Disease Hazards and Immune Responses of Wistar Albino Rat Infected with Streptococcus Mutans

Several studies have attempted to find an explanation for the connection between cardiovascular disease and bacteria. This research aims to clarify this relationship by investigating the experimental induction of cardiovascular diseases exemplified by Infective Endocarditis (IE) by streptococcus mutans in Wistar albino rats, with focus on the host immune response. was followed by examination of certain haematological, biochemical and molecular parameters identified after 1,3 and 7 days post-challenge. The haematological parameters (CBC) showed a substantial increase (P≤0.05) in WBCs, Plts, MCVs, MCHs and N percent and a significant decrease (P≤0.05) in RBC, L% and HCT. The profile of cardiac enzymes (CK, CK-MB, LDH and Troponin) was considerably elevated (P≤0.05). The inflammation biomarkers of the blood (CRP and ESR) were highly elevated (P≤0.05). Rat inflammatory cytokines (IL-1α , IL-12p70, TNF- α , MCP-1 and IFN- γ) achieved significant alteration in their levels with different patterns except for IL-10. Regarding the innate immune response exemplified here by gene expression patterns of the proinflammatory cytokines (IL1-alpha, CKM and LDHA genes) were investigated using the real time (RT-PCR) technique, the results showed high gene expression due to the induced bacterial disease.

CVDs has been the focus of attention in many studies, and several mechanisms have been proposed to explain or support such theories, these theories indicate that the oral lesions are indicators of disease progression and oral cavity can be a window to overall health and body systems [8]. in his study reported that, oral lesions lead to systemic exposure to oral bacteria and that the resulting production of inflammatory mediators is capable of initiating or supporting mechanisms associated with the development of CVDs.
Many scientists have been trying to find a reason for the correlation between dental health and cardiovascular health [9]. By observing what microbes are found at CVD sites, they have found that Streptococcus mutans (Oral pathogen) are often prevalent at the location where the CVDs took place [10], and they refer the reason for the presence of these bacteria to that the bacteria can access these areas by entering the body during dental procedures such as root canals and tooth extractions, or by access through the rotting or infected tooth causing bacteremia [11]. If enough S. mutans enter the bloodstream, the microbes could likely reach the heart tissue. From here the microbes may attach to the endothelial cells. By binding to endothelial cells, they could infect the surface of the heart or create blockages in the heart's inner surface [12].
The fact that S. mutans can remain viable in endothelial cells for prolonged periods indicates that invasive organisms may not be readily cleared by the immune system or antibiotic treatment, potentially contributing to the establishment of chronic disease and the triggering of other pathologies associated with CVDs [10].
Infective Endocarditis (IE) is a bacterial infection of the heart valves or the endothelium of the heart [13]. It often fatal systemic disease and always associated with dental diseases. And it occurs when bacteria in the bloodstream lodge on abnormal heart valves or damaged heart tissue, and occurs rarely in people with normal hearts. So, the people who have certain preexisting heart defects are at risk for developing endocarditis when bacteremia occurs [14].
There are over 1,000 case reports associating dental procedures or disease with the onset of endocarditis [15,16]. Besides, multiple animal models (rats, rabbits and pigs) have shown that oral bacteria and even dental extraction can create endocarditis under experimental conditions [17]. From the other side, when the bacteria enter the bloodstream the immune system forms an inflammatory response to this infection. This inflammatory response can be measured by a variety of inflammatory biomarkers such as nonspecific serum markers of inflammation, including Erythrocyte Sedimentation Rate (ESR), C-Reactive Protein (CRP), and leukocyte count (WBCs) which often elevated but are not used to make the diagnosis of acute endomyocarditis [18]. The pro-inflammatory cytokines have been implicated in cardiac dysfunction as part of the acute phase reaction [19].
Many-body systems affected by exposure to the bacteria [20], which could be investigated by measure many important haematological and biochemical parameters such as liver functions [21], kidney functions [22], cardiac profile [23], inflammatory markers [24], complete blood count [25], and measuring proinflammatory cytokines which act as mediators to immune response [26], these mediators are released initially at the site of inflammation by activated mononuclear phagocytes, lymphocytes, or other differentiated cell types and have potent local and systemic effects and so, and also by studying the change in some inflammatory-cardiovascular related genes expression [27].
The goal of this study is to evaluate some of Wistar albino rats immune responses towards one of the highly pathogenic bacteria (Streptococcus mutans) in concern to the hazardous endocarditis.

Materials and Methods
Biochemical investigations were developed at the biochemistry Labs of Armed Forces Central Labs, Kobry Al-Kobba, Cairo, Egypt.
And the molecular biology investigations were developed in the molecular biology labs in the National Research Center (NRC), Dokki, Egypt.

Experimental Animals
A total of 60 male Wistar albino rats with 120-150 gm body weight were obtained from the animal house in the NRC, housed randomly in twelve stable wire cages (n=5 for each group) with normal temperature and photoperiod. Diets and water were given ad libitum to rats (commercial diet and tap water) and allow acclimating for one week before assay.

Pathogenic Bacteria
The bacterial strain used in this study was Streptococcus mutans pure strains SA53 (serotype k), were kindly obtained from the microbiological archive of the microbiology department, Animal Health Research Institute (AHRI), Dokki, Egypt.It came in the form of an inoculated solution ready to use. This inoculated solution contains 3 X 107 CFU∕ml (nonfatal dose).

Chemicals, Kits and Analyzers
All biochemical analyses were determined using kits purchased

Methods
The experiments were conducted following ethical guidelines for investigations in laboratory animals.

Experimental Design
The 60 rats were distributed in twelve different wire cages with identical physical environments, each cage contains5 rats (n=5) and each two cages were assigned as a group and its replicate, so we had 6 groups. Groups 1-3 were used as negative controls, and groups 4-6 were used as the (challenged) groups. After one week of the conditioning period we applied a process of immune system stimulation by injecting the challenge groups Intravenous (IV) in caudal vein with 0.3 ml (3 X 107 CFU/ml) of the infectious pathogenic bacteria S. mutans. Other control groups were injected with identical volume Phosphate Buffered Saline (PBS) and used as a negative control. Liver and heart tissues and blood samples were collected from the rat's groups through three intervals of time; 1day, 3days and 7days post-challenge to carry out the haematological, biochemical and molecular investigation.

Cardiac Profile
The assays of serum Creatine Kinase (CK) activity and Creatine Kinase-MB (CK-MB) activity was determined following the recommendations of the International Federation of Clinical Chemistry (IFCC) but was optimized for performance and stability.
serum Lactate Dehydrogenase (LDH) activity was determined using UV assay according to standardized method of [28]. The quantitative determination of cardiac troponin-I(cTnI) were determined using Enzyme-Linked Immunosorbent Assays (ELISA).

Biochemical Markers of Inflammation
C-reactive protein is a non-specific biochemical marker of inflammation which was measured here using ultrasensitive latex immunoassay CRP (BIOTEC Laboratories Ltd Co. (Ipswich, Suffolk, UK). Erythrocyte Sedimentation Rate (ESR) is another biomarker of inflammation, ESR was determined according to [29].

Cytokines
The haematological parameters were measured on the day of sacrifice as a Complete Blood Count (CBC). Inflammatory cytokines

Molecular Analyses
Polymerase Chain Reaction (Real-time PCR) was applied using TaqMan Universal Master Mix II, with UNG. Total RNA was extracted from the liver and heart tissues as a pool for the same individual of rats groups, according to the method of (Nicholas and

Probes and Primers
Real time PCR was carried out using Rotor-Gene Q -QIAGEN instrument with 36 wells using Qiagen PCR Tubes for quantification of gene expression in both target and housekeeping genes.
Serial dilution for primer, probe and cDNA samples were made.
Concentrations of 50 picomol primer, 5 picomol prope, 1/100 for HK gene and 1/10 for target genes gave the best results (standard concentrations). PCR cycle consisted of a denaturing step for 15 second at 95ºC and annealing/extension step for 1 minutes at 60ºC. PCR amplification was performed in a final volume of 20 µl.
Relative gene expression was calculated with the 2^-ΔΔCt method [30] relative to the lowest value for each gene in the whole study.

The Statistical Analysis
One-way ANOVA was used to determine differences among the treatment groups for each blood analyte. Considering statistically significant (p≤0.05), the means were compared by the use of Duncan's multiple range test. The analyses were performed using the Statistical Package for Social Sciences Software (SPSS), version 14 (SPSS, Chicago, USA) was used as described by [31]. Experimentally infected rats showed no signs of illness that can be noticed exteriorly except one group that showed about 40% assumed to be weak and ill as signs of disease. No mortalities were recorded along the course of the experiment in all the control and the challenged groups. Investigation of the normal rats' internal organs, showed no significant lesions where there was a prominent splenomegaly observed in the challenged rat groups ranging from 20% to 100% splenomegaly when compared to control rats. The mean spleen length was about 2.4 ml in the control groups while in the challenged groups was ranging from 3.61 ml to 5.03 ml in the challenge groups suggesting splenomegaly with observation that there was a positive relationship between time after injection and the percent of splenomegaly ( Figure 1).

Cardiac Profile
Elevation in cardiac enzymes due to bacterial infections are an early indicator of heart muscle injury in studies of cardiotoxicity, in this study the cardiac profile is exemplified in CK, CK-MB, LDH and cTnI, and the data were reported in Table 2. From cardiac enzymes analyses it was revealed that, There was a highly significant

Biomarkers of Inflammation
CRP and ESR levels are associated with inflammation, they are illustrated in Figure 2. There was an observation that CRP levels have significantly increased in the of infected rats than those in control rats after 1-day, 3-days and 7-days post-challenge where ESR levels increased significantly after the first day and the 3ed day of infection but became normal after the 7 th day of infection.

Discussion
Infectious disease is, by definition; is the consequence of infection by microorganisms such as bacteria, viruses, or parasites [32] Animals respond to the presence of a foreign organism by invoking several physiological changes, including rapid inflammatory changes collectively known as the acute phase response [33]. post challenge but returned to the normal after 3 and 7 days, these elevations may be due to hepatic toxicity as levels of these enzymes are rapidly increased when the liver is damaged by any cause, including hepatitis or hepatic cirrhosis [39]. Increased serum activities of ALP have been explained by pathological processes such as liver impairment, kidney dysfunction, and bone disease [40] which may be systemic response due to bacterial infection.
Our results also match with that of [41] who indicated that the infected patients with bacteremia had significantly higher serum levels of ALT and ALP [42]. indicated that the rats and O. niloticus infected with Colistridium perfringens showed that the liver was other metabolic or nutritional disorders [43], and its levels display reasonably predictable changes in response to acute inflammation, malignancy, trauma, necrosis, infarction, burns, and chemical injury [44]. Laboratory-based evidence and the epidemiological studies have investigated the association between coronary heart disease and plasma protein involved in the acute phase response [45].
In this study, there is a significant increase (P≤0.05) in the serum total protein (Hyperproteinemia) in the infected rats, this may be due to liver total protein synthesis was markedly enhanced due to the increase in the synthesis of exported proteins (acute phase proteins) [34] and may be due to response inflammatory reaction [46], and there is a significant increase (P≤0.05) in the serum albumin level (Hyperalbuminemia) as Albumin is the major protein component of serum, this may be due to hepatic disturbance as a result of tissue damage by the inflammation [47], and also there is a significant increase (P≤0.05) in the calculated globulin in serum, This increase in globulin levels accepted due to the increase in TP as result of the acute inflammatory response to the bacterial infection [48]. These acute inflammatory disorders usually produce an increase of some of the proteins from the α1-globulin fraction such as α1-antitrypsin, ceruloplasmin, haptoglobin, and α1-acid glycoprotein [33,49]. There was a significant decrease (P≤0.05) in the calculated serum A/G ratio, A/G ratios may decrease due to globulin levels rising during chronic inflammation [48], or may be due to the progress of an infection is usually associated with marked changes in the serum proteins, associated with an increase in the percentage of the total protein during some stage of the infection, and there is usually a change in the A/G ratio with an increase in the total globulins , this is in accordance to [50]. In summary, the activities of serum liver enzymes (AST, ALT and ALP) were showed a significant elevation in their activities, and also there is an elevation in the protein levels produced by the liver. So, we can say that the liver was aggressively affected by a bacterial infection in rat challenged groups.
Serum creatinine (Cr) assay is the most commonly used endogenous marker for the assessment of glomerular function [51] and is a decomposing product of creatine phosphate in the muscles, so the increase in Cr is clinically indicative of muscular dystrophy or wasting disease. Increased serum creatinine is also indicative of diseases affecting the heart muscle. In accordance with the normal conditions, creatinine is not reabsorbed by the tubules to any appreciable extent [52]. Urea and serum Blood Urea Nitrogen levels (BUN) may provide additional information on renal function as renal proximal tubular cells may increase BUN reabsorption in the setting of increased neuro-hormonal activation [53]. Measurements of uric acid are used in the diagnosis and treatment of numerous renal and metabolic disorders, including renal failure [54].
In this study, Cr levels were elevated in rats in challenged groups from the first day of the experiment and still high to the last day of the experiment. This change in serum creatinine can be explained by the fact that the increase in serum creatinine is observed only with nephron damage [51]. While urea and BUN levels are significantly increased from the third-day post-challenge.
Findings from this study are in agreement with the study of [55] who reported that, there is hypertrophy of kidneys was observed in rats infected with S. mutans SA31 strain, and they added that the observations from Gram staining showed a considerable bacterial mass in the kidney, this indicates that the kidneys are target to S. mutans bacteria. Results are also in agreement with those of [56] who indicated that bacterial infection induces its toxicity on the kidney with increased serum urea and creatinine levels and also indicates that the distribution rate of S. mutans strains with collagen-binding proteins in the oral cavity is significantly greater in IgA Nephropathy (IgAN) patients than in healthy controls, suggesting that these strains are potentially involved in the mechanism of IgAN development. For clarification; IgAN is primary chronic glomerulonephritis; kidney disease [22]. indicated that bacterial infections cause renal dysfunction which occurs as part of multi-organ dysfunction due to sepsis, hypotension, hemolysis, or hepatorenal syndrome.
In contrast; this work' results contraindicate with those of [57] who indicated that the kidney of rats experimentally infected with S. faecalis was not affected and showed no significant difference in serum urea concentration measured at time 24h between experimental and control groups of rats and remained almost unchanged at 2-days and 7-days post-infection. But in agreement with study of [58] and McCullough and [59] who indicated that there is renal dysfunction has been associated with adverse cardiovascular outcomes, also match with [60] who indicated that the kidneys are commonly affected in infective endocarditis by a variety of complications of clinical significance. McCullough and [59] reported in their study that there is renal dysfunction has been associated with adverse cardiovascular outcomes. From his study [60] indicated that the kidneys are commonly affected in infective endocarditis by a variety of complications of clinical significance.
Calcium results showed a significant decrease in its levels (hypocalcemia). There are many causes of hypocalcemia, such as renal failure, pancreatitis, steatorrhea and hypoparathyroidism [61]. Hypocalcemia may also be due to protein binding because calcium levels vary with total plasma protein [62], and the difference in the concentration of Ca may also be associated to the kidney disease, where, the chronic kidney disease usually results in hypocalcemia and renal dysfunction may occur with hypercalcemia [63]. So we can say that the high elevation in the serum total protein in the challenged groups affected the total calcium level in the serum causing a decrease in its levels (hypocalcemia), and also this hypocalcemia may be due to kidney injury caused by the bacterial infection. Calcium data in this thesis in agreement with a study of [42] who indicated that rats infected with C. perfringens showed a significant decreased in the serum total calcium levels in the challenged groups after 3-days and 6-days post-challenge and with results of [64] who indicated that hypocalcemia associated with severe infections [65]. reported that hypocalcemia might be a risk factor for quinine cardiotoxicity.

Popular causes of serum electrolyte alterations include
inflammation, acid-based status, changes in membrane properties, inflammatory mediators, hemodynamic alterations, and defective renal transport [66]. Potassium (K+) is a major electrolyte in the human body. It is also known as the heart mineral because it directly affects the heart muscle cells, and sodium (Na+) is one of the major cations found in extracellular fluids. Sodium salts are necessary to maintain the balance between calcium and potassium in order to maintain normal cardiac activity [67]. Serum electrolytes (Na+, K+,  [70,71]. Clinically, CK is tested in blood tests as a marker for damage to CK-rich tissue, such as myocardial infarction (heart attack) autoimmune myositides, and acute kidney injury This biomarker is normally not found in the circulation. Injured cardiac muscles release troponin from the contractile proteins into the bloodstream and thus an increased level of serum cardiac troponin-I may suggest myocardial injury [73]. Serum levels of CK, CKMB, LDH and cTnI were found statistically significant to be increased in the three experimental groups of rats compared to the control group by a different pattern referring to cardiac injury. This work' cardiac profile data is matching with the studies of [74] and  [76]. The nonspecific markers of inflammation are often elevated but are not used to make the diagnosis of CVD such as IE, but due to their elevation, they may be helpful to confirm the diagnosis [77,78]. in this study, the nonspecific markers of inflammation (ESR and CRP) were found statistically significant to be increased in the three experimental groups compared to the control groups due to the effect of the infection. Rats serum CRP levels were found statistically significant (P≤0.05) to be markedly of the inflammatory response, and in bacterial infection [79].
So we can say that CRP is a good marker for bacteremia this is in accordance with [80].
The inflammatory markers results here is matching with those of [57] who indicated that rats infected with S. faecalis showed an increase in the ESR levels at 24 hr post-injection, and showed a high elevation in CRP concentrations due to the bacterial infection by about 16 fold increases compared with control levels at 1-day after injection [42]. indicated that rats infected with C. perfringens So the rat's defense mechanisms explain the high production of leucocytes in the challenged groups [68]. Lymphocytes percentage (L%) results showed a decrease in its levels (relative lymphopenia).
The In concern to the inflammatory cytokines investigations on a biochemical base, This study revealed a significant increase in with the uninfected control rats as a response to CVD devolution with the curve peak image characterizes the normal immune response. However, it could be proven that an increase in the levels of expression of genes of the immune system results in enhanced resistance against infection [81]. reported that, to control early bacterial infection before it becomes this severe, IL-1 can help prevent a pathogen from reaching immune-privileged or vulnerable sites, such as the Central Nervous System (CNS), IL-1α is a key mediator of the sterile inflammatory response but is not generally critical for the response to bacterial infection (suggesting that IL-1α contributes to cytokine storm during sepsis, this is in accordance with our results. The increased expression of striated muscle associated creatine phosphokinase as well as the increased release of the cardiomyocyte enzyme CK in the hearts of Wistar rats suggests that exposure to bacterial infection contributes to cardiomyocyte stress. [82], so from our data; (CK) mRNA expressions; could prove to be an important indices of cardiac cellular damage associated with the S. mutans bacterial infection [83][84][85]. LDH enzyme is expressed in body tissues, such as blood cells and heart muscle, it is released during tissue damage, so it is a marker of common injuries and disease such as heart failure [86,87]. Diseases that can cause increased LDH in the blood may include liver disease, heart attack, anemia, muscle trauma, cancers, and infections such as encephalitis, meningitis, and viruses [72]. The increased expression of LDH gene is an indication for the increased serum LDH and this result is consistent with [57] who indicated that any external stressor, such as the bacterial infection even at a sub lethal dose can have a toxicological effect on the liver or the heart and leading to increased level of LDH [88][89][90]. Stephen et al. (2010) in their study indicated that by studying some cases of myocarditis related to acute streptococcal infection [91][92][93], their initial laboratory findings showed an elevated troponin, together with an elevation of LDH and CPK, suggesting cardiac injury due to streptococcal infection [94,95]. However, this study suggests that S. mutans has a direct effects on the heart, liver and kidney, this may be probably explained by the release of several enzymes to the blood (e.g. heart, liver and kidney enzymes) as the cells of these organs were damaged, a phenomenon that provides the basis for clinical diagnosis [96]. Cytokines and other inflammatory profiles are good indicators and immune responses against bacterial infection to keep health in man and animal due to the prominent changes in their parameters which observed in all infected rat groups compared to the control groups [97].