of the embryonic immune system is dependent on adequate concentrations of vitamin A, some of the B vitamins, iron, selenium and linoleic acid (Klasing, 1998). Nutrients that have been shown to influence immune responses include vitamins A, B6, D and E (Blalock et a l., 1984; Aslam et al., 1998; Dalloul et a l., 2002), selenium and zinc (Bartlett et al., 2003).

An important aspect of this is the balance between intakes necessary for optimal immune function and the intakes that are sufficient for maximal feed conversion efficiency and growth rates under ideal conditions. If chickens are overfed with some micronutrients it may have an adverse effect on immune responses (Friedman et a l., 1998).

 Age: Chickens age is a very important factor and the chick is not fully immune-competent before 2 weeks of age and with our wish to get the chickens vaccinated ASAP we tend to forget it. 

Most inflammatory and phagocytic activity appears to be adequate in day old chicks (Bar-Shira et al., 2006). However, the acquired immune response is poor at hatching and continues to develop over the first two weeks of life. Vaccinated day-old chicks, do not strongly respond to a booster vaccination at 28 days of age (Mast et al., 1999) and even Chicks vaccinated at 7 days of age do not respond as well as those vaccinated at 14 days of age.

Infectious agents: Clinical disease or even a sub-clinical situation is relatively easy to detect so infectious factors are getting much more attention than other factors and often blamed by farmers and veterinarians as the main cause of immunosuppression.

In chickens, virus-induced immunosuppression is usually linked to three important pathogens: Marek's Disease Virus (MDV) Infectious Bursal Disease virus (IBDV) and Chicken Anemia Virus (CAV). These viruses can cause apoptosis (cell death) in B lymphocytes (IBDV, MDV) and/or T lymphocytes (MDV, CAV) and in addition these viruses can also affect the cytokine activation pathways and thus impair immune responses.

Viruses are intracellular pathogens, which uses the host biosynthetic systems for production of viral proteins and the replication of the viral genome

During the millions of years of Viral-Host coexistence, hosts have developed immune responses and viruses have learned how to evade it and the evolution balance of mutual survival was created. (The balance between Virus replication and viral clearance by its host)

The goals of viral evasion: 1. it allows persistence in hosts.

Or: 2. to allow numeric increase to an extent that permits propagation of the species.   

Viruses evade the immune systems in many ways that can not be covered during this short paper.

In general, viruses with large coding capacity like DNA viruses (pox, herpes) can express a wide variety of proteins with specific effects on immune recognition and other functions. The small coding capacity viruses like paramyxoviruses (Influenza) constantly change their envelop glycoprotein (HN…) and presenting a "new virus" to the immune system.

Suppressing the immune system is one of the major ways to evade the immune response and one example is the inhibition of complement factors:

The complement cascade activation is a crucial component of both the Innate and adaptive immune responses. However, this is a very dangerous system that must be regulated in order to avoid self destruction and the body is using different proteins to up or down-regulate these responses.

Throughout evolution, viruses have captured cellular genes encoding cellular complement regulators or they have managed to captured regulatory proteins and incorporate them in the viral envelop during budding. These proteins down regulate the complement activation and allow the viruses to escape this aspect of the immune response. ( Favoreel et al,2003).

Unlike the large genome of the Herpes Viruses (e.g. Marek's) the genome of the IBDV is small and simple and therefore it can not contain any complex codes for regulatory proteins. However, the IBDV evades the immune system by creating changes in the VP2 and thus preventing neutralization by the anti VP2 Ab, and by alterations in the apoptosis mechanism: there are evidences that VP5 of the IBDV is both inducer and inhibitor of apoptosis.( (Liu et al, 2006; Yao et al, 2001) and that VP2 has a role in this mechanism as well (Rodriguez-Lecompte et al., 2005)

IBDV is causing immunosuppression by directly destroying IgM+ B cells in the Bursa of Fabricius and by indirect effecting on the CMI (Apoptosis of Macrophages, T cells, IFN-γ and other cytokines etc).

Unfortunately, many of these mechanisms and interactions are incompletely understood and there is some puzzling data like the ability of the chickens to produce an effective high immune response to vvIBDV while other Ab responses are heavily suppressed.

IBDV Pathogenesis and immunosuppressive aspects

General pathogenesis: After oral infection – IBDV replicates in the GALT (Gut Associated Lymphoid Tissue) in Macrophages and Monocytes that rapidly travels via the blood to the Bursa. Within 13h post experimental exposure most bursa follicles are IBDV positive. The virus replication leads to extensive B depletion in the follicles medullar and cortical regions.

At 16h post exposure there is a second Viraemia that leads to disease symptoms and mortality that usually starts 3d PI and decline 6-7 d PI.

The actual cause of direct clinical signs and mortality is unknown.  Bursal lesion, no matter how severe they are, can not explain the acute phase of morbidity and mortality. In addition to that, Mortality rates are variable and the narrow age range of clinical disease has not yet been explained. Septic shock (Cytokines storm) is the suggested explain of clinical signs and mortality but so far it was proved locally and not at a systemic level.

Bursa recovery from filed infection of vvIBDV starts at 5w PI and ends 12w PI

Thymus – Marked atrophy and thymocytes apoptosis that quickly recover (7d)

Spleen, Cecal tonsils – Virus was detected with cellular destruction but without significance.

The acute phase is consists of 3 lines that interact with each other.

1. The macrophages produce the following responses: Interferon (IFN)-α/β production is up-regulated, which has beneficial antiviral effects and stimulate T cells to produce IFN-γ. (Rauw et al., 2007). IFN- γ is clearing viruses and also contribute to the destruction of infected cells.
2. Activated T-Lymphocytes quickly populate the bursa and within 7 days more than 90% of the cells are T Lymphocytes (CD4, CD8) and only 7% are IgM+ B Cells. The T cells were found to have a role in the recovery of the Bursa.
3. The Bursal IgM+ Lymphocytes (Immature!!) are infected and there is a mass necrosis and apoptosis of these cells (also with a by-stander effect) resulting in reduced Ab reaction to immune stimulus.

This up-regulation of T and Mac is followed by down regulation of T-cell that leads to possible cellular immunosuppression.

The severity and duration of immunosuppression is depended on the strain and dose of the virus.

Assessing the level of IBDV immunosuppression:

Bursa size and other physical changes are some times used as field tools to estimate the level of immunosuppression and even to distinguish between vaccine strains.

But, It is quit clear that bursa size alone can not be used due to the fact that it is also related t the size of the chicken and therefore the relation to the body size was added, Bursa To Body Wight Ratio (B2BWR). However, these physical parameters, when tested in commercial broilers, often fail to do the job and there is no parameter that can accurately describe the level of immunosuppression.

Many factors effect bursa size and functionality and the major one is stress.  Simple strong stress like thermal stress (hot cold) or over crowding will results in IBDV like bursal symptoms that is small bursa, low B2BWR because strong stress reduced IgM+ maturation and cause lymphoid depletion by apoptosis! (Muniz et al, 2006 )

To date, the best way to assess the level of immunosuppression is to do vaccination and challenge trials.

An example to that will be the following field trial:

The objective of the study was to do a field comparison study between Gumboro MB train (Intermediate) and ST-12 train (Mild) in the following parameters:

And to estimate the significance of the physical parameters compared to Ab titers.

1,300,000 broilers were raised in two field commercial sites (A & B) each with 10 chicken houses split into 5 pairs of test and control were used. Two cycles were made in each site. Site A: 24,000 chickens/house (open-sided) 240,000/cycle

Site B: 41,000chickens/house (controlled environment) 410,000/cycle

In each site, half of the houses were vaccinated with MB and half with ST-12.

All conditions and vaccination programs were made according to the farm regulations.

ND (HI Method) and IBD (IDEXX Elisa) titers serum samples were tested on 20 chicks from each house 7, 14, 21, 28, 35 days of age, B2BWR - Bursa to Body Weight Ratio was calculated on 5 chickens from each house at 14,21,28,35 days of age (B2BWR=Bursa weight/Body weight x 1000),  Bursal Scoring - was done on 5 chickens from each house at 14,21,28,35 days (0-4 system, modified from that defined by Muskett et al – The Veterinary Record 1979; 104:332-334.

Broilers Performance data – Body Weight, Mortality, Kg/m2, FCR, Age at slaughter, Index

A total of 3200 blood samples and 400 bursa samples were collected and all results were expressed as means SD CV,  statistical analysis was made with JMP software (SAS Institute, 2000) using F-test and t-Test P=0.05

The study showed that there were no differences between the B2BWR in the first cycle and mild differences in the second cycle at day 21. The Bursal Score were different between sites at the 1st cycle and only at 21d between vaccines at the 2^nd cycle. The MB groups got higher scores 7d post vaccination and ST-12 groups got the same levels 7 days posts the 3rd vaccination. As expected, as the B2BWR (representing Bursa size) got lower,  the Bursal scores ( Lymphocytic depletion) were getting Higher.

When the physical parameters were plotted against the NCD titers it was found that at 21 days of age, when the scoring of the MB group was higher then the ST-12,  there were no differences between the NCD titer of the MB group and the ST-12 group.

And there was no correlation between bursal scores and the NCD titers in both groups

In this field trial, Good correlation was found between the B2BWR and lymphocyte depletion but no correlation was found between the bursa size of a flock and the bursa functionality

We have shown that simple B2BWR sampling could not be used as a field tool to estimate the level of the immunosuppressant effect of a given Gumboro vaccine

Possible explanation: The lymphocyte depletion in the Bursa is only with IgM+ B-cell the mature circulating B cells keep producing Ab.

Direct test, like comparing the immune response to other vaccines ( like NCD) might give a more accurate comparison between Gumboro vaccines

The use of ST-12 vaccine strain had no advantage over the use of MB in any of the parameters tested in this field trial. (Titers, Bursa physical parameters Thymus and performance) and vaccination with ST-12 + MB  proved to be as good as 3 vaccinations with ST-12.

In these vaccination programs, results justify the use of a single Intermediate MB strain over a a double vaccination with mild vaccine

The findings from this trial match other field trials showing the benefit of vaccinating with MB :

1. Single dose – Protection also to variant strains! 
2. Low immunosuppression
3. Economic

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