Effect of supplementation with an electrolyte containing a Bacillus-based direct-fed microbial on immune development in dairy calves

Research in Veterinary Science 92 (2012) 427–434

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Effect of supplementation with an electrolyte containing a Bacillus-based direct-fed microbial on immune development in dairy calves K.N. Novak a,⇑, E. Davis a, C.A. Wehnes a, D.R. Shields b, J.A. Coalson b, A.H. Smith a, T.G. Rehberger a a b

Danisco USA, Inc., W227 N752 Westmound Drive, Waukesha, WI 53186, United States Merrick’s, Inc., 654 Bridge Street, Union Center, WI 53962, United States

a r t i c l e

i n f o

Article history: Received 11 January 2011 Accepted 11 April 2011

Keywords: Probiotic Immunity Bacillus Calf

a b s t r a c t Immune characteristics in 65 calves were evaluated in response to a Bacillus-based direct-fed microbial (DFM) provided in electrolyte scour treatment. Blood samples were analyzed for cell surface markers and a1-acid glycoprotein (AGP) concentration. AGP increased in scouring calves given electrolyte containing Bacillus at day 7 post-placement compared to scouring calves administered electrolyte alone and nonscouring calves, enhancing the inflammatory response for pathogen clearance. The Bacillus promotes T cell subsets including greater proportions of activated, mature cells (CD8 CD25+, CD8 CD45RO+, CD8 TCR1+) in calves given electrolyte containing Bacillus than scouring calves administered electrolyte alone and non-scouring calves. Also, the Bacillus may be alleviating inflammation at day 3 post-placement as the proportion of monocytes and granulocytes lacking L-selectin (CD172a+CD62L ) was greater in scouring calves given electrolyte compared to the other groups. Electrolyte containing Bacillus administered at the onset of scours influences components of innate and adaptive immune development during and following the scouring event. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Colonization of commensal bacteria in the gastrointestinal tract (GIT) is vital to appropriate immune development in neonates. The GIT of neonates is devoid of bacteria at birth, but establishment begins during parturition (Mackie et al., 1999). Commensals inhibit pathogens and are integral in promoting activation of immune components to aid in the development of a competent and stable immune system (Borchers et al., 2009). Calves, born with a naïve immune system, come in contact with environmental factors and disease-causing organisms during the first few days of life coinciding with microbial colonization; therefore, this time is crucial for appropriate development of the GIT microbiota and subsequently, a functional immune system (Wilson et al., 1996). Early challenges from the environment aid in the development of the calf immune system (Kampen et al., 2006). However, calves are often unable to cope with these challenges, resulting in dysbiosis of the GIT microbiota. Direct-fed microbial (DFM) products may help with colonization by providing competitive exclusion of undesirable microorganisms and development of a homeostatic host/microbe gastrointestinal environment (Borchers et al., 2009). DFM products have been used to enhance the immune system, as evidenced by in vitro and in vivo systems for the evaluation of

⇑ Corresponding author. Tel.: +1 262 521 1717; fax: +1 262 521 2442. E-mail address: [email protected] (K.N. Novak). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.04.008

both human and animal models (Fuller, 1991; Gill, 2003; Hong et al., 2005). DFM products have demonstrated efficacy in calves, specifically by aiding in the development of rumen microbial populations, leading to a quicker conversion from a milk-based diet to solid feed (Krehbiel et al., 2003; Hong et al., 2005). Other studies have reported a decrease in scours with DFM supplementation, resulting in improvements in growth performance (Abe et al., 1995; Wehnes et al., 2009). However, the immune system response to DFM products has not been well described in calves. Sun et al. (2010) have reported that Bacillus subtilis natto has immunostimulatory properties in calves. Likewise, in humans and other animal species, probiotics and DFM products have exhibited immunomodulatory properties including immune stimulation for pathogen control, immune regulation to control inflammation, and enhancement of cell mediated immunity (Perdigon et al., 1986; Caruso et al., 1993; Isolauri et al., 2001). In addition, probiotics in humans have been demonstrated to be capable of maintaining homeostasis in the GIT by regulating inflammation in the gut caused by bacteria (Isolauri, 1999; Ouwehand et al., 2002). Based on these findings in multiple species, a DFM product may have particular efficacy in the neonatal calf by inhibiting pathogen establishment in the GIT and controlling inflammation associated with bacterial challenges. Bacillus organisms are currently used in DFM products for many species, including calves, despite the fact that they are not considered members of the commensal microbiota of calves (Hong et al., 2005). A commercially available B. subtilis-based DFM product was developed in our laboratory for control of Clostridium perfringens in

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calves (Wehnes et al., 2009). B. subtilis probiotics have been documented to stimulate the immune system of humans (Caruso et al., 1993) and have been demonstrated to have the capacity to activate phagocytes and T cells in mice (Gialdroni-Grassi and Grassi, 1985). These findings suggest that a B. subtilis-based DFM may be beneficial to young calves for enhancement of early immune development. Furthermore, bacilli are able to form a protective spore coat for enhanced stability through feed processing, yet in the hospitable environment of the GIT become vegetative and active (Casula and Cutting, 2002; Hong et al., 2005; Guo et al., 2006; Tam et al., 2006; Wilcks et al., 2008). These qualities of pathogen control, immune enhancement, and stability in product form make Bacillus strains ideal candidates for inclusion in DFM products for livestock. This study was designed to evaluate the effect of a multi-strain B. subtilis DFM product, administered to scouring calves through an electrolyte drench, on subsequent development of immune characteristics within the systemic circulation. 2. Materials and methods 2.1. Animals and treatments All calves were treated in accordance to the guidelines presented in Guidelines for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (available from Federation of Animal Science Societies, 1111 N. Dunlap Ave., Savoy, IL 61874) (Federation of Animal Science Societies, 1999). Animals, treatments, and experimental design were previously described in detail by Wehnes et al. (2009). Briefly, 65 Holstein bull calves were included in the experiment. Calves were estimated to be between 3 and 10 days of age at the time of placement (day 0). Calves were designated into three treatment groups based on the presence of scours, including a control group of non-scouring calves that remained untreated, a group of scouring calves that was treated with a control electrolyte drench supplemented with minerals (Blue Ribbon, Merrick’s, Inc., Union Center, WI), and a group of scouring calves that was treated with the same electrolyte drench containing a B. subtilis-based DFM (3  109 cfu/dose). The etiology of scours was not established in this study. Scouring calves received their treatments for a minimum of two consecutive days. Calves were added to treatments the day after scours occurred. Eight calves were sampled per treatment on each sampling day except for day 3 post-placement. On day 3 post-placement, only six calves had started scouring; therefore, three calves from each electrolyte treatment were available for sampling. By day 7 post-placement, there were enough scouring calves to sample eight calves per treatment on subsequent sampling days. The same calves continued to be sampled throughout the duration of the experiment. Throughout the study, there were 13 non-scouring calves, 26 scouring calves treated with electrolyte only, and 26 scouring calves treated with electrolyte supplemented with Bacillus. 2.2. Blood collection A 20 mL blood sample was obtained by jugular venipuncture from calves in each treatment and collected into tubes containing EDTA (BD Vacutainer, Preanalytical Solutions, Franklin Lakes, NJ) on days 1, 3, 7, 14, 21, 28, and 42 post-placement for the isolation of peripheral blood mononuclear cells (PBMC). An additional 5– 10 mL blood sample was obtained and serum was collected and stored at 80 °C until analysis of acute phase protein concentration.

(Cardiotech Services, Inc., Louisville, KY) according to the manufacturer’s instructions. Briefly, a 5 lL aliquot of calf serum was thawed and dispensed into a test well in the bovine a1-AG plate which contained antiserum to bovine AGP. Plates were incubated at 37 °C for 48 h. The scale provided with the commercial test kit was used to measure the diameter of the precipitin ring that formed. The diameters were plotted and compared to a standard curve that was constructed by measuring the rings of the standard solutions (50–1500 lg/mL) received in the kit. 2.4. Isolation of peripheral blood mononuclear cells PBMC were isolated by density gradient centrifugation using a Ficoll gradient (Histopaque 1077, density = 1.077 g/mL; Sigma Chemical Co., St. Louis, MO). Briefly, the blood was diluted in 15 mL of PBS and then overlaid onto Histopaque. Centrifugation at 400g for 40 min at 10 °C allowed for separation of density gradients. The buffy layer containing the desired cells was removed and diluted in supplemented RPMI (Sigma Chemical Co.) containing 1% antibiotic–antimycotic (Atlanta Biologicals, Lawrenceville, GA; contains 10,000 units/mL penicillin, 10,000 lg/mL streptomycin, and 25 lg/ml amphotericin B) and 5% fetal bovine serum (FBS; Atlanta Biologicals). The cells were harvested by centrifugation at 200g for 10 min at 10 °C. Cell concentration was determined by enumeration on a hemacytometer and cell viability was assessed using Trypan Blue exclusion dye (Sigma Chemical Co.). The cell concentration was adjusted to 5  106 cells/mL for flow cytometric analysis. 2.5. Flow cytometric analysis Mouse monoclonal antibodies used to identify specific bovine cell surface markers are illustrated in Table 1. The primary antibodies were stained with FITC (goat anti-mouse IgG FITC or goat antimouse IgM FITC, Sigma Chemical Co.) or R-phycoerythrin (PE; goat anti-mouse IgG PE or goat anti-mouse IgM PE, Sigma Chemical Co.) secondary antibodies. The flow cytometry panel contained an unlabeled isotype control monoclonal antibody to display nonspecific binding by secondary antibodies. Flow cytometric analysis was performed as described by Davis et al. (2004). Briefly, primary antibodies were administered into appropriate wells of the plate containing cell suspension and the plates were incubated. Following this incubation, unbound antibody was discarded with two washings. After the washings, secondary antibody was added to appropriate wells and plates were incubated. The washing procedure was repeated. Staining, incubations, and washings were repeated with appropriate antibodies to double stain the cells. Data were acquired and analyzed using a BD FACSCalibur flow cytometer with a 488 nm laser (BD Biosciences, San Jose, CA) and CellQuest Pro software (BD Biosciences). After acquisition of samples, populations were gated using the single stained cell surface markers for monocytes, CD45, CD3, and the unlabeled population. Regions were set around these four populations. Multi-color gating was set on the forward scatter/side scatter (FSC/SSC) plots. One region was then drawn around the monocyte and CD3 population on the FSC/SSC plot. Another region was drawn around the CD45 population on the FSC/SSC plot. These two regions were then combined as one gate. This gate was applied to the fluorescent plots and used throughout the analysis on CellQuest Pro. 2.6. Statistical analysis

2.3. Acute phase protein analysis Blood serum was analyzed for a1-acid glycoprotein (AGP) concentration by radial immunodiffusion using diagnostic test kits

Analysis of variance was performed using the GLM procedure of SAS (SAS Institute, Inc., Cary, NC) to analyze flow cytometric data. The model included the effects of sampling day, Bacillus

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Table 1 Monoclonal antibodies specific for bovine leukocytes used to define cell surface molecule expression and differential populations of leukocytes derived from peripheral blood mononuclear cell populations by flow cytometric analysis.

1 2

mAb1

Clone2

Isotype

Cell type(s) expressing molecule

CD3 CD4 CD8a CD25 CD26 CD45 CD45R CD45RO CD62L

MM1A GC50A1 CACT80C LCTB2A CACT114A CACTB51A LCT2A GC44A BAQ92A

IgG1 IgM IgG1 IgG3 IgG2b IgG2a IgG2a IgG3 IgG1

CD172a TCR1-N12 Monocytes

DH59B CACT61A BAQ151A

IgG1 IgM IgG1

T cells CD4+ T helper cells CD8+ cytotoxic T cells; also expressed on NK cells IL-2 receptor; activated lymphocytes (B and T cells) and present on monocytes Activated memory T cells; activated ab and cd T cells, B cells, NK cells, and macrophages; also on small intestinal epithelium All leukocytes Naïve T cells, B cells, monocytes Memory T cells, a subset of B cells, monocytes L-selectin (CD62 ligand), leukocyte endothelial adhesion molecule involved in homing of naïve T cells to peripheral lymph nodes; present on B and T cells, monocytes, granulocytes, and some NK cells Monocytes, granulocytes, SWC3 equivalent in cattle cd T cell receptor Monocytes

Monoclonal antibodies are mouse anti-bovine. All mAb were purchased from Veterinary Medical Research and Development, Inc., Pullman, WA.

supplementation, and appropriate interactions. The mixed procedure of SAS (SAS Institute, Inc.) was performed for the AGP data analysis using calf as the random effect. Differences between treatments were regarded as statistically significant at P 6 0.05. 3. Results Serum AGP concentrations were greater (P 6 0.05) in calves treated with electrolyte supplemented with Bacillus compared to non-scouring calves and calves treated with only electrolyte at day 7 post-placement, whereas no differences were observed in the AGP concentration between treatments on days 3, 14, 21, 28, and 42 post-placement (trt  day interaction, P = 0.05, Fig. 1). The PBMC isolation procedure on blood samples collected on day 14 post-placement failed; therefore, there is no flow cytometry data presented from this sampling day. The proportion of cytotoxic leukocytes (CD4 CD8+, CD8+CD45R ) was greater (P 6 0.05) in calves administered either electrolyte treatment compared to the non-scouring calves (Table 2). Specifically, at day 3 post-placement, the proportion of CD4 CD8+ cells was lower (P 6 0.02) in the non-scouring calves and calves treated with electrolyte containing Bacillus than in calves treated with only electrolyte. However, at day 21 post-placement, the proportion of

AGP Concentration (µg/mL)

1400

a

1200 1000

b

800

b

600 400 200 0 3

7

14

21

28

42

Sampling Day Fig. 1. Serum a1-acid glycoprotein (AGP) concentrations of non-scouring (j) dairy calves, scouring calves treated with electrolyte (h), or scouring calves treated with electrolyte containing Bacillus ( ). Values represent the means of each treatment at days 3, 7, 14, 21, 28, and 42 post-placement and represent the means of eight calves per treatment on all sampling days except day 3. On day 3, values are the means of eight non-scouring calves, three calves administered electrolyte alone, and three calves administered electrolyte containing Bacillus. a,bMeans without common superscripts are significantly different within day (P 6 0.05; trt  day interaction, P = 0.05).

this cytotoxic subset was greater (P = 0.01) in calves treated with Bacillus-supplemented electrolyte compared to calves treated with electrolyte devoid of Bacillus (trt  day interaction, P = 0.02, Fig. 2A). Also, at day 3 post-placement, the proportion of mature cytotoxic T cells (CD8+CD45R ) in systemic circulation was lower (P 6 0.01) in non-scouring calves and calves treated with electrolyte containing Bacillus compared to calves treated with only electrolyte, with no significant differences observed on the other sampling days (trt  day interaction, P = 0.04, Fig. 2B). The peripheral blood of calves treated with electrolyte devoid of Bacillus had a greater (P 6 0.05) proportion of memory cytotoxic lymphocytes (CD8+CD45RO+) than the non-scouring calves (Table 2). Additionally, the proportion of memory lymphocytes exclusive of the cytotoxic T cell population (CD8 CD45RO+) was greater (P 6 0.05) in calves given electrolyte containing Bacillus compared to non-scouring calves and calves given electrolyte alone (Table 2). The proportion of activated memory T helper cells (CD4+CD26+) was greater (P 6 0.05) in the peripheral blood of calves treated with electrolyte alone compared to non-scouring calves (Table 3). The calves treated with electrolyte containing Bacillus had a greater (P 6 0.05) proportion of cells expressing interleukin-2 receptor (IL-2R; CD8 CD25+) in the peripheral blood compared to non-scouring calves and calves given electrolyte alone (Table 3). The proportion of CD8+ cells lacking the cd T cell receptor (CD8+TCR1 ) was lower (P 6 0.05) in calves treated with electrolyte supplemented with Bacillus and non-scouring calves compared to calves treated with electrolyte (Table 4). In addition, calves administered electrolyte containing Bacillus had a greater (P 6 0.05) proportion of cd T cells lacking CD8 expression (CD8 TCR1+) compared to non-scouring and electrolyte-treated calves (Table 4). The proportion of systemic leukocytes expressing L-selectin exclusive of the CD8+ subset (CD8 CD62L+) in calves on either electrolyte treatment was greater (P 6 0.05) than that in non-scouring calves (Table 5). Additionally, calves provided the electrolyte containing the Bacillus and non-scouring calves had a lower (P 6 0.05) proportion of CD8+ cells expressing the adhesion molecule, L-selectin, (CD8+CD62L+) in the systemic circulation at day 3 post-placement compared to electrolyte-treated calves; however, there was no difference between treatments at days 7, 21, 28, or 42 postplacement (trt  day interaction, P = 0.05, Fig. 2C). There was a tendency (P = 0.10) for an effect in the monocyte and granulocyte population lacking L-selectin (CD172a+CD62L ) in the peripheral blood (Table 5). Specifically, the proportion of this population was greater (P 6 0.05) at day 3 post-placement in calves treated with electrolyte alone compared to calves treated with Bacillus-supplemented electrolyte and non-scouring calves. However, at day 28

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Table 2 Percentage of CD4 and CD8 T lymphocyte subpopulations, naivety (as indicated by the presence of CD45R on the cell surface) and memory lymphocytes (as indicated by the presence of CD45RO on the cell surface) within the CD4- and CD8-defined T cell subpopulations within the peripheral blood population of non-scouring dairy calves and calves treated with electrolyte therapy for scours or treated with the electrolyte therapy containing Bacillus.1 Treatments Non-scouring +

CD4 CD8 CD4+CD8+ CD4 CD8+* CD4+CD45R CD4+CD45R+ CD4 CD45R+ CD8+CD45R ** CD8+CD45R+ CD8 CD45R+ CD4+CD45RO CD4+CD45RO+ CD4 CD45RO+ CD8+CD45RO CD8+CD45RO+ CD8 CD45RO+

4

1.31 23.20 50.27b 1.834 21.77 50.15 19.03b 32.09 14.43 0.664 23.01 55.94 10.51 40.46b 20.59b

Electrolyte 4

0.92 26.09 54.83a 2.384 23.81 51.17 23.12a 36.20 12.66 1.264 24.31 57.79 11.72 47.80a 19.51b

Electro + Bacillus2 4

1.00 23.78 54.91a 3.054 22.61 51.22 22.69a 32.09 15.47 1.734 22.64 57.72 9.55 43.92a,b 24.92a

P-value3

SE 4

0.12 1.09 1.45 1.024 1.40 2.46 1.20 1.99 0.91 0.974 1.36 2.68 0.79 1.97 1.58

0.074 0.12 0.04 0.684 0.57 0.94 0.03 0.21 0.07 0.734 0.63 0.85 0.12 0.03 0.03

a,b

Means within a row with different superscripts differ (P 6 0.05). Values represent the means of combined samples at days 3, 7, 21, 28, and 42 post-placement. Values are the means of eight calves per treatment on all sampling days except day 3. On day 3, values are the means of eight non-scouring calves, three calves administered electrolyte alone, and three calves administered electrolyte containing Bacillus. 2 Electro + Bacillus = Electrolyte containing Bacillus. 3 Treatment effect P-value. 4 Population was considered too low to be detectable (<3%). * Trt  day interaction, P = 0.02. See Fig. 2A. ** Trt  day interaction, P = 0.04. See Fig. 2B. 1

post-placement, the calves treated with electrolyte containing Bacillus had a greater (P = 0.02) proportion of CD172a+ leukocytes lacking L-selectin compared to the non-scouring calves. No differences were observed for this population between treatments at days 7, 21, or 42 post-placement (trt  day interaction, P = 0.03, Fig. 2D).

4. Discussion Concurrent work with this immune study found that the addition of Bacillus lowered the cost of therapeutic treatment, reduced the recurrence of scouring events, and reduced C. perfringens levels in the feces of scouring calves treated with electrolyte containing Bacillus compared to scouring calves treated with electrolyte alone (Wehnes et al., 2009). Therefore, the purpose of this study was to evaluate the effect of this Bacillus-based DFM product, administered to scouring calves through electrolyte scour treatment, on subsequent immune development. This study indicated that therapeutic Bacillus supplementation altered peripheral blood immune cell phenotypes. However, as this study reported proportions of leukocyte subsets, it is not known if the treatment altered cell subset proportions due to shifts in leukocyte concentrations or if these changes were differences in cell phenotype proportions within similar leukocyte concentrations. Regardless, these findings demonstrate that the administration of a Bacillus-based direct-fed microbial elicits immunological changes in the systemic circulation of young calves. The concentration of AGP was assessed within the peripheral blood of calves. The presence of AGP, an acute phase protein, is indicative of stress or illness, and in the bovine species, levels of AGP in the blood greatly increase during disease (Tamura et al., 1989). The elevated levels of AGP in scouring calves that received electrolyte supplemented with Bacillus on day 7 post-placement suggests an inflammatory response that could allow the scouring calves given electrolyte containing Bacillus to clear the infection quicker than scouring calves with electrolyte treatment alone.

Wehnes et al. (2009) reported an increase in C. perfringens fecal shedding in scouring calves administered electrolyte supplemented with Bacillus at d 7 post-placement. Although C. perfringens was not determined to be the cause of scours, the presence of C. perfringens could be a possible explanation for the observed increase in AGP. Percentage differences observed in the leukocyte populations in the CD8+ subsets (CD4 CD8+, CD8+CD45R ) demonstrate alterations in immune development due to the scouring event and further alterations due to electrolyte treatment. An increase in bovine CD8+ cells in response to bovine viral diarrhea virus has previously been reported (Beer et al., 1997; Silflow et al., 2005). Although, these data are in response to a virus, they indicate that bovine CD8+ cell populations can increase due to a challenge. In the present study, the cause of scours was not determined, however, we conclude that the development of the CD8+ subset is likely associated with scours as evidenced by its increase in either scouring treatment group overall. Yet, the addition of Bacillus further alters the CD8+ subsets, specifically at day 3 post-placement, where the CD8+ leukocyte populations (CD4 CD8+ and CD8+CD45R ) were similar between scouring calves that received electrolyte containing Bacillus and the non-scouring calves during the time scouring treatments were administered. This implies that while the scouring event influences development of the CD8+-defined T cell subsets, the Bacillus alters immunological development of specific CD8+ subsets analogous to the development of CD8+-defined T cells in non-scouring calves. Additionally, after scouring treatments were administered, the calves treated with electrolyte containing Bacillus demonstrated progressive immune development after supplementation had ceased (day 21 post-placement), as indicated by an enhancement of the CD4 CD8+ T cell population compared to calves treated with electrolyte devoid of Bacillus. This corresponds to a human study in which human PBL were analyzed after treatment with B. subtilis spores and an increase in CD8+ cells was reported (Caruso et al., 1993) indicating that the increase in CD8+ cells could be due to the addition of Bacillus in electrolyte therapy. Additionally, previously reported data by Maue et al.

K.N. Novak et al. / Research in Veterinary Science 92 (2012) 427–434

A 80

b

%CD4-CD8+

60

a

50

b

40 30

a

a,b

70

b

20 10 0 3

7

21

28

42

28

42

Sampling Day

B 40 %CD8 +CD45R -

35

a

30 25 20 15

b

b

10 5 0 3

7

21 Sampling Day

C 90 80

%CD8+ CD62L+

70 60

a

50 40 30

b b

20 10 0 3

7

21

28

42

Sampling Day

D 50 a

%CD172a+ CD62L-

40

a

30 20

a,b b

b b

10 0 3

7

21

28

42

-10 Sampling Day +

Fig. 2. Percent CD4 CD8 leukocytes (A; trt  day interaction, P = 0.02), percent CD8+CD45R leukocytes (B; trt  day interaction, P = 0.04), percent CD8+CD62L+ leukocytes (C; trt  day interaction, P = 0.05), and percent CD172a+CD62L leukocytes (D; trt  day interaction, P = 0.03) within the peripheral blood population of non-scouring (j) dairy calves, scouring calves treated with electrolyte (h), or scouring calves treated with electrolyte containing Bacillus ( ). Values represent the means of each treatment at days 3, 7, 21, 28, and 42 post-placement and represent the means of eight calves per treatment on all sampling days except day 3. On day 3, values are the means of eight non-scouring calves, three calves administered electrolyte alone, and three calves administered electrolyte containing Bacillus. a,bMeans without common superscripts are significantly different (P 6 0.05).

431

(2005) and Hagberg et al. (2008) indicate that the presence of a challenge reduces the number of naïve T cells similar to what was observed in the present study. This suggests that the scouring event contributes to this development and the addition of Bacillus further promotes a mature CD8+ T cell repertoire. The CD8+ memory T cells (CD8+CD45RO+) in the peripheral blood were enhanced due to the scouring event, regardless of scouring treatment. Naïve CD8+ T cells are capable of expanding into memory cells when specifically recognizing antigen (Rocha and Tanchot, 2004). The response to the scour challenge likely generated the development of this memory cell subpopulation in the calves. Yet, another memory subset (CD8 CD45RO+), including memory T helper cells and memory B cells, was further enhanced in scouring calves supplemented with electrolyte containing Bacillus compared to scouring calves receiving electrolyte alone. Memory T cells have the ability to quickly respond to previously encountered pathogens and alleviate that specific disease quickly upon repeat exposure (Rocha and Tanchot, 2004; Pearce and Shen, 2006). Whereas, the presence of scours likely induces development of the CD8+ memory T cells, the addition of Bacillus seems to promote development of a more mature and activated immune system as indicated by the further enhancement of the memory T cell population. The presence of scours stimulates immune activation by promoting the development of activated CD4+ T cells (CD4+CD26+) in the calves administered electrolyte and electrolyte supplemented with Bacillus. The increase in incidence of cells expressing IL-2R (CD8 CD25+) in calves supplemented with electrolyte containing Bacillus also implies immune cell activation and development. The IL-2R is expressed on activated T and B cells as well as monocytes and a rise in CD25 is usually associated with activation (Nonnecke et al., 2003). Caruso et al. (1993) reported an increase in CD25 on human PBL in response to dosing with B. subtilis spores indicating the ability of this organism to activate the immune system. While the scouring event elicits the activated CD4+ T cell response, the induction of the IL-2R demonstrates that the addition of Bacillus specifically activates subpopulations within the peripheral blood that scouring alone does not. Most cd T cells in the bovine species do not express CD4 and CD8 on their cell surface (Hayday, 2000; Meissner et al., 2003) so our finding that scouring calves administered electrolyte containing Bacillus and non-scouring calves have less CD8+ T cells lacking the cd T cell receptor (CD8+TCR1 ) than scouring calves treated with electrolyte alone is not surprising. Bovine peripheral blood cells contain a large proportion of cd T cells (Mackay and Hein, 1989; Hayday, 2000). Specifically, the CD8 cd T cell subpopulation is prevalent in systemic circulation and tends to home to inflammatory locations via adhesion molecules, like L-selectin (Jutila and Kurk, 1996; Wilson et al., 1999; Hayday, 2000). Meissner et al. (2003) suggest that the systemic cd T cell subpopulation lacking CD8 is in an activated/resting state and may have the ability to mount a quick response to challenge. The addition of Bacillus further advances this cd subpopulation (CD8 TCR1+). The cd subset monitors commonly encountered microorganisms and is involved in defense against challenges, as they are able to respond to antigen without presentation by the innate immune system (Jutila and Kurk, 1996; Mak and Ferrick, 1998; Williams, 1998). Because of this ability, cd T cells may be a connection between innate and adaptive immunity (Mak and Ferrick, 1998; Bukowski et al., 1999), and may have a role in regulation of inflammatory responses (Doherty et al., 1991; Fu et al., 1994). The addition of Bacillus promotes the development of the cd population and thereby potentially enhances the calves’ ability to combat commonly encountered pathogens, indicating that administration of bacteria with probiotic properties to neonatal calves has the potential to beneficially enhance immune development and functionality.

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Table 3 Percentage of activated CD4- and CD8-defined T cell subpopulations (as indicated by the presence of CD26 on the cell surface) and IL-2R expression within the CD4- and CD8defined T cell subpopulations (as indicated by the presence of CD25 on the cell surface) within the peripheral blood population of non-scouring dairy calves and calves treated with electrolyte therapy for scours or treated with the electrolyte therapy containing Bacillus.1 Treatments Non-scouring +

CD4 CD26 CD4+CD26+ CD4 CD26+ CD8+CD26 CD8+CD26+ CD8 CD26+ CD4+CD25 CD4+CD25+ CD4 CD25+ CD8+CD25 CD8+CD25+ CD8 CD25+ a,b 1-4

4

Electrolyte 4

0.87 21.85b 55.88 21.38 38.01 11.70 1.934 22.78 49.93 20.46 30.60 13.73b

Electro + Bacillus2 4

0.84 24.81a 55.23 22.60 37.36 10.91 1.664 24.98 52.11 23.54 31.18 13.23b

P-value3

SE 4

0.85 23.14a,b 55.77 22.04 33.77 13.57 1.964 22.78 53.77 21.65 29.35 16.59a

0.984 0.05 0.96 0.81 0.29 0.11 0.474 0.17 0.26 0.22 0.84 0.03

0.12 0.88 1.86 1.36 2.12 0.96 0.204 1.00 1.68 1.30 2.41 1.00

Means within a row with different superscripts differ (P 6 0.05). Refer to Table 2.

Table 4 Percentage of cd T cell receptor (as indicated by the presence of TCR1 on the cell surface) expression within the CD4- and CD8-defined T cell subpopulations within the peripheral blood population of non-scouring dairy calves and calves treated with electrolyte therapy for scours or treated with the electrolyte therapy containing Bacillus.1 Treatments Non-scouring +

CD4 TCR1 CD4+TCR1+ CD4 TCR1+ CD8+TCR1 CD8+TCR1+ CD8 TCR1+ a,b 1-4

4

Electrolyte 4

0.91 15.84 64.61 24.27b 26.64 15.40b

Electro + Bacillus2 4

0.63 17.41 67.29 29.07a 27.62 13.74b

P-value3

SE 4

0.75 16.72 66.33 24.25b 27.23 19.03a

0.054 0.66 0.63 0.01 0.92 0.0003

0.08 1.25 2.05 1.36 1.79 0.93

Means within a row with different superscripts differ (P 6 0.05). Refer to Table 2.

Table 5 Percentage of L-selectin expression within the CD4- and CD8-defined T cell subpopulations and the monocyte population (as indicated by the presence of CD62L on the cell surface) within the peripheral blood population of non-scouring dairy calves and calves treated with electrolyte therapy for scours or treated with the electrolyte therapy containing Bacillus.1 Treatments Non-scouring +

CD4 CD62L CD4+CD62L+ CD4 CD62L+ CD8+CD62L CD8+CD62L+* CD8 CD62L+ CD172a+CD62L ** CD172a+CD62L+ CD172a CD62L+ a,b 1-4 * **

4

0.29 18.20 66.98 7.41 42.41 25.58b 8.31 46.14 22.15

Electrolyte 4

0.16 18.85 70.54 6.93 45.22 31.36a 15.86 43.20 20.31

Electro + Bacillus2 4

0.27 19.01 69.11 6.34 41.12 35.41a 13.38 42.81 25.56

P-value3

SE 4

0.04 1.01 1.52 1.01 2.04 2.20 2.52 3.06 1.84

0.044 0.83 0.25 0.74 0.31 0.01 0.10 0.69 0.10

Means within a row with different superscripts differ (P 6 0.05). Refer to Table 2. Trt  day interaction, P = 0.05. See Fig. 2C. Trt  day interaction, P = 0.03. See Fig. 2D.

The presence of scours, regardless of treatment, promotes the development of the leukocyte populations expressing L-selectin exclusive of the CD8+ subset (CD8 CD62L+). Additionally, the presence of L-selectin (CD8+CD62L+) at day 3 post-placement in scouring calves administered electrolyte alone suggests migration of the CD8+ T cell population to sites of infection. L-selectin, an adhesion molecule, is present on the majority of systemic leukocytes and provides a means for the leukocytes to leave the blood and enter into secondary lymphoid tissue as well as the GIT to the site of infection (Tedder et al., 1995; Reber et al., 2006). The lower

proportion of CD8+ cells expressing L-selectin in calves treated with electrolyte containing Bacillus suggests that any inflammation from scours or stress due to placement may have been alleviated earlier with Bacillus supplementation than with electrolyte treatment alone. Furthermore, memory cell formation can be accompanied with a decrease or loss of L-selectin expression (Lee and Vitetta, 1991; Swain et al., 1991). Therefore, this lower proportion of CD8+ cells expressing L-selectin in calves administered electrolyte containing Bacillus may also indicate an enhancement of immune development within a memory subset associated with

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Bacillus supplementation that is not enhanced in calves given electrolyte treatment alone. DFM products modify immune responses and have a direct effect on various immune cell populations (Delcenserie et al., 2008; Gill and Prasad, 2008) as evidenced in this calf study. Calf immune development is further enhanced by the addition of Bacillus to electrolyte scour treatment in the presence of scours leading to appropriate immune responses and the maintenance of homeostasis. 5. Conclusion Supplementation of a commercial electrolyte product containing Bacillus to scouring calves led to differences in AGP concentration and leukocyte populations compared to their scouring counterparts that received electrolyte treatment alone. The Bacillus promoted development of T cell subpopulations including the cd T cell, memory, and activated subsets and regulated inflammation during the scouring event as indicated by the monocyte population. Furthermore, the treatment of scouring calves with electrolyte containing Bacillus resulted in a divergence in immune development of specific lymphocyte subsets that were distinct from scouring calves that did not receive the Bacillus. Scour treatment containing electrolyte supplemented with Bacillus may provide additional benefits beyond the therapeutic effect, as it may also improve later immune development as evidenced by the enhancement of elements of the innate and adaptive immune systems in calves following the scouring event. Acknowledgements We would like to thank Barry Witmer, Leslie Reeck, Steve Wells, and Joe Goodman of Merrick’s, Inc. for their assistance in the collection of samples and care of the animals and Dr. David Buchanan of North Dakota State University for statistical analysis. References Abe, F., Ishibashi, N., Shimamura, S., 1995. Effect of administration of Bifidobacteria and lactic acid bacteria to newborn calves and piglets. Journal of Dairy Science 78, 2838–2846. Beer, M., Wolf, G., Pichler, J., Wolfmeyer, A., Kaaden, O.-R., 1997. Cytotoxic Tlymphocyte responses in cattle infected with bovine viral diarrhea virus. Veterinary Microbiology 58, 9–22. Borchers, A.T., Selmi, C., Meyers, F.J., Keen, C.L., Gershwin, M.E., 2009. Probiotics and immunity. Journal of Gastroenterology 44, 26–46. Bukowski, J.F., Morita, C.T., Brenner, M.B., 1999. Human cd T cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunity. Immunity 11, 57–65. Caruso, A., Flamminio, G., Folghera, S., Peroni, L., Foresti, I., Balsari, A., Turano, A., 1993. Expression of activation markers on peripheral-blood lymphocytes following oral administration of Bacillus subtilis spores. International Journal of Immunopharmacology 15, 87–92. Casula, G., Cutting, S.M., 2002. Bacillus probiotics: spore germination in the gastrointestinal tract. Applied and Environmental Microbiology 68, 2344–2352. Davis, M.E., Maxwell, C.V., Erf, G.F., Brown, D.C., Wistuba, T.J., 2004. Dietary supplementation with phosphorylated mannans improves growth response and modulates immune function of weanling pigs. Journal of Animal Science 82, 1882–1891. Delcenserie, V., Martel, D., Lamoureux, M., Amiot, J., Boutin, Y., Roy, D., 2008. Immunomodulatory effects of probiotics in the intestinal tract. Current Issues in Molecular Biology 10, 37–54. Doherty, P.C., Allan, W., Eichelberger, M., Carding, S.R., 1991. Heat-shock proteins and the cd T cell response in virus infections: implications for autoimmunity. Springer Seminars in Immunopathology 13, 11–24. Federation of Animal Science Societies, 1999. Guidelines for the Care and Use of Agricultural Animals in Agricultural Research and Teaching Consortium. Federation of Animal Science Societies, Associated Headquarters, Savoy, IL, USA. Fu, Y.X., Roark, C.E., Kelly, K., Drevets, D., Campbell, P., O’Brien, R., Born, W., 1994. Immune protection and control of inflammatory tissue necrosis by cd T cells. Journal of Immunology 153, 3101–3115. Fuller, R., 1991. Probiotics in human medicine. Gut 32, 439–442. Gialdroni-Grassi, G., Grassi, C., 1985. Bacterial products as immunomodulating agents. International Archives of Allergy and Applied Immunology 76 (Suppl. 1), 119–127.

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