Lymphocyte proliferative responses in dairy cows supplemented with an immunomodulatory feed additive and administered polyvalent vaccination

OmniGen-AF is a commercially available, patented, proprietary, branded feed additive that exerts immunomodulatory effects in nonruminant (Branson et al., 2016) and ruminant species (Wang et al., 2009; Nace et al., 2014; Brandão et al., 2016). Therefore, there are many gaps in the understanding of how this feed additive acts on the immune system. In addition, the majority of studies of this additive have focused on investigating its effect on the innate immune response, and its effect on the adaptive immune response is largely unknown. Nonetheless, we hypothesized that its implications are far-reaching and that it may also impact adaptive immunity functions, such as lymphocyte proliferation.

The dairy cow's diet exerts a profound impact on immune function and consequently on its natural ability to resist infections. Thus, if the use of a dietary immunostimulant can enable the host to boost their immunity, it may have effects beyond the health of the individual cow, as it will minimize the undesirable use of antimicrobials, impacting public health and milk chain profitability.
OmniGen-AF ® is a commercially available, patented, proprietary, branded feed additive that exerts immunomodulatory effects in nonruminant (Branson et al., 2016) and ruminant species (Wang et al., 2009;Nace et al., 2014;Brandão et al., 2016). Therefore, there are many gaps in the understanding of how this feed additive acts on the immune system. In addition, the majority of studies of this additive have focused on investigating its effect on the innate immune response, and its effect on the adaptive immune response is largely unknown. Nonetheless, we hypothesized that its implications are far-reaching and that it may also impact adaptive immunity functions, such as lymphocyte proliferation.
Our hypothesis is supported by some previous findings in murine models (Branson et al., 2016) and ruminants (Wang et al., 2009). For instance, this feed additive has the ability to modulate the production of interleukin (IL)-1β (Wang et al., Recebido em 7 de dezembro de 2018 Aceito em 5 de março de 2020 E-mail: nogueirasouza@yahoo.com.br 2009), a pro-inflammatory cytokine, which affords an important linkage between innate and adaptive immune responses by providing an activating signal for lymphocyte proliferation (Murphy and Weaver, 2008). Indeed, our hypothesis was strengthened by the finding of increased expression of genes involved in cellmediated adaptive immune response, such as the induction of CD1D1 and CD80 antigen presenting receptors, and a trend toward up-regulation of CD86, which in turn may activate naïve T cells, resulting in the stimulation of responsive B cells to proliferate and differentiate into plasma cells (Branson et al., 2016). To further investigate this scenario, the present study aimed to evaluate the effect of dietary OmniGen-AF ® supplementation on lymphocyte proliferation in dairy cows under vaccination. Here, we used thirty-two lactating primiparous (n=16) and multiparous (n=16) Holstein x Gir dairy cows. Samples from all dairy cows, which were not exposed to heat stress conditions, were collected for the leptospiral microscopic agglutination test, and samples from sixteen dairy cows (eight control and eight OmniGen-AF ®treated cows) were collected for lymphocyte proliferation. On day -6, cows were uniformly divided based on parity, days in milk, body weight and body condition score to receive or not receive OmniGen-AF ® (Phibro Animal Health, Teaneck, NJ, USA) at 56g/cow, daily as-fed basis, from day 1 to day 56, as previously detailed by Leiva et al. (2017) On days 56 (immediately before vaccination) and 77 (21 days after prime vaccination), blood samples of approximately 20mL from each cow were aseptically collected from the coccygeal vein and harvested into tubes without anticoagulant to obtain serum after centrifugation at 400 x g for 15 min for the leptospiral microscopic agglutination test. At these times, blood samples were also harvested into tubes containing sodium heparin for lymphocyte proliferation tests.
Lymphocyte proliferation was performed as described by Souza et al. (2011) using 5-(6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; 5mM mL -1 ; cat. n. C1157, Invitrogen, Carlsbad, NY, USA) as a probe. Phytohemagglutinin M-form (PHA-M) and concanavalin-A type III (Con-A) were used for mitogen-induced proliferation of T lymphocytes (Soder and Holder, 1999), and lipopolysaccharides (LPS) from Escherichia coli O127:B8 were used to induce the proliferation of B cells (Mehrzad and Zhao, 2008)  A triplicate set of wells containing only 100μL of cells in proliferation medium served as controls. On day 77, heat-killed Leptospira borgpetersenii serovar hardjo at a multiplicity of infection (MOI) of 100 (100 leptospires per PBMC) was also used to induce lymphocyte proliferation, as animals had been vaccinated 21 and 7 days earlier with a Lymphocyte proliferative… polyvalent vaccine containing bacterins from different Leptospira serovar strains, including Leptospira borgpetersenii serovar hardjo. On the lymphocyte proliferation assay on day 77, after the incubation, the B cells were identified using mouse anti-bovine CD21 conjugated with Rphycoerythrin (R-PE; cat. n. MCA1424PE, AbD Serotec, Oxford, England).
First, for flow cytometry analysis, the viable cells were gated according to their side-(SSC-H) and forward scatter (FSC-H) characteristics. Then, lymphocyte proliferation was determined by serially halving the fluorescence intensity of CFSE in total PBMCs and gated CD21 + cells (B cells at 77 d). An unstained control and singlestained samples (CFSE or R-PE CD21) were also prepared as compensation controls. The sample readings were performed using a FACSCalibur™ flow cytometer (Becton Dickinson Immunocytometry Systems™, San Diego, CA, USA) with argon (488nm excitation) and diode (635nm excitation) lasers. For this assay, 50,000 ungated cells were examined in each sample. FlowJo Tree Star Software (FlowJo -Treestar 7.6.5 for Windows, Tree Star Inc., Ashland, OR, USA) was used to analyze the data. The results were expressed as the division index, which represents the average number of divisions for all cells or gated B cells in the culture.
First, the distributions of all the variables were examined using normal probability plots obtained using Shapiro-Wilk tests. All data had gaussian distribution, apart from the PBMC proliferation index stimulated with Con-A and PHA-M at day 77. To identify differences between groups (control vs. OmniGen-AF ® -supplemented; primiparous vs. multiparous cows), Student's ttest for unpaired data was used for data with gaussian distributions, and the Mann-Whitney test was used for nonparametric data. Statistical analysis was performed using graphPad Prism 7.0 software ® (GraphPad Software, Inc., San Diego, CA, USA). The results are reported as the mean ± SEM. Significance was set at P<0.05.
The immune system requires extensive lymphocyte proliferation, ensuring that antigenspecific T and B cells can expand with enough speed to effectively handle infection by pathogenic microorganisms and to enable vaccine-induced protective immunity. To the best of our knowledge, this is the first report that investigated lymphocyte proliferation induced by distinct mitogens in dairy cows supplemented with an immunomodulatory feed additive.
Despite the likelihood that innate and adaptive immunities share many effector mechanisms and the great evidence that supplementation of dairy cows with this commercial feed additive modulates the innate immune response (Wang et al., 2009;Nace et al., 2014;Brandão et al., 2016;Branson et al., 2016) and some aspects of adaptive immunity (Wang et al., 2009;Branson et al., 2016), our data did not support the hypothesis that supplementation with this feed additive robustly affects lymphocyte proliferation.
Overall, we only observed that dairy cows supplemented with this commercial feed additive during lactation showed an increase in spontaneous (unstimulated) PBMC proliferation (P=0.03; Figure 1A) at 21 days after vaccination (day 77), and even worse, a lower B cell proliferation stimulated with L. heat-killed Leptospira borgpetersenii serovar hardjo (P=0.03; Figure 1B) at 21 days after vaccination (day 77). Indeed, no significant difference was observed immediately before vaccination (day 56; data not shown). Furthermore, no alteration of leptospiral antibody titers determined by the leptospiral microscopic agglutination test was found in dairy cows that received this commercial feed additive compared with control cows (data not shown), which supports our lymphocyte proliferation findings. Furthermore, corroborating our data, a previous study showed that daily feeding dairy heifers with this commercial feed additive did not improve the antibody titers in animals vaccinated against Staphylococcus aureus mastitis (Nace et al., 2014).
Vaccination is an important tool to control infectious diseases, and it is somehow expected that immunostimulants often increase vaccine efficacy. However, our data did not show any solid evidence that this feed additive elicited a higher and sustained protective immune response due to vaccination. Nonetheless, as we used healthy mid lactating dairy cows, the possible beneficial effect of this additive on immunocompromised dairy cows, such as periparturient dairy cows, should not be discarded and requires further investigation. While the study size is limited, an intriguing finding of our study is that PBMCs stimulated with LPS behaved differently in primiparous and multiparous cows at 21 days after vaccination (P=0.03; Figure 2); although there were no significant differences, PBMCs were always observed to be numerically higher in multiparous cows, both unstimulated and stimulated with Con-A, PHA-M and Leptospira borgpetersenii serovar hardjo. To the best of our knowledge, apart from one study (Mehrzad and Zhao, 2008), no information is available on lymphocyte proliferation in primiparous and multiparous dairy cows. These previous authors also found that the lymphocyte proliferation induced by distinct mitogens was lower in primiparous cows, and they also suggested that this discrepancy may be a result of the presence of primed and activated lymphocytes from multiparous cows, as they have been much more exposed to antigens throughout their lives.