Streptococcus bovis Contributes to the Development of Colorectal Cancer via Recruiting CD11b⁺TLR-4⁺ Cells.

BACKGROUND An increasing number of studies have demonstrated that Streptococcus bovis and its concomitant inflammatory factors concentrate in the intestine in colorectal cancer (CRC). However, the molecular mechanism of S. bovis on colorectal tumorigenesis remains unclear. This study aimed to explore the role of S. bovis in carcinogenesis and its potential mechanism in CRC of mice orally pretreated with S. bovis. MATERIAL AND METHODS The colons of experimental mice were collected and evaluated for the extent of neoplasm. In addition, comparative feces DNA sequencing was adopted to verify the abundance change of S. bovis during the progression of CRC in patients. RESULTS The results of this study found that S. bovis is more likely to be present at higher levels in patients with progressive colorectal carcinoma compared to those adenoma patients and healthy volunteers (P<0.05). Pretreatment with S. bovis aggravated tumor formation in mice, resulting in more substantial and a higher number of tumor nodes (P<0.05). A cytokine expression pattern with increased levels of IL-6, Scyb1, Ptgs2, IL-1ß, TNF, and Ccl2 was detected in S. bovis pretreated CRC mice (all P<0.05). Furthermore, S. bovis recruited myeloid cells, especially CD11b⁺TLR-4⁺ cells, which could promote pro-tumor immunity in the tumor microenvironment (P<0.05). CONCLUSIONS Collectively, our study indicates that S. bovis may induce a suppressive immunity that is conducive to CRC by recruiting tumor-infiltrating CD11b⁺TLR-4⁺ cells. In conclusion, S. bovis contributes to colorectal tumorigenesis via recruiting CD11b⁺TLR-4⁺ cells.

Effects of the oral administration of viable and heat-killed Streptococcus bovis HC5 cells to pre-sensitized BALB/c mice.

Antimicrobial peptides have been suggested as an alternative to classical antibiotics in livestock production and bacteriocin-producing bacteria could be added to animal feeds to deliver bacteriocins in the gastrointestinal (GI) tract of ruminant and monogastric animals. In this study, viable (V) and heat-killed (HK) Streptococcus bovis HC5 cells were orally administered to pre-sensitized mice in order to assess the effects of a bacteriocin-producing bacteria on histological parameters and the immune response of the GI tract of monogastric animals. The administration of V and HK S. bovis HC5 cells during 58 days to BALB/c mice did not affect weight gain, but an increase in gut permeability was detected in animals receiving the HK cells. Viable and heat killed cells caused similar morphological alterations in the GI tract of the animals, but the most prominent effects were detected in the small intestine. The oral administration of S. bovis HC5 also influenced cytokine production in the small intestine, and the immune-mediated activity differed between V and HK cells. The relative expression of IL-12 and INF-γ was significantly higher in the small intestine of mice treated with V cells, while an increase in IL-5, IL-13 and TNF-α expression was only detected in mice treated with HK cells. Considering that even under a condition of severe challenge (pre-sensitization followed by daily exposure to the same bacterial immunogen) the general health of the animals was maintained, it appears that oral administration of S. bovis HC5 cells could be a useful route to deliver bacteriocin in the GI tract of livestock animals.

Increased exposure to bacterial antigen RpL7/L12 in early stage colorectal cancer patients.

BACKGROUND: Cancer 2010. (c) 2010 American Cancer Society. : Intestinal bacteria have long been implicated in colorectal cancer pathology, and many reports point to a close linkage between Streptococcus bovis biotype I (recently renamed Streptococcus gallolyticus) infections and tumors of the human colon. This work aims to investigate the humoral immune response to this bacterium during different stages of colorectal cancer. METHODS: The presence of serum antibodies against S. bovis antigen RpL7/L12, previously assigned as a potential diagnostic antigen, was evaluated in Dutch (n = 209) and American (n = 112) populations using a newly developed enzyme-linked immunosorbent assay. RESULTS: The analyses consistently showed that an immune response against this bacterial antigen was increased in polyp patients and stage I/II colorectal cancer patients as compared with asymptomatic individuals. This was not paralleled by increased antibody production to endotoxin, an intrinsic cell wall component of the majority of intestinal bacteria, which implies that the humoral immune response against RpL7/L12 is not a general phenomenon induced by the loss of colonic barrier function. Notably, increased anti-RpL7/L12 levels were not or were only mildly detected in late stage colorectal cancer patients having lymph node or distant metastasis. CONCLUSIONS: Cancer 2010. (c) 2010 American Cancer Society. : These findings are indicative of an increased exposure to antigen RpL7/L12 during early stages of colon carcinogenesis and suggest that intestinal bacteria such as S. bovis constitute a risk factor for the progression of premalignant lesions into early stage carcinomas. Clearly, the current findings emphasize the necessity for further studies on the possible etiologic relationship between intestinal bacteria and human colorectal cancer.

Effects of feeding polyclonal antibody preparations on rumen fermentation patterns, performance, and carcass characteristics of feedlot steers.

In a previous study, preparations of polyclonal antibodies (PAP) against Fusobacterium necrophorum (PAP-Fn) or Streptococcus bovis (PAP-Sb) were successful in decreasing ruminal counts of target bacteria and increasing ruminal pH in steers fed high-grain diets. The objective of this study was to evaluate the effects of feeding PAP-Fn or PAP-Sb on performance, carcass characteristics, and ruminal fermentation variables of feedlot steers. In Exp. 1, during 2 consecutive years, 226 or 192 Angus and Angus crossbred steers were fed a high-grain diet containing either PAP-Sb or PAP-Fn, or both. When measured on a BW basis, steers fed only PAP-Sb had a greater G:F (P < 0.05) than those fed no PAP. Nevertheless, when both PAP were fed, feed efficiency was similar (P > 0.10) to steers fed no PAP or only PAP-Sb. Steers receiving PAP-Fn (alone or in combination with PAP-Sb) had a decreased (P < 0.05) dressing percentage. Steers receiving PAP-Fn (alone or in combination with PAP-Sb) had a decreased severity of liver abscess (P < 0.05). No differences (P > 0.10) were observed in any other carcass characteristics. In Exp. 2, sixteen ruminally cannulated Angus crossbred steers (BW = 665 +/- 86 kg) were fed a high-grain diet containing either PAP-Sb or PAP-Fn, or both. Feeding only PAP-Fn or PAP-Sb for 19 d decreased (P < 0.05) ruminal counts of S. bovis when compared with steers fed both or no PAP. The ruminal counts of F. necrophorum in steers fed PAP-Fn alone or in combination with PAP-Sb were decreased by 98% (P < 0.05) after 19 d, when compared with the counts in control steers. Mean daily ruminal pH was greater (P < 0.05) in steers fed both PAP when compared with feeding either or no PAP. Ruminal pH in the first 4 h after feeding was greater (P < 0.05) for steers receiving PAP-Fn alone or in combination with PAP-Sb. Steers receiving either PAP alone or in combination had less (P < 0.05) ruminal NH(3)-N concentrations in the first 4 h after feeding when compared with those of control steers. Polyclonal antibody preparations against S. bovis were effective in enhancing G:F of steers fed high-grain diets, but dressing percentage was decreased. Mechanisms of enhancement of G:F remain unknown but may be related to changes in ruminal counts of target bacteria and associated effects on ruminal fermentation products.

Effects of feeding polyclonal antibody preparations on ruminal bacterial populations and ruminal pH of steers fed high-grain diets.

Three experiments with factorial arrangements of treatments were designed to test the efficacy of avian-derived polyclonal antibody preparations (PAP) against Streptococcus bovis (PAP-Sb) or Fusobacterium necrophorum (PAP-Fn) in reducing ruminal counts of target bacteria in beef steers supplemented or not with feed additives (300 mg of monensin/d and 90 mg of tylosin/d; MT). Feeding increasing doses of PAP-Sb in Exp. 1 or a single dose in Exp. 2 reduced S. bovis counts in a cubic fashion (P = 0.014). In Exp. 1 and 2, inclusion of MT in the diet had no effect (P > 0.05) on ruminal S. bovis counts. In Exp. 2, ruminal pH was increased (P < 0.05) by feeding PAP-Sb, MT, and PAP-Sb plus MT. Ruminal F. necrophorum counts were reduced by feeding PAP-Fn (P = 0.002) and MT (P < 0.001). Reduction in ruminal F. necrophorum counts was greater (P = 0.008) when feeding MT alone than when feeding PAP-Fn and MT together. In Exp. 3, ruminal S. bovis counts were not affected (P = 0.64) by PAP-Fn. Ruminal pH was not affected (P = 0.61) by feeding PAP-Fn, and the total anaerobic bacterial count was not affected (P > 0.05) by either PAP-Sb or PAP-Fn in Exp. 1 or Exp. 3. In conclusion, PAP of avian origin and against S. bovis or F. necrophorum were effective in reducing target ruminal bacterial populations. These PAP could be effective in preventing the deleterious effects associated with these bacteria, and possibly in enhancing animal performance.

Profiling the humoral immune response in colon cancer patients: diagnostic antigens from Streptococcus bovis.

The human bowel contains a large and dynamic bacterial population that is not only essential for intestinal health, but also critical for the development of diseases such as cancer. In this respect, the Gram-positive bacterium Streptococcus bovis has been associated with colon cancer for many years. To investigate the clinical importance of this association, an immunocapture mass spectrometry assay was developed that can generate infection-related protein profiles. The composition of these profiles is governed by the capture of specific antigens by serum antibodies from colon cancer patients. This assay showed that S. bovis antigen profiles could distinguish 11 out of 12 colon cancer patients from 8 control subjects, whereas antigen profiles derived from the gut bacterium Escherichia coli were not diagnostic for colon cancer. Moreover, S. bovis antigen profiles were also detected in polyp patients, indicating that infection with this bacterium does occur early during carcinogenesis. Highly accurate tandem mass spectrometry was used to identify one of the diagnostic antigens as a surface-exposed heparin-binding protein, which might be involved in attachment of S. bovis to tumor cells. Together, these findings corroborate the hypothesis that colonic lesions provide a specific niche for S. bovis, resulting in tumor-associated "silent" infections. These infections, however, only become apparent in colon cancer patients with a compromised immune system (bacteremia) or coincidental cardiac valve lesions (endocarditis). This makes profiling of the humoral immune response against "silent" S. bovis infections a promising diagnostic tool for the early detection of human colon cancer, which is crucial for the effective treatment of this disease.

Antibody response in sheep following immunization with Streptococcus bovis in different adjuvants.

Recent studies have shown that immunization with Streptococcus bovis using Freund's complete adjuvant (FCA) may confer protection against lactic acidosis in sheep. The major objective of this study was to compare the antibody responses to S. bovis in a practically acceptable adjuvant (Freund's incomplete adjuvant (FIA); QuilA; dextran sulphate (Dex); Imject Alum; or Gerbu) and in FCA. Thirty-five sheep were randomly allocated to 7 treatment groups. Six groups were immunized with S. bovis in an adjuvant; the other group served as the non-immunization control. The primary immunization was administered intramuscularly on day 0. followed by a booster injection on day 28. Immunization with FCA induced the highest saliva and serum antibody responses. The saliva antibody concentrations in the FIA and QuilA groups were significantly higher than those in the Alum, Dex and Gerbu groups (p < 0.01). The serum antibody concentration in the FIA group was significantly higher than those in the QuilA, Alum. Dex and Gerbu groups (p < 0.01). Immunization enhanced the antibody level in faeces (p < 0.05), but there was no significant difference between the different adjuvant groups (p > 0.05). Seven and 14 days following booster immunization, the saliva antibody levels induced by QuilA and/or FIA were comparable with the level stimulated by FCA (p > 0.05). There was a strongly positive correlation (R2 = 0.770, p < 0.01) between the antibody concentrations in salival and serum. Compared with the controls, a higher faecal dry matter content was observed in the animals immunized with either FCA or QuilA. The change in faecal dry matter content was positively associated with the faecal antibody concentration (R2 = 0.441, p < 0.05). These results indicate that FIA and QuilA were effective at inducing high levels of antibody responses to S. bovis, and suggest that either Freund's incomplete adjuvant or QuilA may be useful for preparing a practically acceptable vaccine against lactic acidosis.

Effects of various adjuvants on efficacy of a vaccine against Streptococcus bovis and Lactobacillus spp in cattle.

OBJECTIVE: To determine efficacy of vaccines incorporating QuilA, alum, dextran combined with mineral oil, or Freund adjuvant for immunization of feedlot cattle against Streptococcus bovis and Lactobacillus spp. ANIMALS: 24 steers housed under feedlot conditions. PROCEDURE: Steers were randomly assigned to 4 experimental groups and a control group. Animals in experimental groups were inoculated on days 0 and 26 with vaccines containing Freund adjuvant (FCA), QuilA, dextran combined with mineral oil (Dex), or alum as adjuvant. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were measured, along with fecal pH, ruminal fluid pH, and number of S bovis and Lactobacillus spp in ruminal fluid. RESULTS: Throughout the study, serum anti-S bovis and anti-Lactobacillus IgG concentrations for animals in the Dex, QuilA, and alum groups were similar to or significantly higher than concentrations for animals in the FCA group. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were significantly increased on days 26 through 75 in all 4 experimental groups, and there was a linear relationship between anti-S bovis and anti-Lactobacillus IgG concentrations. For animals in the QuilA and Dex groups, mean pH of feces throughout the period of experiment were significantly higher and numbers of S bovis and Lactobacillus spp in ruminal fluid on day 47 were significantly lower than values for control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that immunization of feedlot steers against S bovis and Lactobacillus spp with vaccines incorporating Freund adjuvant, QuilA, dextran, or alum as an adjuvant effectively induced high, long-lasting serum anti-S bovis and anti-Lactobacillus IgG concentrations. Of the adjuvants tested, dextran may be the most effective.

Immunization with a Streptococcus bovis vaccine administered by different routes against lactic acidosis in sheep.

Streptococcus bovis is an important lactic acid bacterium in the rumen, which contributes to the development of lactic acidosis. This study was designed to test the efficacy of immunization with S. bovis primed either intramuscularly (i.m.) or intraperitoneally (i.p. ) against lactic acidosis. Forty-five wethers were allocated to three treatment groups. Two groups were injected with a S. bovis vaccine by either the i.m. or i.p. route for primary immunization; both groups were further immunized by the same route(s) (oral and/or i.m.) for boosters. The third group was not immunized (control). Antibody concentrations were measured in saliva prior to and following animals being fed a grain diet, and also in the rumen fluid, before the animals were suddenly introduced to a grain diet. The average antibody concentration in the animals of the i.m. group was higher than the i.p. group (P< 0.05). The antibody concentration in the rumen fluid of immunized sheep was higher than the control animals (P< 0.01). The difference in the rumen fluid antibody concentration between the i.m. and i.p. groups was not statistically significant (P> 0.05). In the i.m. group, there was a significantly greater feed intake, higher rumen pH, lower diarrhoea scores, and less increase in blood packed cell volume following grain feeding than in the animals of the control group. The severity of diarrhoea and the increase of blood packed cell volume in the animals of the i. p. group were also less than in the animals of the control group. The results suggest that the risk of lactic acidosis can be reduced by immunization against S. bovis, and that the immunization primed i. m. is more effective than the immunization primed i.p.

Immunization with Streptococcus bovis protects against lactic acidosis in sheep.

Lactic acidosis is a gastrointestinal disorder resulting from the rapid overgrowth of lactic acid-producing bacteria when ruminants are suddenly introduced to grain feed. The present study has investigated the ability of live and killed bacterial vaccines to reduce lactic acidosis in sheep, via a stimulation of specific antibody production against lactic acid-producing bacteria. Forage-fed sheep were immunized with live or killed Streptococcus bovis Sb-5 vaccine, with or without adjuvant, via intramuscular injection. After the primary immunization, three boosters were given at 2-4 week intervals. Sheep were subsequently challenged by a sudden switch to a grain-based diet. Following challenge, vaccinated sheep maintained significantly higher feed intake, and had higher rumen pH, lower L-lactate concentrations, and less severe diarrhoea scores than non-vaccinated control sheep. Higher rumen pH, lower mortality and less severe diarrhoea were found in the animals immunized with live vaccine compared to the animals immunized with killed vaccines. Significant increases in mucosal and systemic antibody responses were observed after boosting; the S. bovis-specific antibody concentrations were significantly higher in samples of saliva, rumen fluid and serum from sheep immunized with live vaccine than with killed vaccines. These results demonstrate that lactic acidosis can be reduced by immunization against S. bovis, and that live Sb-5 vaccine is effective in invoking mucosal as well as systemic antibody responses.

Identification of virulence associated markers in the cell wall of pigeon Streptococcus gallolyticus strains.

The cell wall protein profiles of 56 isolates of Streptococcus gallolyticus of differing virulence for pigeons were compared by SDS-PAGE. Additionally, Western blot analysis was performed on the cell wall proteins of 14 strains using sera of pigeons, experimentally infected with A(+)T1 or A(-)T2 strains of S. gallolyticus. The profile of silver stained gels exhibited a complex array of 20-50 bands ranging from less than 6.5-210kDa. A band with molecular mass of 114kDa was only observed in isolates that belonged to the highly virulent A(+)T1, A(+)T2, A(+)T3 and A(-)T1 culture supernatant groups. A band with a slightly higher molecular mass (115kDa) as well as a 207kDa band were only detected in isolates that belonged to the moderately A(-)T3 or low A(-)T2 virulent culture supernatant groups. The 114 and 115kDa band were recognised by all homologous and heterologous pigeon sera used whereas the 207kDa band was only recognised by sera of pigeons infected with a A(-)T2 strain. These findings may indicate that the 114, 115 and 207kDa bands are useful as additional virulence associated markers for pigeon S. gallolyticus strains.

Immunological cross-reactivity between the vaccine and other isolates of Streptococcus bovis and Lactobacillus.

Recent studies have showed that immunisation with Streptococcus bovis (Sb-5) and Lactobacillus (LB-27) may confer protection against lactic acidosis in sheep and cattle. The present study was designed to determine the degree of immunological cross-reactivity between Sb-5 and eight other strains of Streptococcus bovis; and between LB-27 and four other isolates of Lactobacillus. The cross-reactivity index (CRI, a low CRI indicates a high degree of immunological cross-reactivity) ranged from 7.3 to 56.1% between the strains of S. bovis (the encapsulated strains with CRIs ranging from 7.3 to 12.4%). For isolates of Lactobacillus the CRIs ranged from 11.5 to 72.2%. The results indicate that all the isolates tested have a certain degree of immunological homology with Sb-5 and LB-27, and suggest that the vaccine may cross-react with a large number of strains of S. bovis and Lactobacillus which may cause lactic acidosis. As most of the S. bovis strains in the rumen are encapsulated, the high degree of homology between Sb-5 and encapsulated S. bovis strains further suggests that the vaccine containing Sb-5 may be effective against a wide range of strains of S. bovis in sheep and cattle.

Immunisation against lactic acidosis in cattle.

The present study was designed to investigate the efficacy of control of lactic acidosis by immunisation against lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus. Ten steers were allocated to two treatment groups. One group was immunised with a vaccine containing S. bovis (strain Sb-5) and Lactobacillus (LB-27) cells, and the other was a non-immunised control group. The vaccine, using Freund's complete adjuvant for primary immunisation and Freund's incomplete adjuvant for boosters, was administered intramuscularly. After primary immunisation, boosters were given at 2 to 4 week intervals. Both anti- S. bovis and anti- Lactobacillus IgG levels in saliva increased significantly (P < 0.01) after the 1st booster which were lower (P < 0.05) than the IgG levels after the 2nd and 3rd boosters, but were not significantly different (P > 0.05) from the IgG levels prior to a grain challenge (after the 4th booster). There were positive correlations between the anti- S.bovis and anti- Lactobacillus IgG in serum and saliva. Compared with the control group, steers in the immunised group had higher (P < 0.05) feed intakes, lower (P < 0.05) rumen concentrations of lactate and lower numbers of S. bovis and Lactobacillus. Three of the control animals were withdrawn from the grain challenge due to their rumen pH persisting below 5.2, while only one animal in the immunised group was withdrawn. These results suggest that the risk of lactic acidosis can be reduced by immunisation against S. bovis and Lactobacillus.

Immunity in pigeons against homologous and heterologous serotypes of Streptococcus bovis after infection.

Groups of 20, 20 and 10 pigeons were intravenously inoculated with 1 x 10(9) CFU of a Streptococcus bovis serotype 1, 2 and 3 strain, respectively. Groups which received the highly virulent serotypes 1 or 2 strains were treated with antibiotics starting from 2 days post inoculation in order to prevent the development of clinical signs. Pigeons inoculated with the low virulence S. bovis serotype 3 strain were left untreated. Another group of 10 non-inoculated pigeons was used as challenge control. Four weeks later, pigeons were challenged intravenously with S. bovis serotype 1. Morbidity after challenge infection was 19%, 36% and 70% in groups previously inoculated with serotypes 1, 2 and 3, respectively, whereas it was 100% in the control group. Results demonstrate that pigeons developed significant protective immunity to S. bovis serotype 1 septicaemia following infection with S. bovis serotype 1 and 2 strains but not after infection with the serotype 3 strain. Protection did not correlate with the presence of antibodies as detected in ELISA.

Serological investigation into the association between Streptococcus bovis and colonic cancer.

AIMS: To determine if there was an increase in antibody titre to Streptococcus bovis in patients with colonic cancer, and if this might be a useful marker of the presence for colonic cancer. METHODS: Serum samples from 16 patients and 16 age matched controls were tested by immunoblot and enzyme linked immunosorbent assay (ELISA) against antigen preparations from two strains of S bovis and one strain of Enterococcus faecalis. RESULTS: No distinction between cancer patients and controls could be made using immunoblots. ELISA did show an increase in antibodies to S bovis, but there was a greater increase in antibodies to E faecalis. The increase in antibody titres was greatest with antigens which had been treated with periodate, and was therefore thought not to be caused by antibody to the shared group D carbohydrate antigen. CONCLUSION: It may be possible to construct a test for the detection of colonic cancer based on the detection of antibody to S bovis or E faecalis, though considerable further development of this concept is required.

Prevalence of antibodies to Streptococcus bovis serotype 1 in racing pigeons.

Indirect ELISA techniques for the detection of antibodies to S. bovis serotypes 1, 2 and 3 in pigeon plasma were developed. Whole formaldehyde-inactivated bacteria were used as coating antigens. Bound antibodies were detected with peroxidase-conjugated rabbit anti-pigeon serum and OPD and H2O2 as chromogen and substrate, respectively. ELISA was used to determine the prevalence of antibodies to S. bovis serotype 1 in healthy Belgian racing pigeons. Antibodies were demonstrated in 83 (= 37%) of 225 samples tested. Results indicate that pigeons may build up plasma antibodies in the absence of clinical signs resembling septicaemia.

Adherence of glucan-positive and glucan-negative strains of Streptococcus bovis to human epithelial cells.

Adherence to buccal epithelial cells (BEC) and the role played in the binding by lipoteichoic acid (LTA) and other superficial components have been studied in reference and clinical strains of Streptococcus bovis either glucan-positive biotype I or glucan-negative biotype II. To avoid the synthesis of glucan by biotype I strains, adherence was studied in bacteria grown in Todd-Hewitt broth, a sucrose deficient medium. Both biotypes were shown to bind to BEC and clinical isolates, irrespective of biotype attached to the same degree but in greater numbers than reference strains. Inhibition studies suggest that at least two mechanisms,--LTA and protein-mediated--are responsible for the adherence of both glucan-positive and negative strains of S. bovis. Moreover, in glucan-positive strains capsular polysaccharides may be also involved.

Biochemical and antigenic properties of Streptococcus bovis isolated from pigeons.

Biochemical and serological properties of 60 strains of Streptococcus bovis isolated from healthy pigeons and from pigeons that died from S. bovis septicemia were determined. On the basis of the hemolysis of bovine erythrocytes, the production of polysaccharides on saccharose-containing media, and the fermentation of mannitol, inulin, trehalose, and L-arabinose, the isolates were classified in five biotypes and two subbiotypes. Slide agglutination and microagglutination tests using monospecific rabbit antisera allowed the classification of all isolates in five serotypes.