The use of direct fed microbials (DFM) in livestock is not new. However, there is a lack of information regarding the safety of bacteriocin-producing bacteria during passage through the GI tract of ruminant and monogastric animals. In this regard, it is of special interest to study the histological alterations and immunostimulatory response induced by orally administered microbes. Previous in vivo
studies indicated that viable and non-viable Lactobacillus plantarum
cells showed a similar cytokine production when orally administered to mice 
. Nonetheless, humoral immune response appears to vary when animal models are exposed to viable and non-viable Streptococcus
. No studies so far have addressed the effects of the administration of the bacteriocin-producing bacterium Streptococcus bovis
HC5 to an animal host. In the present study, we determined the local and systemic effects of the oral administration of S. bovis
HC5 to pre-sensitized BALB/c mice. Viable and heat-killed cells of S. bovis
HC5 were used in order to determine the differences in response induced by these cells in our in vivo
The treatment with S. bovis HC5 has not impaired the weight gain of BALB/c mice, but the animal gastrointestinal physiology was influenced by the viability of S. bovis HC5 cultures. Upon oral challenge with heat-killed S. bovis HC5 cells, the gut permeability to proteins was indeed altered, as evidenced by greater uptake of the bystander protein β-LG, when compared to the negative control group. In the V group, some β-LG was detected after 0.5 h of administration, but the β-LG passage was still 50% lower compared to the animals of the HK group.
Balzan et al.
demonstrated that greater intestinal permeability can increase the risk of bacterial translocation. However, hyperpermeability is not a determinant factor for bacterial translocation. Bacterial translocation was initially defined as the passage of viable intestinal bacteria through the gut mucosa into normal sterile tissue 
. This concept was expanded to include the translocation of endotoxins or antigens from the intestinal lumen into the circulation, causing systemic inflammation and distant organ injury 
. Bacteria translocate via intestinal epithelial cells (transcellular route) or by using the tight junctions (paracellular route) 
. In healthy hosts, with an intact intestinal barrier and a competent immune system, indigenous bacteria “spontaneously” translocate across the intestinal epithelial barrier at a low rate, which is considered a physiologic event. In this situation, the majority of bacteria that translocate are destroyed by innate immune defenses of the host, without deleterious consequences 
Although bacterial translocation to the mesenteric lymph nodes were not determined in our in vivo
assays, which would be considered a direct evidence of bacterial translocation 
, extensive histological analysis did not reveal the passage of bacterial cells from the gastrointestinal lumen to the lamina propria or other tissues. The rate of translocation varies among bacterial species, and previous work demonstrated that facultative anaerobic Enterobacteriaceae
translocate most readily from the GI tract 
. The Gram-positive, oxygen-tolerant bacteria, such as Staphylococcus epidermidis
, translocate at an intermediate rate, while strict anaerobes, such as Bacteroides
, and Fusobacterium
, translocate at the lowest rate, even though they normally occur at very high levels in the GI tract (1010−11
/g caecum). In addition, evidences suggest that oxygen sensitivity may be a limiting factor in the translocation of anaerobic bacteria 
In the present work, the sensitization of the animals probably triggered the immune system to respond to S. bovis HC5 antigens, with the majority of bacterial cells being phagocytosed. This activity may contribute to the intestinal inflammatory response and prevent bacterial translocation. This idea is supported by the absence of histological alterations in the extra-intestinal sites (e.g. spleen, liver and heart) of the animals treated with S. bovis HC5.
The significant reduction on the total number of splenocytes observed when the animals were treated with S. bovis
HC5 cells may be due to the migration of immune cells to the intestine, 
, where the main histological alterations were observed. Edema and congestion in the small intestine were often similar for both V and HK groups. The administration of S. bovis
HC5 caused the flattening of the villi in the small intestine, but a more prominent reduction of the villous diameter was detected among the animals of the V group. These alterations were probably caused by the direct effect of the bacteria or their components on the small intestinal epithelial cells. Similar results were obtained when viable cells of Lactobacillus delbrueckii
UFV-H2b20, a probiotic lactic acid bacterium, were administrated to mice 
In general, the presence of substances in the gastrointestinal tract influence the expression and activity of key proteins involved in the regulation of cell proliferation, differentiation and apoptosis 
. In both V and HK groups, a hypertrophy of Paneth cells and also an increase in mitotic activity were observed, indicating that despite the loss of villi architecture, the processes of antimicrobial compounds secretion, cell turnover and tissue repair remained activated, in an attempt to counteract the injuries caused by S. bovis
HC5 cells in the intestine. Again, the effects on the Paneth cells and mitotic division were more prominent in the mice that received viable cells.
The number and the secretory activity pattern of goblet cells can be affected by a variety of stimuli, including the exposure to bacteria, alterations of the normal microbiota and inflammatory mediators 
. It has been suggested that acidic mucins are less degraded by bacterial glycosidases and host proteases, thus protecting against bacterial translocation 
. In this sense, an increase in acid mucin producer goblet cells (AB+
cells) were expected in response to the exposure to S. bovis
HC5. However, cells secreting exclusively acid mucins were not observed in this study. In addition, a reduction in the number of PAS/AB+
goblet cells (producing neutral and acid mucins) was observed following the oral administration of S. bovis
HC5 cells to BALB/c mice. This effect cannot be fully explained by the reduction of mucus secretion, but could have been affected by the limited number of counting fields, which was caused by partially destructed villi in the small intestine of animals from the V and HK groups.
In contrast with the effects observed for the small intestine, animals that received viable or heat killed cells of S. bovis
HC5 did not show epithelium degeneration and congestion in the large intestine. However, edema and mucosal atrophy were detected in the large intestine of the animals and did not differ between the V and HK groups. In the large intestine, the passage rate and physical-chemical conditions favor bacterial colonization, with a dominance of anaerobic bacteria (1011
. This dense microbial community and the production of antimicrobial substances by resident bacteria can limit the growth and colonization of alloctone bacteria arriving in the large intestine. Additionally, the microbial populations in the large intestine can influence the proliferation and differentiation of epithelial intestinal cells and also contribute to the maintenance of the mucosal immune system 
. Based on these observations, it appears that orally administered S. bovis
HC5 cells will be limited in their ability to cause epithelial degenerative changes in the large intestine compared to the small intestine.
Despite of similar histological and morphometric alterations caused by viable and heat-killed S. bovis
HC5 cells to the GI tract of BALB/c mice, a reduced epithelial barrier function was observed only for the HK group, as demonstrated by the increased β-LG concentration in the sera of animals receiving HK cells. Based on the degenerative epithelial alterations observed among the animals of the V group, a reduced barrier function caused by the administration of viable S. bovis
HC5 cells could be expected. However, an increase in intestinal permeability was not observed in the V group, which could be explained by a different pattern of intestinal cytokine expression between the V and HK groups. The proinflammatory cytokines TNF-α and IFN-γ appear to induce a pathological opening of the intestinal tight junction (TJ) barrier, a mechanism that contributes to intestinal inflammation 
. In this study, the TNF-α expression was increased in the HK group, while the levels of IFN-γ were higher in the V group. Additionally, the administration of heat killed cells induced T-helper type 2 cytokines (IL-5 and IL-13), which are also related to the increasing of intestinal permeability 
Some LAB can affect the host's systemic and mucosal immunity 
, although the intensity and characteristics of these immune-stimulatory activities can vary among species and even among strains, being affected by the ecological niche of the microorganism under evaluation, the growth phase of the bacterium, the animal model adopted, and the doses administrated 
. Moreover, the autolysis of bacterial cells 
, the structural modification or degradation of the effective components from bacterial cells affect the immune-stimulatory effects induced by bacteria, and have been described as an important issue for recognition by the phagocytes 
To determine if the events triggered by chronic exposure to viable and heat-killed S. bovis HC5 cells were restricted to the intestinal mucosa or whether they were part of a systemic response, the expression of cytokines was analyzed in the intestine and spleen of the BALB/c mice. In our study, systemic immune-mediated effects were not been detected by the administration of S. bovis HC5 cells to BALB/c mice, but local immune-mediated effects could be observed on the jejunum of these animals, and, in this case, the immune response was affected differentially by the viability of the S. bovis HC5 cells. We have shown that viable cells of S. bovis HC5 were a potent inducer of the TH1-type cytokines IL-12 and INF-γ by immune-competent cells in the intestinal mucosa, and, on the other hand, heat-killed S. bovis HC5 cells induced the production of the pro-inflammatory cytokine TNF-α, and also type 2 cytokine, IL-5 and IL-13.
Certain lactic acid bacteria induce the production of TH
1-type cytokines, such as IL-12 and IFN-γ, and shift a TH
2-dominant condition to a TH
1-dominant condition 
. In agreement with our results, the administration of the halophilic LAB Tetragenococcus halophilus
Th221 promoted TH
1 immunity, inducing the IL-12 production 
, and a heat-killed Lactobacillus plantarum
L-137, a strain isolated from fermented food, was also a potent inducer of TNF-α 
. Mohamadzadeh et al.
has also shown the effectiveness of viable cells of Lactobacillus
1 response, in which activation of human dendritic cells by Lactobacillus
skews T cells toward TH
1 polarization. Lactobacillus gasseri
OLL2809 stimulated the production of IL-12 (p70) by murine splenocytes, and suppressed serum antigen-specific IgE levels via the TH
2 balance 
However, Maeda et al.
did not detect pro-inflammatory cytokines in the serum of the C57BL/6 mice after oral administration of heat-killed Lactobacillus plantarum
L-137. According to Pochard et al
, in the presence of both viable and non-viable bacterial cells of Lactobacillus plantarum
NCIMB8826, Lactococcus lactis
MG1363, Lactococcus lactis
ATCC393 and Lactobacillus rhamnosus
GG, an increased secretion of INF-ã and a reduction in the synthesis of IL-4 and IL-5 were observed in mononuclear cells from healthy and allergic patients. In a similar way, Kalliomaki et al
reported that after oral supplementation with Lactobacillus rhamnosus
GG, children with atopic dermatitis and allergy to cow's milk exhibited a transient increase in the production of INF-γ and a reduction 1in the production of IL-4.
In summary, the administration of S. bovis HC5 cells to pre-sensitized BALB/c mice induced major effects at local level, altering the permeability, morphometric characteristics and the pattern of cytokine production on the intestine of the animals, and the viability of the bacterial cells differentially affected some of the features evaluated. The mechanisms by which S. bovis HC5 interacts with intestinal cells and stimulate immune response, as well as the possible effects of this bacterium on the microbial community composition in the intestine, will be the subject of future studies.