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Adjuvant Activity of Vero Cell on Cellular and Humoral Immunity Responses Against E Research iMedPub Journals Archives of Clinical Microbiology 2021 www.imedpub.com Vol.12 No.1:137 ISSN 1989-8436 Department of Microbiology, Razi Vaccine and Serum Research Institute, Shiraz Branch, Agricultue Razi Vaccine and Serum Research Institute, Shiraz Branch, Agriculture Research, Education and Extension Organization (AREEO), Shiraz, Iran Adjuvant Activity of Vero Cell on Cellular and Humoral Immunity Responses against E. coli O157:H7 Infections in Balb/c Mice Alborzi Research Center, Namazi Hospital, Shiraz, Iran 2 Department of Microbiology, Science and Research Branch, Islamic Azad University, Tehran, Iran Hajar Molaee1, Yahya Tahamtan1* and Nahid Haidari 2DepOrganizationartment of Microbio l(AREEO),ogy, Razi Vaccin Shiraz,e and Seru mIran Research Institute Shiraz Branch, Agricultural Research, Education and Extension 1Department of Microbiology, Razi Vaccine and Serum Research Institute, Shiraz Branch, Agriculture Research, Education and Extension Organization (AREEO), Shiraz, Iran Organization 2Department of Microbiology, Alborzi Research Center, Namazi Hospital, Shiraz, Iran 3 *Corresponding author: Yahya Tahamtan, Department of Microbiology, Razi Vaccine and Serum Research Institute Shiraz Branch, Agricultural Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran Research, Education and Extension Organization (AREEO), Shiraz, Iran, E-mail: [email protected] Received date: December 18, 2020; Accepted date: January 01, 2021; Published date: January 08, 2021 Citation: Homayoon M, Tahamtan Y, Kargar M (2021) Adjuvant Activity of Vero Cell on Cellular and Humoral Immunity Responses against E. Coli O157:H7 Infections in Balb/c Mice. Arch Clin Microbiol Vol.12 No.1:137. Introduction Abstract Eenterovirulent Escherichia coli rank among the most common relevant agents of bacterial diarrhea in humans and Enterohemorrhagic Escherichia coli constitute an important several animal species. Five different pathotypes or groups of current problem of public health and animal production. the E. coli strain were recognized which include Extensive efforts have been made to fight the infections Enterohaemorrhagic E. coli (EHEC), Enteropathogenic (EPEC), caused by the path types which responsible for diseases such as hemorrhagic colitis and hemolytic uremic Enterotoxigenic (ETEC), Enteroaggregative (EAEC) and syndrome. Different vaccines have been tested against Enteroinvasive (EIEC) [1]. Enterohemorrhagic Escherichia coli these bacteria. Here, the current study was undertaken to (EHEC) is an important zoonotic pathogen in humans which evaluate the Humoral and cellular immune responses and causing severe diarrhea and Haemolytic-Uremic Syndrome (HUS) protective immunity conferred by an E. coli O157:H7 [2]. vaccine with vero cell as adjuvant in mice. Konowalchuk et al. [3] found that one group of Escherichia E. coli O157:H7 was grown in the Tryptic Soy Broth (TSB), coli strains produced a toxin that is very similar to the one inactivated with formalin and adjuvant with vero cell and produced by Shigelladysenteriae type 1, this group was named alum. Mice were immunized with inactivated E. coli Shiga Toxin-Producing E. coli (STEC). Also, these toxins may be O157:H7 vaccine to study the splenocytes suspension and identified by Vero Cell Toxicity Test [4] so these bacteria are serum cytokines proliferation. The serum antibody titers called Verocytotoxin or Verotoxin-Producing E. coli (VTEC). Most and cytokines Interleukin-1a (IL-1a) and TNF-y were identified by ELISA. At 28 days post immunization, the of the verotoxigenic strains were made anything from studied groups were challenged with the LD50 dose of the uncomplicated diarrhea to hemorrhagic colitis, which can led to live bacteria. the progress into Hemolytic Uremic Syndrome (HUS) [1]. The firmly attached bacterial colonies to the epithelial cells cause the Mice immunized with vero cell adjuvant E. coli O157:H7 (VO damage to microvilli and rearrangement of host cytoskeleton group) were observed to produce high levels of serum IgG proteins [5]. The basic virulence factors in EHEC are encrypted at antibodies, also inducing increases in the production of the Locus of Enterocyte Effacement (LEE) [6]. LEE is responsible lymphocyte proliferation and secretion of cytokines. Besides, this immunized group produced complete for the attaching and effacing intestinal histopathology exhibited protection after challenge. Our findings introduce that VO by Enterohemorrhagic (EHEC) E. coli and related pathogens [7]. immunized mice is capable of releasing an immunological Among the STEC serotypes, O157:H7 serotype is by far the response with several cytokines and produce complete protection after challenge. These obtained results most frequently involved in human illness [1,8]. The Centers for suggested that VO group has the potential as a candidate Disease Control and Prevention (CDC) has predicted 75,000 for vaccine against E. coli O157:H7. illnesses and 61 deaths annually in the United States which caused by E. coli O157:H7 infections [9,10]. Keywords: E. coli O157:H7; Vero cell; Adjuvant activity; Vaccine; Humoral and cellular immunity The Shigatoxigenic (STEC or VTEC) strains produce Shiga toxins 1 and/or 2 (stx1, stx2) while EHEC produce stx1 and/or stx2 and intimin (eaeA) [11].The Shiga toxin (Stx) is the main virulence factor of STEC/EHEC which is an optimal target to elicit neutralizing antibodies [12]. © Copyright iMedPub | This article is available from: https://www.acmicrob.com/archive.php 1 Archives of Clinical Microbiology 2021 ISSN 1989-8436 Vol.12 No.1:137 Since the cattle are the widespread reservoirs of EHEC Materials and Methods O157:H7 strains, reduction of the prevalence of contamination in cattle are the best method for reducing the risk of EHEC infections in the man [13]. Animals Unfortunately, the risk of HUS has been increased by In this experiment, 40 female Balb/c mice (weight 18-22 g) antibiotics [14] because of the induction of the bacterial SOS were randomly chosen from the Laboratory Animal Center of response (an abundant response to DNA damage) which induce Razi Vaccine and Serum Research Institute (RVSRI), Shiraz, Iran. the transcription of type 2 Shiga toxin genes [15,16] and The mice were randomly divided into 4 groups each containing increase the extracellular concentrations of Shiga toxin [17]. of 10 mice. All experiments have been performed in accordance There are no definitive treatments for EHEC human infections, with the guideline for care and use of experimental animal of and vaccines to prevent the disease are not yet accessible other the National Counsel of Animal Experimentation Control. than common supportive procedures [9]. Cultivation and purification of E coli O157:H7 Global efforts are underway to develop vaccines for a potential EHEC infection and to reduce the contamination of Frozen E coli O157:H7 were cultured in defibrinated sheep cattle and its environmental dissemination of EHEC O157:H7 blood agar 6% (Merck). The purity of the isolates were [18]. Different factors make complicate the development of a confirmed by morphological characteristics on smears stained vaccine to prevent EHEC/STEC human infections include; the with Gram's staining and by growth on the Eosin Methylene Blue type of immune response may confer protection, multiple Agar (EMB) (Merck) and MacConkey agar (MA) (Merck). pathways of infection include cattle-containing food products, Afterward, this strain were inoculated in the Tryptic Soy Broth green vegetables, drinking water [19]. (TSB) (Merck) and incubated at 37°C for 24 hr. According to other our studies, multiplex variation polymerase chain reaction (MV- Several vaccines with different immunogenic, adjuvants, PCR) was applied for molecular analysis of E. coli isolates inoculation pathways, number of doses have been synthesized [27,28]. [18]. Besides, other efforts continue to develop new adjuvants to improve the immunogenicity and efficacy of mucosal delivered vaccines. So far different adjuvants were used for this DNA extraction and PCR procedure purpose, such as Freund’s incomplete adjuvant [20], aluminum DNA extraction for molecular techniques has been done by hydroxide, oil-based adjuvant, lipids, peptidoglycans, different boiling method [29]. 25 μl reaction mixtures included 0.25 μl toxins [21] and protein based adjuvant [9] which lacks the TaqDNA polymerase, 1.25 μl MgCl2, 0.5 μl deoxynucleotide capability to produce both Humoral and cellular immunity. triphosphates 10 mM, 2.5 μl PCR buffer (10x), 1 μl primer 20 and For example, as mentioned before, one of the most important 2 μl of the DNA template. Thermocycler (Master cycler proteins for the vaccination of ruminant targets is the LEE- Eppendorf, Germany) parameters included 94°C (5 min) one encoded type III secreted proteins. Several researchers cycle and 94°C (55 s), 55°C (30 s), and 72°C (60 s) for 35 cycles, investigated the vaccine based on this protein. Immunization of followed by a final extension time for 72°C (4 min). The the cattle with E. coli O157:H7 type III secreted proteins oligonucleotides used as primers in PCR to detect E. coli O157 motivate the inhibition of attachment of E. coli O157:H7 to and Stx1, Stx2 with variants are shown in (Table 1). HEp-2 cells by the immunized cattle sera [22,23]. Base pair Primer Primer Generation sequence Until now, current vaccination strategies only partially successful in reducing E. coli O157:H7 excretion. Awareness of 420 CGT GAT GAT F O157 the host cell interactions of E.
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