Effects of two probiotic spores of Bacillus species on hematological, biochemical, and inflammatory parameters in Salmonella Typhimurium infected rats.

Salmonella infections have become a major health concern in recent decades. This pathogen has evolved to become resistant to antibiotics, which has caused problems in its treatment. As such, finding a novel preventive method is important in the treatment and management of this infection. In recent years, uses of probiotics, especially spore-former genera such as Bacillus spp. has become increasingly popular. In this study spores of two probiotic bacteria, Bacillus subtilis and Bacillus coagulans were fed to rats for three weeks through their daily water intake after which Salmonella Typhimurium was gavaged to the rats. On days 1, 3, 5 and 7 after gavaging, the number of Salmonella was counted in liver, spleen, mesenteric lymph nodes, feces and content of ileum and cecum. Hematological and biochemical parameters, inflammatory mediators, total antioxidant capacity and malondialdehyde were also measured. The results showed that B. subtilis and B. coagulans caused delation in infiltration of Salmonella into the lymph nodes, spleen and liver, reduction of the inflammatory mediators, and decreases in oxidative stress, hematological and biochemical changes. The overall count of Salmonella in the above mentioned parameters has also decreased and a faster return to normal base were also witnessed. The results showed that the use of B. subtilis and B. coagulans can potentially help boost the body’s immune system, to combat the effects of exposure to the Salmonella pathogen.

. Number of Salmonella (Log CFU/gr) in liver, spleen and mesenteric lymph nodes of rats with different treatments in 1, 3, 5 and 7 days after gavaging Salmonella. Values are mean ± standard deviation of 5 independent replicates. The different small letters in columns indicate significant differences among groups (P < 0.05). The different capital letters in each row for each parameter indicate significant differences among post infectious sampling days (P < 0.05).   Table 2. Number of Salmonella (Log CFU/gr) in the ileum, cecum and feces of rats with different treatments in 1, 3, 5 and 7 days after gavaging Salmonella. Values are mean ± standard deviation of 5 independent replicates. The different small letters in columns indicate significant differences among groups (P < 0.05). The different capital letters in each row for each parameter indicate significant differences among post infectious sampling days (P < 0.05).

Groups
As previous studies have shown, probiotics have the beneficial effect of preventing the colonization of pathogenic bacteria in the digestive tract, competition for nutrients and adhesion receptors, and stimulation of host immunity 20,21 . As the number of Salmonella was at the highest levels on the 5 th day post gavaging and the differences between the two groups, it can be said that B. subtilis and B. coagulans reduce the S. Typhimurium colonization and invasion. Vila et al. (2009)  Hematological parameters. The results showed that the packed cell volume (PCV) percentage, red blood cell (RBC) count (until the 5 th day) and hemoglobin (Hb) concentration (until the 3 rd day) were greater in the Salmonella group than the other groups (P < 0.05). While, these values returned to the normal range by day 7 (Table 3). These increments were a result of severe dehydration in the Salmonella group, which occurred due to diarrhea 22 . In Salmonella group, the stool was looser than the Blank Control group for 3 days after gavaging. Previous studies have shown that the Salmonella Typhimurium increase the Hb, PCV and RBC amount of piglets 23 and calves 24 . In the Salmonella group the RBC count had a decreasing trend from the 1 st day to the 7 th day of infection (P < 0.05). However, in the Probiotic+Salmonella group there was no increase in the PCV, RBC count and Hb concentration and these parameters were similar to the Blank Control group. In Probiotic+Salmonella group, Salmonella showed lower pathogenicity due to the presence of active probiotics that prevented the colonization of Salmonella and competed for nutrients and adhesion receptors 20,21 .
The results show that no significant difference was observed in platelet counts (P > 0.05) (Data not shown). The number of white blood cell (WBC) and neutrophils in the Salmonella group was significantly higher than the other groups from the first day after gavaging. The WBC and neutrophils count were increased during three days of infection and then started to come down from day 5 onwards. The use of probiotics before gavaging Salmonella, reduced the increasing trend of the WBC and neutrophils levels and eventually brought up the levels of these two indicators to the normal range by day 5. On day 7, there was no significant differences in the number of WBC and neutrophils between Salmonella and Probiotic+Salmonella groups (P > 0.05). The counts were greater than Blank Control and Probiotic Control groups ( Table 4).
The introduction of Salmonella significantly increased the number of lymphocytes compared to the Blank Control group (P < 0.05). While using probiotics before gavaging Salmonella, significantly reduced the increasing trend of lymphocytes (P < 0.05). These results held true from days 1 to 5 but on the 5 th day, no significant difference between Salmonella and Probiotic+Salmonella groups (P > 0.05) were observed. On the 7 th day, the number of lymphocyte in Probiotic+Salmonella group returned to normal levels and there were no differences in levels compared to the Blank Control group. However, the Salmonellal group had greater levels of lymphocytes compared to the other three groups (P < 0.05) ( Table 4).
No significant differences were observed in the levels of monocytes, eosinophils and basophils among the four treatment groups (P > 0.05) (Data not shown).
The correlation coefficient between the number of Salmonella in the ileum, cecum, feces, liver, spleen and mesenteric lymph nodes with the number of WBC was 0.32, 0. 36 Table 3. The PCV, Hb and RBC of blood samples of rats with different treatments in 1, 3, 5 and 7 days after gavaging Salmonella. Values are mean ± standard deviation of 5 independent replicates. The different small letters in columns indicate significant differences among groups (P < 0.05). The different capital letters in each row for each parameter indicate significant differences among post infectious sampling days (P < 0.05).
www.nature.com/scientificreports www.nature.com/scientificreports/ In this experimental study, the number of WBC in the Salmonella group was significantly more than other groups from the first day after gavaging due to the increase in neutrophiles and lymphocytes. This was also confirmed before that Salmonella caused neutrophile and lymphocyte increases resulted in the WBC count increase 24,25 . Also, the increase in the number of Salmonella in the ileum, cecum, feces, liver, spleen, and mesenteric lymph nodes were accompanied by an increase in the number of WBC, neutrophils and lymphocytes (P < 0.05). However, in the Probiotic+Salmonella group, these levels rose after a delay. These results would indicate that B. subtilis and B. coagulans decrease the effects of S. Typhimurium. Additionally, in the Probiotic+Salmonella group, the complications from S. Typhimurium are seen in a shorter period of time and the changes returned to the normal base quicker than the Salmonella group. Havelaar et al. (2001) showed that S. Enteritidis in rats cause detectable changes in neutrophile counts from doses of 10 4 CFU upwards and large increases in WBC and neutrophils levels were observed after five days. These results showed that all leukocyte types, except eosinophils, have positive dose response relationships 26 .

Biochemical parameters. There was no significant difference in biochemical parameters between Blank
Control and Probiotic Control groups (P > 0.05). The Salmonella infection increased the total protein and globulin amounts in the blood (P < 0.05), but had no significant effect on blood serum albumin (P > 0.05). Consumption of probiotics in Salmonella group, significantly reduced the total protein and globulin levels (P < 0.05) and brought these levels close to the control values ( Table 5). The amount of globulin and total protein on the 1 st , 3 rd and 5 th days was significantly higher in the Salmonella group compared to the Probiotic+Salmonella group, and both were higher than the Blank Control group. On the 7 th day with Probiotic+Salmonella group, the amount of globulin and total protein was similar to the Blank Control group. Oral inoculation of Salmonella, stimulates the immune responses, including humoral and cell-mediated responses, which increases the serum level of immunoglobulin and leads to an increase in total protein 27 . As the results show, no differences were observed in the amount of albumin among the groups. Therefore, the changes in the total protein are due to the change in the amount of globin levels. The increase in the number of Salmonella in the liver, spleen and mesenteric lymph nodes were accompanied by serum globulin and total protein increases (P < 0.05). The correlation coefficient between the number of Salmonella in the liver, spleen and mesenteric lymph nodes with the amount of total protein of blood was 0.25, 0.28 and 0.35; globulin was 0.25, 0.35 and 0.43, respectively. The B. subtilis and B. coagulans reduce the globulin levels and also the total protein levels in comparison to the Salmonella group. This indicates that B. subtilis and B. coagulans reduce the pathogenic effect of S. Typhimurium. Abudabos et al. (2016) showed that Bacillus as probiotic had no effect on total protein, globulin and albumin levels. When exposed to Groups WBC (10 4 /μL) Neutrophile (10 3 / μL) Lymphocyte (10 3 /μL) www.nature.com/scientificreports www.nature.com/scientificreports/ Salmonella, probiotics inhibit albumin decreases and globulin increases which resulted in positive control of Salmonella 28 .
There was no significant difference in the amount of alkaline phosphatase (ALP) between four groups during the sampling days (P > 0.05) (Data not shown). No significant differences were observed in the amount of alanine transaminase (ALT) and aspartate transaminase (AST) among four groups until the 5 th day of sampling (P > 0.05), while, on the 7 th day, Salmonella caused a significant increase in the ALT and AST activity (P < 0.05). Probiotic consumption kept the level of these two enzymes in the normal range throughout the sampling period (Table 6). Increase in ALT and AST on the 7 th day post oral inoculation in Salmonella group, indicate the inflammation and damage in the liver 29 . However, in the Probiotic+Salmonella group, no increase was observed, which means B. subtilis and B. coagulans inhibit the pathogenic effect of S. Typhimurium and inflammation in the liver. Also lesser changes in hepatic factor in Probiotic+Salmonella group is due to the lesser count of Salmonella in this group in comparison to Salmonella group.
During infections, S. Typhimurium disseminates systemically from the Peyer's patches to the liver and spleen, where it continues to grow within macrophages 30,31 . Glycolysis is required for intracellular replication and survival of S. Typhimurium in macrophages 32 . The lower glucose could be due to a greater blood glucose uptake when the metabolism is responding against the Salmonella infection 23 . In the Salmonella group, the glucose amounts increased on the 1 st and 3 rd days post inoculation (Table 6), but stayed within the normal range in the Probiotic+Salmonella group, which means B. subtilis, and B. coagulans reduce the effects of Salmonella. The increase in the number of Salmonella in the ileum, cecum, feces, liver and spleen was accompanied with serum glucose reduction (P < 0.05). The correlation coefficient between the number of Salmonella in the ileum, cecum, feces, liver and spleen with the serum glucose was -0.28, -0.31, -0.26, -0.22 and -0.22 in order (P < 0.05). The Salmonella infection increased the level of lactate dehydrogenase (LDH) significantly (P < 0.05). LDH is recognized as a marker for cell toxicity and the lower amount indicates lower cell injuries 33 . The use of probiotics in Salmonella infected rats, lead to smaller increment in the LDH level, however, it was still higher than the Blank Control and Probiotic Control groups (P < 0.05) ( Table 7). This indicates that B. subtilis and B. coagulans decrease the cell injuries resulted by Salmonella. The increase in the number of Salmonella in the ileum, cecum, feces, liver, spleen, and mesenteric lymph nodes were accompanied by LDH increases (P < 0.05).
In the Salmonella group, creatine levels increased from the 5 th day of gavaging. The same trend was seen in the blood urea nitrogen (BUN) on the 5 th and 7 th days of sampling, which indicated kidney dysfunction. It was also mentioned by Doorn et al. (2006) that in Salmonella infection, renal dysfunction occurred and this causes BNU and Creatinin increases 34 . In the Probiotic+Salmonella group, the amount of creatine and BUN were similar to the Blank Control and Probiotic Control groups (P > 0.05) on all sampling days (Table 7). It indicates that B. subtilis and B. coagulans inhibit the pathogenic effect of S. Typhimurium on kidneys.  www.nature.com/scientificreports www.nature.com/scientificreports/ The increase in the number of Salmonella in the liver, spleen and mesenteric lymph nodes were accompanied with BUN and creatine increases (P < 0.05). The correlation coefficient between the number of Salmonella in the liver, spleen and mesenteric lymph nodes with the amount of creatine was 0.28, 0.40 and 0.45; and BUN was 0.41, 0.59 and 0.53, respectively (P < 0.05).
The level of total iron in the Salmonella group increased significantly on days 1, 3 and 5, and then returned to the normal range on the 7 th day. S. Typhimurium secretes siderophores to bind to the ferric iron 35 . However the use of probiotics maintained the total blood iron level within normal range in all sampling days (Table 7). In the Probiotic+Salmonella group, it has been higher just on the 1 st day and returned to the normal range after that. This would suggest that, however Salmonella causes an increase in the amount of serum iron, but probiotics inhibit this mechanism. Geith et al. (2015) reported beneficial role of Lactobacillus acidophilus on the modification of acute phase parameters such as decreased fibrinogen, ESR, TIBC (total iron-binding capacity), UIBC (unsaturated iron-binding capacity), and ceruloplasmin and on the other hand increased albumin, total protein, iron, and transferrin saturation percentage during the S. Typhimurium infection in albino rats 4 .

Inflammatory mediators determination.
The level of inflammatory mediators, including TNF-α, serum amyloid A (SAA) and IL-10 in the Probiotic Control group and the Blank Control group was similar (P > 0.05). Salmonella infection significantly increased the level of TNF-α and SAA in all days of sampling (P < 0.05). With the Probiotic+Salmonella group, the level of these two inflammatory mediators significantly decreased compared  www.nature.com/scientificreports www.nature.com/scientificreports/ to the Salmonella group (P < 0.05), but was still higher than the Blank Control and Probiotic Control groups (P < 0.05) (Figs. 1 and 2).
Salmonella infection significantly increased IL-10 levels in all days compared to the Blank Control group (P < 0.05), while probiotic consumption prevents the increase of IL-10 level up to 3 rd day after Salmonella gavaging. However, on day 5, the level of IL-10 increased significantly and decreased again on the 7 th day compared to the Blank Control group (Fig. 3). The correlation coefficient between the number of Salmonella in the ileum, cecum, feces, liver, spleen and mesenteric lymph nodes with the level of TNF-α was 0. Systemic infection and bacterial colonization in the intestine induce SAA proteins in the liver 36 . TNF-α is produced early in the response to Lipopolysaccharide (LPS) and is believed to be a major mediator of the diverse pathophysiologic responses to LPS 37 . Also, IL-10 works as an essential molecule to allow S. Typhimurium infection by reducing the soluble inflammatory determinants that can activate both innate and adaptive immune responses that restrict S. Typhimurium dissemination 38 . In the current study, S. Typhimurium caused the increase in IL-10, TNF-α and SAA but B. subtilis and B. coagulans decrease these parameters. These probiotics reduce the number of Salmonella in the intestine and inhibit the colonization of this pathogen. Also, lower LPS levels are witnessed resulting in SAA, TNF-α and IL-10 showing lower changes in comparison to the Salmonella group. The decrease in the number of Salmonella in the ileum, cecum, feces, liver, spleen and mesenteric lymph nodes was accompanied with SAA, TNF-α and IL-10 reductions (P < 0.05).

Total antioxidant capacity (TAC). Salmonella infection reduced the amount of TAC significantly
(P < 0.05), while the use of probiotics during exposure to Salmonella, increased the TAC (P < 0.05) (Fig. 4). The correlation coefficient between the number of Salmonella in the ileum, cecum, feces, liver, spleen and mesenteric lymph nodes with the amount of TAC was -0.36, -0.37, -0.34, -0.39, -0.43 and -0.48 in order (P < 0.05).
During the inflammatory processes, the phagocyte activation and bacterial products enhance the multicomponent flavoprotein NADPH oxidase, which catalyzes the production of high amounts of the superoxide anion radicals. As a result, the body requires adequate levels of antioxidant to avoid the reactive oxygen species harmful effects. Hence, antioxidants are necessary to regulate the reactions that release free radicals 39 . S. Typhimurium enhances oxidative stress. LPS of S. Typhimurium can induce H 2 O 2 generation and a concomitant decline in TAC 40 . In this study, the TAC decreased more in the Salmonella group than Probiotic+Salmonella group. The increase in the number of Salmonella in the ileum, cecum, feces, liver, spleen and mesenteric lymph nodes was accompanied with TAC reduction (P < 0.05). This shows that probiotics reduce the destructive effects of Salmonella induced stress. Also, B. subtilis and B. coagulans as probiotics cause increases in TAC amounts in comparison to the Blank Control group. So these probiotics prepare the body for exposure to the pathogen and the stress that is caused by them.

Measurement of malondialdehyde (MDA).
The amount of MDA during the sampling period was higher in the Salmonella and Probiotic+Salmonella groups compared to the Blank Control and Probiotic Control groups. Using probiotics during Salmonella infection, reduced the MDA, but the reduction was not significant (P > 0.05). The MDA content in the Probiotic Control group was similar to the Blank Control group (Fig. 5).
The correlation coefficient between the number of Salmonella in the ileum, cecum, feces, liver, spleen and mesenteric lymph nodes with the amount of MDA was 0.32, 0.32, 0.31, 0.31, 0.47 and 0.53 in order (P < 0.05). Malondialdehyde has been the most frequently used biomarker of lipid peroxidation and oxidative stress in many health problems 41 . MDA is one of the products that increased in Salmonella disease. The MDA in the Probiotic+Salmonella group has no significant difference with the Salmonella group. We can conclude that B. subtilis and B. coagulans as probiotics do not affect on MDA concentration. However, the correlation results showed a lesser number of Salmonella in the ileum, cecum, feces, liver, spleen and mesenteric lymph nodes was accompanied a lesser amount of MDA (P < 0.05).

Conclusion
B. subtilis and B. coagulans as probiotics show no significant changes in serological parameters in comparison to the Blank Control group. However, if these probiotics used as a preventative measure against Salmonella infection, they compete with this pathogen in the intestine and enhance the mucosal immune system, delaying the infiltration of Salmonella into lymph nodes, spleen and liver, reduce the inflammatory mediators, decrease oxidative stress, and decrease any hematological and biochemical changes. The beneficial effects listed above, changes the parameters to return to normal ranges faster.

Methods
Animal ethics. All methods were performed in accordance with the relevant guidelines and regulations by the Ethical Committees of Shiraz University and approved by the governing body (1395/9234308). The concentration of probiotic spores fed to the rats was determined based on our previous study 42 26,43,44 .
For confirmation that the rats are Salmonella-free, on days 0, 7, 14 and 21 of the study, fecal samples from each subgroup were taken and examined. One gr of pooled fecal sample from each subgroup was added into 9 ml lactose broth (Merck, Germany) and incubated at 37 °C for 24 h. After incubation, 1 ml of that was added to 10 ml Selenite Cystine broth (Merck, Germany) and in parallel, 0.1 ml was added to 10 ml Rappaport Vassiliadis broth (Merck, Germany). They were incubated at 37 °C and 42 °C, respectively, for 24 h. After which a loop of the selective enrichment broth was cultured on Xylose Lysine Deoxycholate agar (XLD agar, Merck, Germany) and Brilliant Green agar (BG agar, Merck, Germany). The plates were incubated at 37 °C for 24 h and checked for Salmonella colonies 14,45 . Sampling. On days 1, 3, 5 and 7 after intragastric gavage of S. Typhimurium, one rat was chosen randomly from each subgroup (replicated independently five times for each treatment group) and blood samples were obtained from the heart in an anesthetic state. After that, the rats were euthanized and their liver, spleen, mesenteric lymph nodes and intestines were removed under aseptic condition.
Blood samples were collected into tubes with and without anticoagulant (EDTA). The sera were separated by centrifugation of the blood sample without anticoagulant at 3,000 × g for 10 min and stored at -20 °C until used. The blood samples with anticoagulant were used for hematological parameters and differential WBC count.
Enumeration of Salmonella. The liver, spleen, mesenteric lymph nodes, the content of ileum and cecum and about 2 gr of feces were weighed and transferred into stomacher bags along with 9 times sterile phosphate buffer saline (PBS, pH: 7.2). They were then homogenized in a stomacher (Seward Stomacher 400 Blender BA6021, UK) for 2 min and then tenfold serial dilution was made in PBS. The dilutions were then surface plated on BG agar and incubated at 37 °C for 48 h. This process was duplicated for all the cultured samples. The Salmonella enumeration was expressed as CFU/gr 5,46 . Also, on the first day of sampling, one gr of liver, spleen and mesenteric lymph nodes were separately crushed using a scalpel and added into 9 ml lactose broth (Merck, Germany) and incubated at 37 °C for 24 h. After incubation, 1 ml of that was added to 10 ml Selenite Cystine broth (Merck, Germany) and in parallel, 0.1 ml was added to 10 ml Rappaport Vassiliadis broth (Merck, Germany). They were incubated at 37 °C and 42 °C, respectively, for 24 h. Then a loop of the selective enrichment broth was cultured on XLD agar and BG agar. The plates were incubated at 37 °C for 24 h and checked for Salmonella colonies 14,45 . Measurement of hematological parameters. Hematological parameters including PCV, Hb, RBC, platelets and WBC concentration, were measured using cell blood counter (Celltac α MEK6550, Nihon Kohden Company, Japan).