Background
Campylobacter jejuni is the leading cause of bacterial food-borne gastroenteritis in humans in industrial countries. Poultry is considered as the main reservoir for
C. jejuni, with high bacterial loads in the gastrointestinal tract.
C. jejuni has been regarded as a commensal for chickens [
1]. However, recent studies have reported that
C. jejuni may induce a mild inflammatory response and affects the gut morphology in colonized chickens [
2,
3]. It is therefore suggested that
C. jejuni may have a substantial impact on the chicken’s health and welfare [
2].
Different risk factors may affect the colonization pattern of
C. jejuni in chickens including strain to strain variation, the inoculation dose, host genotype, management as well as water and feed composition [
3,
4]. Poor gut health and compromised immunity are considered to negatively influence the chicken’s health [
2].
Different pathogens may modify the functionality of the immune system [
4]. Infectious bursal disease virus (IBDV) is one of the most important immunosuppressive viruses affecting the chickens worldwide [
5]. Both humoral and cell-mediated immune responses (CMI) are affected. IBDV-infected birds show systemic as well as local depletion of B cells, infiltration of T cell subsets in the bursa of Fabricius (BF) and modulation of innate immune parameters [
5‐
7]. IBDV infection leads to a strong upregulation of proinflammatory mediators and cytokines, a so called ‘cytokine storm’, in the acute phase and even cause death during this period [
6,
7]. Surviving chickens may suffer from permanent immunosuppression when they were infected early in life [
5,
8]. Immunosuppressed chickens are more susceptible to secondary infections, which was experimentally demonstrated after IBDV-co-inoculation with
Salmonella typhimurium (ST) and
Escherichia coli [
9,
10]. Inoculation of specific-pathogen-free (SPF) birds with an IBDV Del-E strain led to an increase in ST shedding, and anti-ST immune reactions were dramatically impaired in co-infected birds [
10]. Increased
C. jejuni colonization and shedding was demonstrated in chickens co-inoculated with a Del-E strain of IBDV and
C. jejuni [
11]. Another study showed that vaccination of chickens with an intermediate IBDV strain led to lesion development in the gut and liver when birds were also inoculated with
C. jejuni in comparison to
C. jejuni mono-inoculated birds [
12]. However, the mechanisms leading to an exacerbation of
C. jejuni colonization are not fully clear. We speculate that IBDV may modify the local
C. jejuni colonization pattern. It may compromise the induction of
C. jejuni-specific humoral immunity and may have possibly other direct or indirect effects on gut immunity [
13] and microbiota composition. It was suggested that maternal antibodies may protect against
C. jejuni colonization [
14]. However, the role of humoral immunity in
Campylobacter control has been discussed controversially [
15]. Recent studies with chemically B cell-compromised chickens indicated that humoral immunity may be important in the clearance of
C. jejuni from the small intestine [
16].
In the present study, commercial broilers were inoculated with a very virulent (vv) IBDV strain at 2 weeks post hatch, when maternally derived anti-IBDV antibodies were below the break-through level of the virus. IBDV-induced suppression of circulation B cells was confirmed starting at 3 days post virus (IBDV) inoculation (pvi) in both experiments lasting up to 9 days pvi. Subgroups of vvIBDV-inoculated and virus-free birds were subsequently orally inoculated with C. jejuni at two different time points expecting birds to be at different stages of IBDV-pathogenesis and induced immunosuppression: In experiment (Exp.) A, birds were C. jejuni-inoculated at seven and in Exp. B at 9 days pvi. Lesion development, replication of pathogens as well as gut associated immune parameters and microbiota composition were determined.
Discussion
In this study we investigated the effect of vvIBDV-induced immunosuppression on
C. jejuni colonization. Two experiments were conducted using broiler chicks from the same genotype with comparable IBDV-maternally derived antibody levels.Broiler chickens were inoculated with vvIBDV at 14 days post hatch. Virus replication, as indicated by high numbers of IBDV-antigen positive cells in the BF, was confirmed [
6]. Monitoring of circulating B cells showed a significant decrease in relative B cell numbers from 5 days pvi onwards (
P < 0.05) compared to the virus-free controls. We also observed bursal atrophy with B cell depletion. vvIBDV-antigen-loads, sero-conversion as well as bursa lesion development were comparable between Exp. A and B. These results are consistent with previous studies confirming vvIBDV infection and immunosuppression [
5,
7,
8]. Although previous studies indicated an effect of IBDV on
C. jejuni-colonization (Subler et al. [
11]; Stojanov et al. [
12]), the influence of the time interval between the inoculations of the pathogens on the outcome of infections was not investigated. Co-inoculation experiments with various other pathogens in vitro and in vivo demonstrated clearly a significant influence of the time interval on the pathogenesis of either infectious agent [
27,
28]. Two different time points after vvIBDV-inoculation were selected for
C. jejuni-inoculation. Seven and 9 days pvi were chosen, which was after the acute phase of vvIBDV infection, when the effects on B cells were expected to be most prominent and numbers of B cells were clearly depressed but the vvIBDV-induced cytokine storm may have waned [
5,
20]. It would have been desirable to investigate both time points of
C. jejuni-inoculation within one experiment, which was not possible in this experiment due to logistic constrains. But the comparable outcome of vvIBDV-infection and the comparable
C. jejuni colonization rate between three and 14 days pbi in mono-inoculated groups in both experiments allows the direct comparison of both experiments.
Interestingly, co-inoculation with C. jejuni may delay follicular recovery, although the mechanisms behind this observation that are not clear.
Two days difference in
C. jejuni-inoculation after vvIBDV-infection had a significant effect on the outcome of
C. jejuni colonization. In Exp. A the early phase of
C. jejuni colonization was clearly impacted by vvIBDV, leading to 2.8- to 8.1-fold higher CFU in co-inoculated birds compared to
C. jejuni mono-inoculated ones. In Exp. B vvIBDV affected mainly the colonization pattern at 21 days pbi, which might correlate with the clearance of
C. jejuni. Co-inoculated birds had significant higher CFU with all six birds having a CFU range between log
10 of 3.2 and 8 in the caecum compared to the mono-inoculated group, in which two birds were
C. jejuni negative by culture of caecal content and four had CFU of 4.3 ± 1.7 at 21 days pbi (
P < 0.05). Possibly this effect would also have been seen in Exp. A, if the experiment would have been extended. Interestingly this significantly higher bacterial load in co-inoculated birds at 21 days pbi coincided with a reduced
C. jejuni-specific antibody response compared to the
C. jejuni mono-inoculated groups (
P < 0.05). Antibody levels were significantly lower at 21 days pbi in co-inoculated birds compared to
C. jejuni mono-inoculated ones in Exp. B. A trend of a compromised anti-
C. jejuni-antibody response was already visible at 14 days pbi in both experiments. These results provide circumstantial evidence that
C. jejuni-infection may be controlled by humoral immunity as suggested before [
14]. In Cyclophosphamide-immunocompromised birds the same trend was observed that antibodies may play a significant role in the bacterial clearance phase later after inoculation [
16]. Further studies need to be done, to evaluate the local
C. jejuni-specific IgA-response to confirm the role of local humoral immunity in
C. jejuni-colonization.
An additional confirmation of vvIBDV-induced B cell depletion, and the lack or reduced stimulation of the B cell-mediated anti-C. jejuni-response is provided by the reduced number of LP B lymphocytes in co-inoculated birds compared to C. jejuni mono-inoculated ones (P < 0.05). While C. jejuni mono-inoculation led to an upregulation of LP B lymphocytes starting at three days pbi in Exp. A, and on days three and seven pbi in Exp. B, there was only a detectable B cell upregulation in the co-inoculated groups at seven days pbi in both experiments. At 14 days pbi local B cell numbers were reduced in co-inoculated groups compared to most other groups.
This effect of vvIBDV on the
C. jejuni-specific antibody response is interesting, as this effect was most prominent at 21–23 days pvi, when virus clearance in the BF was very much advanced and beginning bursal recovery was observed in both vvIBDV-inoculated groups. This clearly shows that B cell immunosuppression lasts beyond the acute phase and the effects on secondary infections may be detectable over 14 days post vvIBDV-infection. Previous studies demonstrated that bursal recovery with two different types of follicle structures starts 5 weeks post IBDV infection [
8,
29,
30]. The larger follicles are correlated with partial recovery of the antibody responsiveness, while the smaller follicles appear unable to produce antigen-responsive B cells [
29,
30], which suggested that immunosuppression was maintained. In Exp. A other immunosuppressive or immunomodulatory effects beside the depression of the humoral immunity may have additionally affected
C. jejuni colonization as already early bacterial replication was exacerbated in co-inoculated groups at 3 and 7 days pbi. It was shown in a variety of other studies, that during the early phase of IBDV infection not only the humoral immunity may be compromised in IBDV-infected birds but also macrophage activity, and indirectly the cell-mediated immunity [
5,
7].
There was no information available regarding the effect of vvIBDV infection on the gut microbiota in chickens. It may be speculated that due to its immunosuppressive effects on the gut-associated immune system [
13] IBDV may indirectly modulate the composition of the microflora. Effects like that were previously seen in chickens infected with Marek’s disease virus (MDV) [
31], and also in humans after infection with human immunodeficiency virus (HIV) [
32]. Interestingly, vvIBDV modified the gut-microbiota composition in both experiments compared to control birds as indicated by the PCoA analysis (Fig.
6). While in Exp. A, there was a clear separation between all groups in the composition of the microbiota composition, in Exp. B, non-inoculated controls and
C. jejuni-mono-inoculated chickens grouped together as well as the vvIBDV-infected and co-inoculated ones. This suggests that at least in Exp. B vvIBDV-effects on the microbiota dominate also in co-inoculated groups, which is interesting and the meaning for the host has to be investigated further. We observed that
C. jejuni-infected birds had a higher abundance of
Clostridiaceae in the caecal content in both experiments, which confirms previous studies [
3,
33].
C.
jejuni colonization results in hydrogen consumption, which promotes the growth of some
Clostridium sp. through increased fermentation, leading to an increased organic acid production, which subsequently allows
C.
jejuni to use organic acids as an energy source [
34].
Faecalibacterium, a butyrate producer, dominates in the caecal microbiota of chickens at approximately 3 weeks of age [
35]. High numbers of
Faecalibacterium may be detrimental for
C.
jejuni since butyrate may inhibit replication of
C.
jejuni [
36]. Different colonization rates between
C.
jejuni mono-inoculated and co-inoculated birds might have been due to the different abundance of
Faecalibacterium in the gut microbiota. In Exp. B, a higher abundance of
Faecalibacterium (13.95%) was correlated with a lower abundance of
Campylobacter (8.83%) in the
C. jejuni mono-inoculated birds compared to co-inoculated birds, which showed a lower abundance of
Faecalibacterium (11.07%) with a higher abundance of
C. jejuni (10.72%). Further studies on the interaction of the gut microbiota and
C. jejuni need to be conducted to elucidate the exact mechanisms being involved in the control of
C. jejuni in the gut. This may subsequently help to develop better control strategies and ultimately reduce the colonization rate and therefore the risk for infections of humans after chicken meat consumption [
37].
The role of T cells in the control of
C. jejuni was demonstrated in mice and humans, but little is known about T cell responses in chickens. In our study a significant up-regulation of T lymphocytes was observed locally in the caecum of
C. jejuni mono-inoculated and co-inoculated birds compared to control birds. This observation coincides with previous studies suggesting that the presence of these CD4
+ and CD8
+ LP T lymphocytes is due to an intraluminal antigenic stimulus [
3]. It was suggested that activated T cells may play a protective role in the host defense against
C. jejuni infection [
3,
38]. Especially regulatory T cells (Tregs) may be import in the regulation of inflammatory versus anti-inflammatory conditions in the gut [
39,
40]. vvIBDV did not affect this
C. jejuni-mediated T cell response in the gut. Co-infected groups showed a comparable number of LP T lymphocytes as
C. jejuni-mono-inoculated ones, which did not correlate with the increased number of CFU in these groups suggesting a neglectable role of these cells in the control of
C. jejuni under our experimental conditions.
Cytokine expression pattern were either influenced by C. jejuni or vvIBDV. Co-inoculations only had an enhancing effect on IL-8 mRNA expression at 3 days pbi compared to the other groups, but the consequences for the pathogenesis are not known. The expression pattern of IL-6 is dominated by vvIBDV during the acute phase of C. jejuni-colonization, and later on by C. jejuni suggesting that this cytokine may not play a key role in the early control of C. jejuni.