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1 Available online freely at Bioscience Research Print ISSN: Online ISSN: Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, (2): OPEN ACCESS Immunological trial of formalized inactivated Salmonella Typhimurium vaccine for poultry Gehad A Yousef 1, Nasser A Sherif 1, Mahmoud Elhariri 2 and Nashwa Ezzeldeen 3 1 Central Laboratory for Evaluation of Veterinary Biologics, Abbasia, Cairo, Egypt 2 Microbiology Dept., Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt 3 Microbiology Departments, Faculty of Veterinary Medicine, Cairo University, Taif University, Kingdom of Saudi Arabia *Correspondence: mahmoud_elhariri@cu.edu.eg Accepted: 16Apr Published online: 27 June This study is a trial to prepare a formalized inactivated Salmonella Typhimurium vaccine and study its immunological effect in vaccinated birds. To achieve this purpose, 25 chickens were vaccinated with the prepared vaccine and another 25 chickens were kept as a non-vaccinated control. Fifteen days post 1st dose, another dose (booster dose) was injected intramuscularly in the vaccinated group. After another 15 days of booster dose, the vaccinated and control groups were challenged with virulent Salmonella Typhimurium microorganism orally and kept under observation for one month. During this period, shedding, protection and re isolation percentages were recorded. The results revealed that vaccinated group gave better percentages for shedding (16.67%), protection (88%) and re isolation (6.7%) than those obtained in control one (50%, 20% and 73.3%) respectively. Finally, it could be summarized that the prepared formalized inactivated Salmonella Typhimurium vaccine is potent and can be used to vaccinate the birds to protect against infection with Salmonella Typhimurium. Keywords: Salmonella Typhimurium, Formalized vaccine, challenge, shedding INTRODUCTION Salmonella species is considered one of the major food borne pathogen that may colonize the gastrointestinal tract (GIT) of chickens. They can be associated with processed poultry (Elhariri et al., 2017b) and may cause severe illness and even death in humans (Tauxe, 1991). Consequently, great attention and efforts are being looked for these entero-pathogens in poultry farms (Barrow and Tucker, 1986). There are many sources of Salmonella species in poultry farms, which could be the deep litter (Ahmed et al., 2012), baby chicks (Saad et al., 2017), workers, and even broiler breeder. In many cases broiler breeder could be the major source of Salmonella and Mycoplasma infection (Liljebjelke et al., 2005, Khalifa et al., 2014 and Hossam et al., 2016) The emergence of antibiotic resistance inbetween Enterobacteriaceae group and other bacterial pathogens has a great concern in Egypt & worldwide (Osman et al., 2012, Abdel-Moein et al., Saad et al., 2017, El-hariri et al., 2017a) Control of Salmonella infections in poultry is posing itself as one of the difficult problems because of the fact that most of Salmonella serovars, which poultry harbor act as potential pathogens for man (Schroeder et al., 2005). Vaccination with live or inactivated Salmonella vaccines was able to provide protection against challenge. Vaccination plays a good role in a management control of Salmonella in layer flocks (Groves et al., 2016). In India, the most successful killed vaccine was made from formalin killed, alum precipitated

2 (Singh, 2009). Salmonella reduction in broilers from commercial broiler breeders vaccinated with live and killed Salmonella vaccines. Broiler breeders were vaccinated with Poulvac ST live S. Typhimurium vaccine at 1 day of age, and this was repeated at 2 and 6 weeks of age. The breeders were then administered a killed autogenous oil emulsion adjuvanted vaccine containing S. Kentucky, S. Heidelberg, and S. Hadar, at 10 and 18 weeks of age. (Young et al., 2007) Vaccination of broiler and breeder increased humoral immunity and reduced Salmonella prevalence and loads in their broiler progeny, but did not significantly decrease Salmonella in the breeder farm environment (Berghaus et al., 2011). The duration of immunity of difference vaccination schemes was examined and found that the vaccinated groups shows a significant different in the ceacal swaps and the organs re- isolation compared with the non-vaccinated control group (Springer et al., 2011). So, this study was designed to study the immunological effect of formalized inactivated S. Typhimurium vaccine in chickens. MATERIALS AND METHODS Salmonella Typhimurium Strain: Local field isolate of pathogenic strain of Salmonella Typhimurium was kindly obtained from Sera and Antigens Department, Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo. It was used for vaccine preparation and challenge of vaccinated chickens. Preparation of Salmonella Typhimurium inactivated formalized vaccine: Preparation of bulk culture from S. Typhimurium: It was performed by using the method described by Charles et al., (1994). Salmonella Typhimurium was grown on Salmonella - Shigella ager for 24hrs at 37 C. Separate colonies were selected and inoculated in10ml of tryptose soya broth and incubated for 24hrs at 37 C. After that the culture was inoculated in100ml of tryptose soya broth and incubated for 24hr at 37 C.Then the 100ml were inoculated in 250ml of tryptose soya broth and inoculated for 24hrs at 37 C. After that 250ml were inoculated in one liter of tryptose soya broth and incubated for 24hr at 37 C. Then each bacterial suspension was centrifuged at 5000 rpm at 4 C for 30mins to pellet the bacterial strain. A separate final suspension from S. Typhimurium was prepared and the count was adjusted for each type to CFU/0.5ml using colony count technique Colony count technique: It was carried out according to (Arnon et al., 1983). The culture was harvested in the 10 ml of the diluent (sterile saline), then, using a syringe 1 ml of the vaccine was withdrawn and was added to 99 ml of the diluent (sterile saline). This is the 10-2 dilution. The bottle was shaken for 1 minute, then, using a fresh syringe1 ml of the vaccine was withdrawn and was added to 99 ml of the diluent (sterile saline). This is the 10-4 dilution. The bottle was shaken for 1 minute, then, using a fresh syringe1 ml of the vaccine was withdrawn and was added to 99 ml of the diluent (sterile saline). This is the 10-6 dilution. The bottle was shaken for 1 minute, then, using a fresh syringe1 ml of the vaccine was withdrawn and was added to 99 ml of the diluent. The pervious steps were repeated till dilution Using 5 Tryptone soya agar medium (TSA) plates for each dilution, 0.1 ml was inculcated to TSA plates and spread by a sterile glass from each dilutions. The plates were incubated at 37 ºC for 1-2 days. The number of colonies was counted and the number of viable organisms per ml of vaccine was calculated as follow: 0.1 1/dilution number of colonies = CFU/ml Inactivation of S. Typhimurium culture: It was done according to (Charles et al,.1994). The inactivating agent (formaldehyde solution 37%) was added to the bacterial suspension in 0.3% of final concentration. The inactivation was carried out under stirring for 24 hrs at 24ºC to complete the inactivation process. The inactivated culture was neutralized with Sodium meta-bisulfite then stored at temperature of 5-7ºC. Preparation of the gel adjuvanted S. Typhimurium formalized inactivated vaccine: Each 0.5 ml of vaccine (equal to 1 vaccinal dose) was adjusted to contain S. Typhimurium inactivated bacterial cells. The amount of formalized inactivated bacterial cells suspension from each strain that represents 500 doses was calculated and centrifuged at 5000 rpm for 20 minutes at 4ºC. The supernatant was discarded and the bacterial cell pellets were collected. Bioscience Research, 2018 volume 15(2):

3 The bacterial cell pellets were re suspended in 200 ml PBS containing 500 doses of the 4 inactivated Salmonella strains were gently mixed and 50ml of aluminum hydroxide gel was added and thoroughly mixed a total of 250 ml containing 500 doses of each of vaccine immunogens (0.5 ml=1 vaccine dose). Potency Test: This test was carried out according to (Egyptian Standard Regulations for Evaluation of Veterinary Biologics 2009), where 25, 14-day-old, SPF chickens were inoculated intramuscularly with 0.5 ml of the prepared inactivated Salmonella Typhimurium vaccine ( CFU/0.5ml) and a booster dose was injected after 2 weeks of the first dose. Another group containing 25 of the same age and source of vaccinated group was kept as control negative group. The Challenge test with virulent Salmonella Typhimurium strain (1 x 10 9 CFU/0.5ml) was performed two weeks post boostering of vaccinated group and after 4 weeks after onset of the experiment for the control group. Both vaccinated and control groups were kept under observation for 4 weeks post challenge. The mortality rate was recorded during the observation period. Challenge test against Salmonella Typhimurium: Following the method described by (Paiva, et al,. 2009), challenge test was done using 0.5 ml of Salmonella Typhimurium containing 1 X 10 9 CFU of Salmonella Typhimurium. The chicks were challenged orally by dropper at four weeks after vaccination, and observed for one month. The degree of protection was assessed according to the severity of the clinical signs, the mortality rate and the reisolation of the challenge organisms from post mortem materials. Intestinal shedding: Cloacal swabs were collected from five birds / group at 1, 2, 3, 4, 5, 6, 7, 11, 14, 18, 21, 27 and 30 days post challenge with virulent Salmonella Typhimurium strain for exploring the frequency of Salmonella Typhimurium fecal shedding (Nahed, 1998).All samples were cultured in tubes of tryptose soya broth (TSB) and incubated for 24 hours at 37 o C. A loopful of broth was then streaked onto Salmonella-Shigella (S-S) agar for 24 hours at 37 o C for Salmonella isolation. Re-isolation of Salmonella Typhimurium (Clearance test): Four weeks post challenge, five chickens from both vaccinated and control groups were slaughtered and the liver, spleen and heart were collected for re- isolation of Salmonella Typhimurium. RESULTS AND DISCUSSION Worldwide, salmonellosis is a serious medical and veterinary problem and raises great concern in the food industry. In recent years, Salmonella enterica serovar Enteritidis has replaced serovar Typhimurium as the primary etiologic agent of Salmonella infections in many countries (Rabsch et al., 2001). Many researchers all over the world has been trying to control and eradicate salmonellosis in poultry by vaccination. Live attenuated Salmonella vaccines may be hazardous because the residual virulence due to insufficient attenuation (Arnon et al., 1983). Inactivated vaccines for the prevention of avian salmonellosis have been reported by several authors (Liu et al., 2001). The current study was designed to study the immunological effect of formalized inactivated Salmonella Typhimurium vaccine in chickens. Regarding to the results of shedding of Salmonella Typhimurium from chickens vaccinated with prepared inactivated formalized Salmonella Typhimurium vaccine after challenge with virulent Salmonella Typhimurium strain as shown in Table (1), it is noticed that out of 60 samples (5 samples per day), 10 samples were positive with a percentage of %. The previous results are nearly similar to those obtained by (Rafik, 2010) who stated that the shedding percentage in the vaccinated group after 4 weeks of vaccination was 10.5%. While, in case of control group (nonvaccinated but infected with virulent Salmonella Typhimurium), out of 60 samples, 30 samples were positive with a percentage of 50%. This result is in agreement with that obtained by (Gehad et al., 2011) who recorded that the shedding percentage in control chickens after infection with Salmonella Typhimurium was 55.4%. Concerning with the results of protection test applied to chickens vaccinated with the prepared inactivated formalized Salmonella Typhimurium vaccine and chickens served as a control against challenge with virulent Salmonella Typhimurium strain as shown in Table (2), it is clear that the percentage after 4 weeks post challenge was 88% in case of vaccinated group, while it was 20% in case of control group. Bioscience Research, 2018 volume 15(2):

4 Table.1 Salmonella Typhimurium shedding from chickens (vaccinated and control) after challenge with virulent Salmonella Typhimurium strain Group Table.2 Protection against Salmonella Typhimurium of chickens vaccinated with formalized inactivated Salmonella Typhimurium vaccine and control groups after challenge with virulent Salmonella Typhimurium strain No. of dead birds /Week Table.3 Clearance of Salmonella Typhimurium from Liver, Spleen and Heart of chickens vaccinated with formalized inactivated Salmonella Typhimurium vaccine and control groups after challenge with virulent Salmonella Typhimurium strain Groups No. of samples Liver Spleen Heart Total positive / Total No. % of positive Group (1) 15 0/5 1/5 0/5 1/ % Group (2) 15 4/5 5/5 2/5 11/ % Group (1): No. of samples Days post challenge (five samples per day) Chickens vaccinated with formalized inactivated Salmonella Typhimurium vaccine. Group (2): Control negative group. somewhat higher (4.2%) in chicken group which was vaccinated with live attenuated Salmonella Typhimurium vaccine and in non-vaccinated control group was (83.3%). The above mentioned results is in coincidence with the previous observations reported by (Hazem, 2013) who stated that the protection rate four weeks after challenge of chicken vaccinated with local formalized gel adjuvant vaccine was (88%). As well as the Egyptian Standard Regulations for Evaluation of Veterinary Biologics (2009) stated that the inactivated Salmonella Typhimurium vaccine will be satisfactory if the protection % in vaccinated birds is not less than 70% and not more than 20 % in case of control birds. Concerning with the results of S. Typhimurium isolation (clearance test) from liver, spleen and heart shown in Table (3), it was found that the re isolation of S. Typhimurium organism from liver, spleen and heart after 4 weeks post challenge were (6.7 %) in chicken group which was vaccinated with the prepared inactivated formalized Salmonella Typhimurium vaccine and (73.3%) in case of non vaccinated and infected group. These results were somewhat supported by previous researchers (Gehad et al., 2011) who found that the percentages of re isolation showed No of positive / Total No / /60 50 Group No. of chickens 1 st post challenge 2 nd 3 rd 4 th week No of Positive / Total No. Mortality % % of +ve Protection % week week week / / CONCLUSION From the aforementioned results, it could be concluded that the prepared inactivated formalized Salmonella Typhimurium vaccine is potent where it reduced both the re isolation and shedding percentages and induced good protection in comparing with the non-vaccinated control group. And it is recommended to use this vaccine in birds to protect them against infection with Salmonella Typhimurium. CONFLICT OF INTEREST The authors declared that present study was performed in absence of any conflict of interest. ACKNOWLEGEMENT All members of Central Laboratory for Evaluation of Veterinary Biologics, Abbasia, Cairo, Egypt AUTHOR CONTRIBUTIONS NE and NAS designed the experiments and ME Bioscience Research, 2018 volume 15(2):

5 wrote and reviewed the manuscript. NAS and GAY performed all the experiments. All authors read and approved the final version. Copyrights: author (s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. REFERENCES Ahmed Z.A.M., Sedik M., Alharery M.D, Khalaf M.A., Nasr S. A. and Abdelrahman H.A (2012) Microbial ecology of composting dead poultry and their wastes Global Veterinaria 9 (6): Abdel-Moein K.A., El-Hariri, M.D., Wasfy, M.O., Samir, A. (2017) Occurrence of ampicillin-resistant Enterococcus faecium carrying esp gene in pet animals: An upcoming threat for pet lovers J Glob Antimicrob Resist. 9: Arnon, R.M.; Shapira, M. and Jacob, C.O. (1983): Synthetic vaccines. J. Immunol. Methods, 61: Barrow, P.A. and Tucker, J.F. (1986): Inhibition of colonization of the chicken caecum with Salmonella Typhimurium by pre-treatment with strains of Escherichia coli. J. Hyg., 96: Berghaus, R.D.; Thayer, S.G.; Maurer, J.J. and Hofacre, C.L. (2011): Effect of vaccinating breeder chickens with a killed Salmonella vaccine on Salmonella prevalences and loads in breeder and broiler chicken flocks. J Food Prot. 74(5): doi: / X.JFP Charles,S.D.; Hussein, I.; Nagraja,K.V. and Sivanadan, V. (1994): Adjuvanted subunit vaccines for the control of Salmonella enteritidis infection in turkeys. Am. J. Vet. Res., 55 (5): Clifton-Hadley, A.F.; Breslin, M.; Venables, M.L.; Sprigings, A.K.; Cooles, W.S.; Houghton, S. and Woodward, J.M. (2002):A laboratory study of an inactivated bivalent iron restricted Salmonella enterica serovars Enteritidis and Typhimurium dual vaccine against S. Typhimurium challenge in chickens. Vet. Microbiol.,89 (2-3): Egyptian Standard Regulations for Evaluation of Veterinary Biologics (2009): Central Laboratory for Evaluation of Veterinary Biologics (CLEVB), Second Edition, Volume (I), Pages Elhariri, M., Hamza, D., Elhelw, R., & Dorgham, S. M. (2017a). Extended-spectrum betalactamase-producing Pseudomonas aeruginosa in camel in Egypt: potential human hazard. Annals of Clinical Microbiology and Antimicrobials, 16(1), 21. Elhariri M., Aleslamboly Y.S., Elshater M. A., Elhelw R. and Refai M.K. (2017b) Rapid Salmonella Detection in Different Food Samples by Direct-PCR. Bioscience Research, 14(4): Gehad A. Youssef, Nashwa A. Ezzeldeen, Ashgan M. Mostafa and N.A. Sherif (2011): Effects of Isolated Lactobacillus acidophilus as a Probiotic on Chicken Vaccinated and Infected with Salmonella typhimurium. Global Veterinaria 7 (5): Groves, P.J.; Sharpe, S.M.; Muir, W.I.; Pavic, A. and Cox, J.M. (2016): Live and inactivated vaccine regimens against caecal Salmonella Typhimurium colonization in laying hens. Vet J., 94 (10): doi: /avj Hazem Mohammed Ibrahim Mostafa (2013): Immunological studies on polyvalent Salmonella vaccines in poultry. PhD. Thesis, Department of Microbiology, Fac. Vet. Med., Cairo Univ. Hossam, Mahmoud, Armanious Wagih, Elenbawy Mona, Elhariri Mahmoud, Elhelw Rehab, and El-Din Taher Salah."The Recovery and Molecular Diagnosis of Mycoplasma gallisepticum Infection in Commercial Poultry Flocks in Egypt."Indian Journal of Science and Technology 9, no. 29 (2016). Khalifa R., Eissa S., El-Hariri, M. and Refai M. (2014) Sequencing Analysis of Mycoplasma gallisepticum Wild Strains in Vaccinated Chicken Breeder Flocks.J. Mol. Microbiol. Biotechnol. 24: Liljebjelke, K. A., C. L. Hofacre, T. Liu, D. G. White, S. Ayers, S. Young, and J. J. Maurer Vertical and horizontal transmission of Salmonella within integrated broiler production system. Foodborne Pathog. Dis. 2: Liu, W.; Yang, Y.; Chung, N. and Kwang, J.; (2001): Induced of humeral immune Bioscience Research, 2018 volume 15(2):

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