Antibody response of growing German Holstein bulls to a vaccination against bovine viral diarrhea virus (BVDV) is influenced by Fusarium toxin exposure in a non-linear fashion

Deoxynivalenol (DON) is a Fusarium toxin and a highly prevalent feed contaminant. It often co-occurs with zearalenone (ZEN) and an array of further Fusarium toxins but constitutes the major part of the toxicological potential of such multi-contaminated feedstuffs (Binder et al. 2007; Nesic et al. 2014). Cattle is regarded as quite resistant to DON due to its capability to degrade DON to the less toxic metabolite de-epoxy-DON (de-DON) pre-systemically as mediated by ruminal micro-organisms (Dänicke and Brezina 2013). Irrespective of this general detoxification capability, a guidance value for critical concentration of 5 mg DON per kilogram complete feeding stuff at a reference dry matter (DM) content of 88% for ruminants was established by the European Commission (European Commission 2006) based on literature evaluation performed by European Food Safety Authority (European Food Safety Authority 2004). Due to the limited data base, nearly exclusively performance parameters such as DM-intake and live weight gain were used as toxicological endpoints. On the other hand, on a molecular level, DON is known to inhibit protein synthesis and to trigger a so called ribotoxic stress response (Iordanov et al. 1997; Pestka et al. 2004; Pestka 2007) which is characterized by an activation of mitogen-activated protein kinases (MAPKs). These effects were particularly demonstrated for immune cells giving rise to conclude immuno-modulating effects of DON.

Bovine viral diarrhea virus (BVDV) is an enveloped single-stranded, positive sense RNA pestivirus of the family Flaviviridae that occurs in two genotypes (BVDV-1 and BVDV-2) and two biotypes (non-cytopathogenic [ncp] and cytopathogenic [cp]) (Brackenbury et al. 2003). BVDV is capable of infecting granulocytes, macrophages, antigen-presenting myeloid cells, CD4+ and CD8+, and T cells and B cells. Thus, it affects both innate and acquired immune responses (Chase et al. 2004). Infection with BVDV is highly prevalent worldwide in cattle, is characterized by a number of clinical manifestations, and is therefore subject to various prevention strategies, including vaccination (Lanyon et al. 2014).

Considering the possible interference of DON and its known and putative derivatives with immune cells, we hypothesized that exposure of bulls to DON modulates immune-related traits, including the antibody response to BVDV vaccination.

To verify this hypothesis, increasing dietary DON concentrations was tested either in unvaccinated or BVDV-vaccinated bulls. The results of the present paper complete the findings on general growth performance and toxin residues in physiological specimens as reported recently (Winkler et al. 2016).

BVDV vaccines usually contain either modified-live virus (MLV) or inactivated virus. While MLV stimulates both cell-mediated and humoral immunity-inactivated vaccine results in a weaker T cell response but a strong humoral immunity as mediated by adjuvants (Stevens et al. 2009). Vaccination with MLV is known to induce transient immunosuppression due to replication of the MLF in immune cells (Ridpath 2013). As we used an inactivated vaccine, such effects were not expected. However, vaccination interactively influenced several leucocyte parameters. The time-constancy of monocyte counts in BVDV-vaccinated bulls compared to the final decline in their mock-treated counterparts might be interpreted as a general stimulation of the immune system probably mediated by the adjuvant and further triggered by the interactions between vaccine antigen and antigen-presenting cells. Similarly, the higher lymphocyte proportions in vaccinated bulls, particularly after initial and booster vaccination, might be a reflection of a generally stimulated proliferative response. In interpreting the effects of vaccination on the immune system, it needs to be considered that the readouts represented both an antigen-specific response, i.e., the BVDV-antibody titers, but also unspecific immune reactions. These rather antigen-unspecific effects might result from the adjuvant components of the BVDV vaccine. According to the declaration of the manufacturer, the vaccine preparation used in the present experiment contained aluminum (III)-ion (Al³⁺) as aluminum phosphate and aluminum hydroxide as adjuvant and methyl-para-hydroxy-benzoate as preservation agent. One vaccination dose of the used vaccine contained 6–9 mg Al³⁺ and corresponded to a single dose of approximately 12–19 μg of Al³⁺ per kilogram b.w. at day 0 and 21 of experiment.

While the proportion of CD4+ T cells increased in the vaccinated control group over time, the percentage of this phenotype remained constant in the group exposed to the highest levels of Fusarium toxins (FUS III). Opposite relationships were observed for cytotoxic T cells (CD8+) with a decrease in FUS III group and a time-constancy in the control group resulting both in a comparable CD4+ to CD8+ ratio and total counts of both phenotypes in peripheral blood. As aluminum adjuvants trigger humoral immunity by activating Th2 type cells (Gupta 1998) the proportional increase in CD4+ T cells of the control group might be due to an increase in Th2 type cells supporting antibody response by B cells. In addition, recent research on hepatitis B vaccines suggests that aluminum-based adjuvants improve both Th1 and Th2 cellular responses to antigens (He et al. 2015). Therefore, the discussed increase in CD4+ cells as observed in the present experiment could be due to increases both of Th1- and Th2-type cells.

Obviously, such an effect was prevented by high exposure to Fusarium toxins resulting in a lower BVDV-antibody response in group FUS III compared to the control group. However, the dose-response relationships between Fusarium toxin or DON exposure and BVDV-antibody formation appeared to be non-linear since the medium exposure level (FUS II) induced the most strongest BVDV-antibody response which additionally was paralleled by mean CD4+ T cell proportions lower than in the control group but higher than in group FUS III. Although the relationship between DON exposure and the proportion of CD4+ T cells appeared to be linear (Fig. 3), the BVDV-antibody response was non-linear (Figs. 1 and 4) suggesting additional mechanisms other than the involvement of CD4+ T cells. In interpreting the relationships between DON-exposure and BVDV-antibody formation, it has to be considered that we used a competitive ELISA which detects all antibody isotypes. Therefore, the net outcome of total antibody response might be influenced by DON exposure mediated linear and non-linear responses of specific isotypes.

It is interesting to note that a decrease in CD4+ T cells was also reported for early lactating dairy cows exposed to increasing dietary DON concentrations up to 5.2 mg/kg DM (Dänicke et al. 2017). These and present results are anyway surprising since ingested DON is known to be largely metabolized to de-DON in the rumen resulting in serum de-DON levels substantially higher than that of non-metabolized DON (Dänicke and Brezina 2013). Also, for the present experiment, nearly exclusively de-DON was detected in blood of bulls in a linear dose-response-related fashion. The ratio between DON and de-DON amounted to approximately 1 to 40 corresponding to a de-DON proportion of 95% of the sum of DON plus de-DON in plasma (Winkler et al. 2016). With regard to comparative in vitro toxicity using bovine PBMC, an IC₅₀ (i.e., the concentration where the proliferation was inhibited by 50%) of 0.5 μM was titrated for DON while the maximum tested de-DON concentration of 18.29 μM inhibited proliferation just by 24% underpinning the low toxicity of this rumen-originating DON metabolite on this particular immune cell population. For cows exposed to a Fusarium-toxin contaminated diet for several weeks maximum serum de-DON levels of 52 ng/mL (0.19 μM) were detected while DON concentrations reached maximum levels of 9 ng/mL (0.03 μM) (Dänicke et al. 2011). PBMC isolated from these cows were characterized by a slightly lower viability ex vivo compared to PBMC of non-exposed control cows although blood toxin levels were markedly lower than the effective doses found in the in vitro trial. Besides the presence of Fusarium toxins other than DON and ZEN in the contaminated diet, further associated observations were discussed as possible reasons for the unexpected effects. Intake of DM and consequently of energy, nutrients, and mycotoxins was increased in cows fed the contaminated diet (Keese et al. 2008a) and suspected to have influenced the vulnerability of PBMC (Dänicke et al. 2011). Bulls of groups FUS I, II, and III consumed approximately 2–4%, 3–6%, and 8–10%, respectively, more DM, metabolizable energy and crude protein compared to animals of the CON group (Winkler et al. 2016) supporting the view that an increased metabolic supply with nutrients, energy but also with mycotoxins interferes with immunological responses. The linear relationships between blood cholesterol and GGT activity, total protein, and albumin, but also between DON-exposure and hematocrit (Fig. 3) as well as the higher blood urea concentration in all FUS groups further support the view that a higher DM intake influences metabolic processes and presumably has consequences for the immune system. This aspect becomes particularly obvious in group FUS III where the increase in energy and nutrient intake was most pronounced. Metabolic parameters further support this view as this group exhibited a high-blood level of BHB compared to the CON group which might indicate a higher rumen turnover due to higher feed intake which results in higher levels of ruminal butyrate subsequently metabolized to BHB. Moreover, NEFA levels were lower in group FUS III suggesting a lower level of lipolysis compared to CON group. However, these changes were particularly observed in BVDV-vaccinated groups whereas the time-dependent alterations in mock-treated groups were rather subtle. In addition, for group FUS III, the highest activities of GGT, GLDH, and ASAT in blood were detected leading to conclude a higher degree of liver lesions caused by the highest exposure to DON and/or by the stimulated DM intake compared to the other groups. However, it needs to be stressed that enzyme activities of all treatment groups were generally lower than the reference limits of 50, 30, and 80 U/L for GGT, GLDH, and ASAT (Moritz 2014), respectively. Thus, the treatment effects were most probably without pathological relevance. Another experiment with growing German Holstein bulls fed rations with a DON concentration of approximately 2.2 mg/kg DM did not reveal adverse effects on ASAT and GLDH activity either (Dänicke et al. 2002).

Unspecific effects of BVDV-vaccination become obvious when the red blood count indices are considered. These effects might be related to immune-related impacts in some publications discussed as immunotoxic effects (Batista-Duharte et al. 2014; Zhu et al. 2014). These effects are based, amongst others, on stimulation of the pro-inflammatory NLRP3 pathway, including the induction of pro-inflammatory cytokines such as TNF-α spreading from the site of i.m. injection to the systemic circulation and consequently also to the liver where an acute phase reaction might be initiated. Under these conditions, many metabolic effects might occur (Batista-Duharte et al. 2014; Zhu et al. 2014; He et al. 2015).

Based on the laboratory findings of the red blood count, a microcytic normochromic condition can be diagnosed for BVDV-vaccinated bulls relative to their mock-treated counterparts since erythrocyte counts, hematocrit, and MCV were all significantly decreased, particularly at day 70 of experiment, while hemoglobin concentration and MCH remained statistically uninfluenced. These changes are quite similar to toxic aluminum effects on red blood count in laboratory animals characterized by a decrease in erythrocyte count, hematocrit, and hemoglobin concentration and an increase in MCH (Zaman et al. 1993). In interpreting the significant treatment effects, it has to be considered that red blood count traits were within the corresponding reference ranges which are given at 5–10 T/L for erythrocytes, 28–38% for hematocrit, 9–14 g/dL for hemoglobin, 11–17 pg for MCH, and 46–65 fL for MCV (Moritz 2014). Interestingly, a time delay of approximately 7 weeks between the last (booster) vaccination and the effects on hematocrit and erythrocyte counts was required in the present experiment with bulls while the MCV appeared to be influenced earlier (Fig. 2d). A regenerative component of the erythrocyte drop was not detected in the present experiment when red cell distribution width (RDW) is evaluated as an indicator for anisocytosis. Rather, the average RDW varied between 14.1 and 14.5% and is lower than the reference range of 16–20% (Roland et al. 2014) suggesting a higher degree of erythrocyte size-uniformity and a lower presence of precursor cells.

Besides the effects of BVDV vaccination on red blood count, there appeared to be an interaction between DON exposure and vaccination, particularly for hematocrit at day 70 of experiment. While BVDV-vaccinated bulls of the CON and FUS I group clearly showed a lower hematocrit compared to their mock-treated counterparts, this distinct difference disappeared or was less clear at higher DON exposures (groups FUS II and III) generally characterized by dose-related increases in hematocrit (Fig. 3a). The phenomenon of a DON-dose related increase in hematocrit was also observed in cows (Dänicke et al. 2017) and pigs (Prelusky et al. 1994) and was discussed as a myelotoxic or a haemo-concentrating effect induced by treatment-associated variations in water intake. However, both water intake and RDW remained uninfluenced by increasing DON exposure in the present study making myelotoxic or haemo-concentrating effects less probable. Viability of human erythrocytes was not influenced at high concentrations of 10 μM DON (Parent-Massin and Parchment 1998) while erythrocyte progenitor cells were shown to respond quite sensitive with a cytotoxic DON level of 0.25 μM and a no effect level (NOEL) of 0.075 μM (Froquet et al. 2001). These concentrations would correspond to 74 and 22 ng DON per milliliter, respectively. Although DON was detected only in traces in blood of bulls of the present study (Winkler et al. 2016), experiments with dairy cows suggest that such DON concentrations in systemic circulation are within the realms of possibility in the ruminating bovine (Keese et al. 2008b; Winkler et al. 2014). Despite these obvious in vitro myelotoxic effects of unmetabolized DON, no information is available on cytotoxic effects of de-DON on these progenitor cells. The increase in erythrocyte counts and hematocrit as observed in the present experiment would rather suggest stimulating effects of feeding the FUS-contaminated diets. It has been discussed that bone marrow effects cannot be excluded because of the co-presence of other Fusarium toxins, i.e., modified forms of DON and ZEN such as DON-3-glucoside (4426 μg/kg), 3- and 15-acetyl-DON (545 and 3657 μg/kg), and ZEN-4-sulfate (183 μg/kg) besides the free DON and ZEN and other toxins like butenolid (2714 μg/kg), aurofusarin (25,904 μg/kg), culmorin (13,625 μg/kg), and 15- and 5-OH-culmorin (2350 and 12,081 μg/kg) (Dänicke et al. 2017). While modified forms of DON and ZEN are systemically active in their free forms due to rumen metabolism (Dänicke and Brezina 2013), much less is known about bone marrow effects of other co-contaminating mycotoxins. In addition, discussed possible interfering effects of the FUS-associated increase in DM-intake have to be borne in mind in an overall interpretation of treatment effects.

The PCA revealed that DM intake formed a cluster with water intake, blood urea concentration, BHB, GLDH, GGT, cholesterol and others suggesting associations and supporting the discussed results of the variance analyses. Antibody titers to BVDV were less close situated to this cluster but appeared to be associated with some traits of granulocytes and red blood count supporting the discussed treatment effects. The treatment effects can even be more distinctively distilled when all 42 variables recorded for each bull are condensed (Fig. 5). With a few exceptions, all bulls of group CON were situated in the lower right quadrant. Moreover, the vaccinated animals of this group appeared to form a sub-cluster. FUS-fed animals formed another large cluster distantly situated from the CON group with bulls of groups FUS III and FUS I separating in distinctive sub-clusters while bulls of group FUS II were located in between. The effects of vaccination appeared to be less clear compared to the CON group. This visual integrative representation of all the data makes clear that treatments clearly influenced the metabolic, hematological, and immunological status of the animals.

The total antibody response to BVDV-vaccination was influenced by oral exposure to Fusarium toxin-contaminated feed containing mainly DON in a non-linear manner which might also be due to linear and non-linear specific isotype responses. A dietary DON-concentration of 5.66 mg/kg DM was shown to result in the strongest total antibody response while a concentration of 8.31 mg/kg DM lowered total antibodies.

Based on total BVDV antibody response, DON concentrations higher than 5.66 mg/kg DM corresponding to a DON concentration of 5.0 mg/kg at a reference DM content of 88% have to be regarded as critical. This concentration matches the guidance value for critical dietary DON concentration of 5 mg/kg (88% DM) according to the European Commission recommendation (European Commission 2006).

Future studies should consider the detection of isotype-specific BVDV antibodies to clarify the nature of the DON exposure related non-linear response of total antibodies. Moreover, BVDV challenge experiments in combination with BVDV vaccination and DON exposure could be useful in examining the DON-related effects on BVDV-antibody protection to infection.

The marked effects of BVDV-vaccination on red blood count indices are probably due to the aluminum-containing adjuvant. These effects as well as the interactions with DON-exposure require further elucidation.