Genotype effects

8.1.1 Variations in the development of the immune system

The immune system of chickens starts developing during embryogenesis (FELLAH et al. 2014). Several studies demonstrated that structural changes within the lymphoid organs and tissues in the first weeks post hatch are necessary for complete functionality JEURISSEN et al. 1994; MAST and GODDEERIS 1999). Immune organ weights, the distribution and numbers of immune cell populations as well as the development of important functional compartments can be used as indicators of maturation (JEURISSEN et al. 1994; MAST and GODDEERIS 1998, 1999; OLÁH et al. 2014). In both conducted studies, we measured the body weight development, the relative BF and spleen weights as well as intrabursal CD4+, CD8+ T-cell numbers at different time points ph. In the first study we also determined splenic CD4+, CD8+ T-cell, B-cell and macrophage numbers, which define the B-cell (PELS) and T-cell compartments (PALS) and finally the numbers of GC in the spleen and CT.

The body weight as well as the relative bursal and splenic weight development differed significantly between the genotypes in both conducted studies (P < 0.05). In all experiments, BT chickens showed significantly higher body weights compared to LT and DT chickens at all investigated time points (P < 0.05). In contrast, the relative bursal and spleen weights were significantly lower in BT chickens at all investigated time points of both studies compared to LT chickens (P < 0.05). DT chickens took an intermediate position in body and relative immune organ weight development.

Interestingly, female DT chickens showed a comparable relative spleen weight

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development to female BT chickens, while the relative spleen weight development of male DT chickens was more similar to male LT chickens. The introduction of a dwarf gene on the female side of DT chickens has an effect on the body size and weight of the hens and might therefore be related to the similarities in relative lymphoid organ weights to female BT chickens.

It was suggested that the selection towards a fast growth and high body weight in BT chickens led to a decrease in relative growth of the immune organs and might therefore indicate a loss of immunological function (PARMENTIER et al. 1995; CHEEMA et al.

2003). The data of our studies showed that BT chickens mounted the most vigorous immune responses compared to LT and DT chickens during the first five weeks ph, which will be discussed in the next chapter. Therefore, we suggest that the relative growth of immune organs might not be the only indication for immune responsiveness.

As mentioned above, the numbers of immune cell populations and the structural lymphoid organ organization should be taken into account.

The spleen represents the most important secondary lymphoid organ, which harbors important structures, which are necessary for the development of humoral immune responses (OLÁH et al. 2014). The number and size of GC, the structural organization of the PELS and PALS indicate the developmental status of the spleen (JEURISSEN et al. 1994). A varying manifestation of these structural units was suggested for different chicken lines selected for a high or low antibody response to SRBC (PARMENTIER et al. 1995; KREUKNIET et al. 1996). We demonstrated that BT chickens developed significantly higher numbers of GC in the spleen and CT with increasing age compared to age-matched LT and DT chickens. The structural development of the PELS and PALS was comparable between all genotypes. No differences were observed in the size of the structures and in the numbers of immune cells (CD4+, CD8+, B-cells and macrophages) between the genotypes. Furthermore, the age had a significant impact on the intrabursal CD4+ T-cell and splenic macrophages numbers in all genotypes. Intrabursal CD4+ T-cell numbers decreased from 14 to 28 dph and stayed a consistent level until 63 dph, which was comparable between all genotypes in both conducted studies. The numbers of splenic

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macrophages also decreased with the age from 14 to 28 dph in all genotypes. We may speculate that the development of the investigated immune cell populations and structures was already finished as early as 14 dph. Therefore, we strongly suggest including earlier time points during the first week after hatch in the future.

8.1.2 Variations in innate and adaptive immune responses

In the present study, isolated spleen leukocytes were used to investigate the in vitro immune responsiveness of macrophages and T-cells after stimulation with either LPS or ConA. Differences in the CMI between LT and BT birds were assessed, using various LT (White Leghorn, Brown Nick) and BT chicken lines (Avian x Avian, Hubbard x Hubbard, Ross) at different ages by in vitro ConA and PHA stimulation of spleen cell cultures or intradermal PHA injection (CORRIER 1990; LESHCHINSKY and KLASING 2001; KOENEN et al. 2002). These studies achieved opposing results. In our studies, we could confirm that the magnitude of the CMI was significantly influenced by the genotype (P < 0.05). BT chickens showed the highest CMI after in vitro ConA stimulation of spleen cell cultures followed by the spleen cells of DT and finally LT chickens at all investigated time points. Significant differences were only observed at 35 dph. Several studies showed that immune responses in general also vary within different BT and LT chicken lines and might even depend on the B-haplotype of the chickens (GEHAD et al. 1999; KRAMER et al. 2003; PARMENTIER et al. 2006;

KJAERUP et al. 2017). Therefore, variations between experiments should be considered with respect to the used BT or LT chicken lines and the age of the birds.

Macrophages as cells of the innate immune system are important for the detection and phagocytosis of invading pathogens but also fulfill regulatory functions by initiating and directing the immune responses in the beginning of an infection (KLASING 1998;

QURESHI 2003). The magnitude of macrophage responses was shown to depend on the genetic background of the chickens. Studies using chickens with different B-haplotypes showed that the magnitude of iNOS activity and NO production was associated with the respective B-haplotype of the chicken lines (HUSSAIN and QURESHI 1997, 1998; DIL and QURESHI 2002). It was demonstrated that macrophages, which derived from a BT chicken line (MQ-NSCU) and a LT chicken line

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(B¹⁵B¹⁵), were classified as hyper-responders to LPS compared to macrophages from two other layer-type strains (GB1 B¹³B¹³ and GB2 B⁶B⁶). In our studies, ex vivo NO production of spleen cell cultures differed significantly between BT, LT and DT chicken lines (P < 0.05). The chicken genotypes used in our studies might possess different B-haplotypes. BT chicken spleen cell cultures released the highest amounts of NO compared to the other genotypes, whereas LT and DT chickens showed a comparable NO release after LPS spleen cell stimulation. Again DT chickens took an intermediate position in the macrophage activity, measured by NO release after LPS stimulation.

The genotype-associated differences in the development of the immune system and innate and adapted immune responses provided the basis for the interpretation of differences in IBDV-specific vaccine immune responses between the chicken lines.