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Pathological And Immunological Study On Infection With Escherichia Coli In ale BALB/c mice

Escherichia coli bacteria is considered as one of the common responsible for the frequency and severity of infections that it hospitalized patients. E. coli simultaneously carries a harmful side in which only a slight genetic recombination can bring about a highly pathogenic strain that most frequently causes the scourge of bacterial infections worldwide including sepsis, neonatal meningitis, pneumonia, bacteremia and traveler’s diarrhea. This study was carried out to assess Escherichia coli infection induced pathologically and immunologically. Following Escherichia coli isolation, identification and counting, the lethal dose (LD-50) was determined before infection. Twenty-two mice were used in this study for 21 days infection, the animals were sacrificed at 3, 6, 9, 12, 15, 18 and 21 days, and tissues of different tissue were collected, examined for bacterial infection. Bacteria and mice immunization and ELISA were used to detect immunoglobulin G level in serum as well. For histological study, different infected organs were used. The results indicated that the LH50 was 1×109 cell; and all organs were infected after 3 days followed by decreased in infection level shown in brain at day 12, lung, kidney and intestine at day 15 and in liver, spleen and heart at day 21. Moreover, ELISA results revealed that concentration 1:200 of serum in positive and negative state and optimum concentration of Ag 1:40 dilution and compact dilution is 1:1000. In addition, diversity of histopathological alteration occurs in tissue on time-depended manner. This study concluded that the ability of activated E.coli to stimulate the intestinal secretory immune system of germ might result from a retardation of immunological maturity.


Introduction
Escherichia coli are a gram negative bacterium which is responsible for the frequency and severity of infections that it causes hospitalized patients. E. coli simultaneously carries a harmful side in which only a slight genetic recombination can bring about highly pathogenic strain that most frequently causes the scourge of bacterial infections worldwide including sepsis, neonatal meningitis, pneumonia, bacteremia, and traveler's diarrhea [1,2].
There are many physiological changes accrue during infection with EPEC such as transport of iron [3], inflammatory responses initiation, and increase in the cellular permeability [4]. These physiological events are similar to the changes accrue by EPEC of host signaling pathways, such as inositol phosphate fluxes [5], protein kinase C, mitogen-activated protein kinases [6], tyrosine kinase enzyme, myosin light-chain kinase [7], and NF-KB [8].
The motivation of some these lanes have been proven to the biological changes induction of EPEC. Though of achievement over the last 25 year for understand the EPEC illness, there is still needed more studies to comprehend the EPEC activation of biological changes. Earlier studies with different animal models have been reported to investigate host immune reactions such as modulated rabbits and mice [9,10].

IHSCICONF2017
IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 1003 (2018) 012009 doi : 10.1088/1742-6596/1003/1/012009 The diseases which are induced by REPEC in animal is comparable to EPEC causes infections [9] but, there are confines to the same model, like genetic and immunological difficulties. However, the present study was done to examine of Escherichia coli for BALB/c mice was used for animal immunological and histological studies.

Isolation and identification of Bacteria
In the present study, the samples of bacteria were collected aseptically in sterilized labeled containers from Al-Yarmook Hospital, Baghdad, Iraq, and were brought to the Microbiology Laboratory, Division of Biotechnology, Department of Applied Science, University of Technology. The E. coli were isolated by following standard protocol using sterile bacteriological media, including Nutrient agar and MacConky agar. The specimen were aseptically inoculated on the plates and incubated aerobically at 37 °C for 24 hours. The morphological investigation of Bacterial strains have been done using Gram's staining. In the other hand biochemical tests were also included such as , catalase, (MR-VP), gelatin liquefaction test, starch hydrolysis, nitrate reduction test, indole, motility, coagulase, urease, oxidase, TS,I sugar fermentation and Bacterial counting as described by [11].

Laboratory Animals
Male BALB/ mice were the tested animals, which were 8 week old at the beginning of experiments, and their weight was 22 ± 0.5 gram. They had free excess to water and food during experiments. The mice were divided into 2 groups.

Lethal Dose -50 (LD50)
The lethal dose-50 was determined in mice before carrying infection efficacy experiments by injecting intraperitoneally (i.p.) different doses of viable E. coli suspended in phosphate-buffered saline (PBS). The doses were 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , and 1×10 7 CFU per mouse. Five mice were utilized for each dose group. Another group of five mice were used as control and administered with PBS only. The living and dead mice were detected after 28 days for LD-50 and calculated using the formula as below:

Infection of Mice
Approximately 1 × 10 7 of E. coli cells in 250 μL of PBS were injected interaperitonial into 33 mice. Control animals of 11 mice were received 250 μL of PBS. During the experimental time of treatment, animals were detected every 24 h for observing action and aquatic intake, and mass was measured as well. Following infection, the animals were sacrificed at 3, 6, 9, 12, 15, 18 and 21 days, and tissues of liver, spleen, kidney, lung, brain intestines and intraperiton were collected, examined for bacterial infection and processed for further analysis.

Preparation of E. coli Ag for Immunization
E. coli cells were inoculated into nutrient broth for 24 h at 37°C and then washed with PBS by centrifugation at 3000 rpm for 5 min at 4°C; and then re-suspended to the appropriate density in PBS. Bacterial count were 60 were killed by heating 60°C for l strain CFU/ml. Bacteria 7 to verify the number of bacteria at 1˟10 done min. The sterility of antigens was tested before use according to [12]. Protein concentration has been measured by biuret protein assay according to standard protocol.

Immunization of Mice
The mice were divided equally into two groups, 10 mice of each. The group one was injected subcutaneously 2 times for two weeks with 250 μL containing 1x10 7 CFU/ml. Second group (control group) was injected subcutaneously with 250 μL of PBS. Serum were collected at 3, 6, 9, 12, 15, 18 and 21 days. Serum samples were stored at -20 ºC until use for analysis by ELISA.

Enzyme-Linked Immunosorbent Assay
ELISA technique are used to measured the level of immunoglobulin G (IgG) in the serum as described techniques by [13]. 200 μl prepared as described above. Diluted 1.5 μg mL -1 in carbonate bicarbonate buffer as coating antigen which kept overnight in refrigerator. Plates were washed three times with washing solution (PBS containing 0.1% Tween 20). Blocking solution 200 μl well -1 was added (1% BSA in PBS), plates were incubated at 37 °C for 60 min. Then washed three times with washing solution. Diluted 100 μl of each serum samples 1:100 in 1% BSA was distributed into appreciate well, positive and negative control sera were added and then incubated at 37 °C for 60 minutes. Washing the plates three times, 100 μl of anti-IgG peroxidase conjugate diluted 1:1000 in diluents buffer containing 1% Bovine serum albumin were added to each well and plates were incubated at 37 °C for 60 minutes. Washing 3 times again and then add 100 ml of diluted OPD (1 tablet plus 75 ml deionized distilled water and immediately H2O2 30% for each diluted OPD) incubated 20 minutes in dark place at room temperature. Absorbance values at 490 nm were measured using ELISA Reader.

Histological study
The histological examination selected organs of treated mice were washed with PBS, fixed in 10% formalin, followed by use of paraffin dispensing module EG 1150H (Leica, Germany), and embedded in paraffin. Sections were prepared by use of a microtome RM2255 (Leica, Germany) and followed by hematoxylin and eosin (H&E) staining. The processing and staining of sections were performed according to a standard procedure used in histopathological laboratories [14].

Results
The biochemical evaluation for E. coli showed that colonies growing in MacConkey agar have a ping color due to the presence of fermented lactose sugar and give a positive test in catalase and negative test in urease as shown in Table (1):

Histopathological changes
Sections of infected organs were stained with haematoxylin and eosin and then evaluated for histological changes. The tested organs were liver, lung, spleen and kidney.  In spleen, increase in growth of white core of spleen tissue and contact between with other core and congestion in red core of spleen tissue and eccentric artery, in third week amyloidosis type sago spleen was seen in the infected sections. No abnormal changes were observed in control sections ' Figure (

ELISA result
To detect level of immune response against E.coli in detect time table to differential optimum dilution between positive and negative control and detect optimum quantity of Ag we found that concentration 1:200 of serum in positive and negative state and optimum concentration of Ag 1:40 dilution and compact dilution is 1:1000 Table (4).

Discussion:
The results of present study improved that immunization of male mice by E. coli results in activation of immunity in treated group compared with control. These results is in contract with a study of [15] which reported mice immunity stimulation with two doses of the same bacteria vaccines containing O157 or O157:H7; K99 to antibodies in serum. Other study has been reported different immune response parameters such as lymphocyte numbers and spleen weight where the infection with E. coli increase the spleen weight and number of lymphocytes after infected with 36× 106 CFU/ml [16]. Other studies have been demonstrated that continuous injection of E. coli increases number of immune cells such as intestinal monocytes when we compare the number produced through infection with a live E. coli [17].
Several studies have been reported the LD50 dose in mice; for example, Tzong et al. [18] informed that dose of 250μg subcutaneous administrated was sub-toxic to mice, while control PE was toxic at 0.5 and 1μg. Moreover, Armstrong and Merrill [19] have reported the inhibition of protein synthesis by activation of ADP on factor 2 and the lethal dose was at 1μg in in vivo and in vitro study. Susan et al., [20] documented that toxin had median lethal in mice at i.p injection dose of 0.15μg /22 g. However, Mohawk et al., [21] reported that there is no one reviews all E. coli O157:H7 infection features, and many respected mouse prototypes have been established to authorization examination of E. coli O157:H7 pathogenesis and disease can be measured.
Histopathological investigation in administrated mice in this study reveal that toxin had a marked effects on different tissues characterize by the presence of inflammatory cells such as neutrophils, lymphocyte and macrophage. Vulcano et al., [22] investigated the histopathological and immunological alteration in mice infected with E. coli and suggested that EPEC activates immune action and intestinal histological changes with instruction to use mice as an appropriate model for the proposal and experimental of immuno-biological products for active or passive immunization. Not many in vivo researches have studied the differences between E. coli improved from patients with renal clinical signs and patients with signs of pyelonephritis. One of the mechanism is that E. coli has LPS in its wall, which is consider as a possible elements that generate an inflammatory reaction by motivating cytokine producing inflammatory mediators such as interleukins, TNF- and CAM. [23]. While LPS stimulation through Toll-Like Receptor-4 (TLR-4) causing cytokines production [24]. Thus in view of the B-cell mitogenicity of bacterial lipopolysaccharide it may well be the Gram-negative component of the intestinal flora which most significantly effects expansion of immunocytes and thereby plays a more fundamental role in the activation, differentiation and maturation of the local immune system [25]. Furthermore, Ogawa et al. [26] have reported that STEC infected infant rabbit model shown intragastric epithelial changes including; vacuolation in epithelial cells, detached and necrosis due to increase growth of STEC; cecum and colon epithelial changes were exfoliation of epithelial cells, infiltration of immune cell and mitotic activity. Other studies have been demonstrated that continuous injection of E. coli increases number of immune cells such as intestinal monocytes when we compare the number produced through infection with a live E. coli [17]. Additionally, higher IgG level in mucosa in cattle has been observed by Martorelli et al., [27] in response to E. coli O157 where the level of IgA was low in serum. Hoffman et al. [28] explanation was that production of Stx2 by E. coliO157:H7 throughout infection time might diminish lympho-proliferative responses in intra-gastric epithelial cells and peripheral lymphocytes. It would be interesting if further examination of other protein response in serum and mucosa study.
This finding suggests a relationship between immunization, immune response and histology. Nevertheless, further studies are necessary to investigate the role of antibodies and cellular immunity in the E. coli infection. In addition, immune responses improvement can be proposed to control E. coli affects.