_____________________________________________________________________________________________________ *Corresponding author: E-mail: johnisa66@yahoo.com; Asian Journal of Immunology 2(1): 18-25, 2019; Article no.AJI.52667 Immunomodulatory Effects of Honey in Wistar Rats Infected with Salmonella typhimurium Justinah F. John-Isa1*, Tinuola T. Adebolu1 and Victor O. Oyetayo2 1 Department of Microbiology, Federal University of Technology Akure (FUTA), P.O.Box 704, Akure, Ondo State, Nigeria. 2 Department of Microbiology, Federal University of Technology, Akure, Nigeria. Authors’ contributions This work was carried out in collaboration among all authors. Author JFJI designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors TTA and Author VOO managed the analyses of the study. Author TTA managed the literature searches. All authors read and approved the final manuscript. Article Information Editor(s): (1) Dr. Jaffu Othniel Chilongola, Department of Biochemistry and Molecular Biology, Kilimanjaro Christian Medical University College, Tumaini University, Tanzania. Reviewers: (1) Asma Bouasla, University Souk-Ahras, Algeria. (2) Oyiborhoro Onoriode, University of Medical Sciences, Nigeria. (3) Syed Umer Jan, University of Balochistan, Pakistan. Complete Peer review History: http://www.sdiarticle4.com/review-history/52667 Received 10 September 2019 Accepted 14 November 2019 Published 19 November 2019 ABSTRACT Aim: To evaluate the immunomodulatory effects of honey on Wistar rats infected with Salmonella typhimurium. Place and Duration of Study: Research laboratory of The Federal University of Technology Akure (FUTA), Ondo State, Nigeria between July, 2019 and September, 2019. Methodology: A total thirty – nine (39) apparently healthy Wistar rats, three (3) rats per group were used in this study. Twelve (12) out of the rats were used to determine the infectivity dose of S. typhimurium on the rats and twenty – seven (27) rats for infection and treatment assay. The rats were divided into nine (9) groups of 3 rats per group, the first 8 groups were infected with S. typhimurium and treated for seven (7) days with honey, augmentin and oral rehydration solution (ORS) (different treatment for different groups) except group 1 that was infected and not treated and group 9, that was not infected, not treated. The blood samples of all the rats was collected after treatment to study the effect of honey on the haematological parameters of the rats. Original Research Article John-Isa et al.; AJI, 2(1): 18-25, 2019; Article no.AJI.52667 19 Results: Honey administered at 2ml and 3ml twice daily to the S. typhimurium infected rats exerted good therapeutic potential in combating diarrhoea in the animals. Also, in these group of rats, honey caused an increase in the PCV, RBC, HB and lymphocytes which displays honey to be a good immunostimulator and immunomodulator. Conclusion: Honey exerted therapeutic, haematinic and immunomodulatory potentials in rats infected with S. typhimurium. These findings therefore could be exploited in the treatment of diarrhoeal diseases caused by this bacterium. Keywords: S. typhimurium; wistar rats; honey; augmentin; Oral Rehydration Solution (ORS); immnuomodulation. 1. INTRODUCTION Salmonella typhimurium is a Gram-negative, flagellated, aerobic (oxygen-consuming) bacteriium, the major cause of human salmonellosis [1], a type of gastroenteritis, or inflammation of the intestine [2]. S. typhimurium is also a frequent cause of acute, self-limiting food borne diarrhoea. It is spread primarily by contaminated food and drink, but it can come in contact with human via direct contact with an infected animal or pet [1]. Salmonella typhimurium induces a systemic infection in rats, so S. typhimurium-infected rats have been extensively used as models for the understanding of the immunological and antibacterial effect of honey. Diarrhoeal diseases are among the leading causes of morbidity and mortality in young children in developing countries [3]. It is characterized by frequent, loose and watery stool which may result in dehydration and in severe cases, death. Each year, an estimated 2.5 billion cases of diarrhoea occur among the children under five years of age, and estimates suggest that overall incidence has remained relatively stable over the past two decades. Although, diarrhoea is self-limiting however when it is as a result of bacterial infection, antibiotic therapy might be required but because of the high resistance rate of bacteria to available antibiotics, administration of antibiotics may not result in recovery of patients. Moreover, some of these antibiotics can also induce diarrhoea known as “antibiotic induced diarrhoea” [4]. Therefore, it becomes imperative to search for alternatives to conventional antibiotics to treat this disease. In most ancient cultures, honey has been used for both nutritional and medical purposes. The belief that honey is a nutrient, a drug and an ointment has been carried into our days, and thus, an alternative medicine branch, called ‘‘apitherapy’’, has been developed in recent years, offering treatments based on honey and other bee products against many diseases including bacterial infections. Honey has been reported to have immunomodulatory and antibacterial activity on bacteria found in wounds [5], responsible for food spoilage [6], common diarrhoeagenic bacteria such as S. typhimurium [7] and many other bacterial species. It becomes worthwhile therefore to investigate whether honey has therapeutic and immunostimulatory potentials in Wistar rats infected with S. typhimurium in addition to nits antibacterial potential. 2. MATERIALS AND METHODS 2.1 Location and Duration of the Research The research was carried out in the Graduate Research Laboratory of Department of Microbiology, Federal University of Technology, Akure, Ondo State, Nigeria between July and September, 2019. 2.2 Honey Sample The honey sample used was collected from FUNAAB, Abeokuta Ogun State. It’s of wild flora source. 2.3 Test Organism The test organism used was Salmonella typhimurium. 2.4 Isolation and Identification of the Test Organism S. typhimurium was isolated from poultry droppings, the droppings was serially diluted in sterile distilled water using the method of Boateng and Diunase [8]. The dilutions were plated on Salmonella - Shigella agar to isolate the bacterium and was identified based on morphological and biochemical characteristics according to the method of Rozanska [9]. 2.5 Experimental Animals A total of 39 female Wistar rats of weight range 60-90 g were used for the study. The animals John-Isa et al.; AJI, 2(1): 18-25, 2019; Article no.AJI.52667 20 were purchased at Animal Production and Health Dept. of Federal University of Technology Akure, Ondo State. They were brought to the animal house of Microbiology Department, FUTA and acclimatized for 7 days before the commencement of this work. The animals were fed with broiler starter and clean water twice daily. 2.6 Determination of Infectivity Dose (ID) of Salmonella typhimurium A total of twelve (12) female apparently Wistar rats was used to determine infectivity dose. The rats were divided into four groups of 3 rats per cage. This was done using standard method described by Adebolu et al. [10]. A colony of S. typhimurium of 24 hrs old was inoculated into 100 ml of Nutrient agar, incubated at 37ºC for 18 – 24 hrs. The cells were harvested by centrifuging at 3000 rpm for 15 minutes. The supernatant was decanted and 10ml of sterile normal saline was poured into the tube and was further centrifuged to wash the cells, this was done three times. Serial dilution was carried on the harvested cells and 1ml was taken from each of the different concentrations already prepared to infect different groups of the experimental animals respectively. The dilution that produced the symptoms of illness in all of the animals was taken as the infectivity dose (ID) of the organism. 2.7 Experimental Design A total of 27 female apparently healthy Wistar rats were assigned into nine (9) treatment groups designated as 1 – 9. i.e. 3 rats per cage. Rats in group 1 were infected with the ID of S. typhimurium and not treated, rats in group 2 were infected and treated with 1 ml raw honey 12 hourly, group 3 infected and treated with 2 ml raw honey 12 hourly, group 4 infected and treated with 3ml raw honey 12 hourly, group 5 infected and treated with 0.5 ml Augmentin (30 mg/kg/day) 12hourly, group 6 infected and administered honey – ORS 12 hourly, group 7 infected and administered 1ml commercial ORS 12 hourly, group 8 infected and administered 1ml homemade ORS 12 hourly and group 9 not infected, not treated (control group). 2.8 Infection of Rats with the ID of S. typhimurium The infection of the animals was done using the infectivity dose (ID) of the organism by orogastrically dosing them according to the method of Adebolu et al. [10]. The infectivity dose used in this study was calculated to be 1.5 x10 8 cfu/ml. 2.9 Treatment of Infected Rats Treatment begins 24 hours after which infection has set in, specific volume of honey, augmentin, honey – ORS, ORS both commercial and home - made variants were administered to the infected rats for 7 days according to Oladunmoye [11]. 2.10 Isolation, Identification and Enumeration of S. typhimurium in the Faeces of Infected Rats One gram (1 g) of faeces of the infected rats were aseptically collected, serial dilution was done on them and plated on salmonella shigella agar in order to isolate the Salmonella typhimurium present in the rats and monitor their bacterial count throughout the experiment [12]. 2.11 Weighing of Animals The weight of the animals were taken throughout the pre and post ingestion period using the method of Momoh et al. [13]. 2.12 Haematological Assay The blood of the infected and uninfected rats was collected weekly into EDTA bottles after which the Packed Cell Volume (PCV), Haemoglobin (HB), Red blood Cell (RBC), White Blood Cell (WBC) and differential leukocytes counts of the collected blood samples were evaluated according to the method described by Baker et al. [14]. 2.13 Statistical Analysis All experiments were done in triplicates, Mean, Standard deviation were calculated for all data using Descriptive Statistics and difference between means was determined by Duncan’s New Multiple Range Test at p≤.05. 3. RESULTS AND DISCUSSION All the rats infected with S. typhimurium and treated with honey recovered by the 3 rd day while those ones that was administered honey – ORS John-Isa et al.; AJI, 2(1): 18-25, 2019; Article no.AJI.52667 21 and the ones with homemade ORS recovered by day 4 and those treated with augmentin and those administered commercial ORS recovered by the 5 th day, those that were infected but not treated started to show signs of recovery by the 6 th day. The recovery without treatment by the 6 th day confirms that acute diarrhoea is self limiting according Chen et al. [15]. The mean recovery times of rats infected with Salmonella typhimurium and treated with honey –ORS was significantly reduced when compared with infected and not treated group (Table 1). This is agreement with the work of Beretta et al. [16]. There was no evidence of S. typhimurium in the faeces of rats treated with 1 ml, 2 ml, 3 ml of honey and administered 1 ml of honey –ORS 12 hourly for 7 days (Fig. 1). There was no evidence of Salmonella typhimurium in the faeces of rats treated with 1 ml, 2 ml, 3 ml of honey and administered 1 ml of honey – ORS 12 hourly for 7 days. (Fig. 1). Table 1. Physical observations of the wistar rats during infection with Salmonella typhimurium and treatment Group of rats Treatment Interval (Days) 1 2 3 4 5 6 7 1 Infected and not treated with honey RA, EL, UF,PM,SS RA, EL, UF, PM, SS RA, EL, UF, PM, SS RA, EL, UF, PM, SS RA, EL, UF, PM, SS A, EW, FS, NM, SF A, EW, FS, NM, SF 2 Infected and treated with 1 ml honey (12 hourly) RA, EL, UF, PM, SS RA, EL, UF, PM, SS A, EW, FS, NM A, EW, FS, NM A, EW, FS, NM A, EW, FS, NM A, EW, FS, NM 3 Infected and treated with 2 ml honey (12 hourly) RA, EL, UF, PM, SS RA, EL, UF, PM, SS A, EL, UF, PM, SS A, EL, FS, NM, SF A, EL, FS, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF 4 Infected and treated with 3 ml honey (12 hourly) RA, EL, UF, PM, SS RA, EL, UF, PM, SS A, EL, UF, PM, SS A, EL, FS, NM, SF A, EL, UF, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF 5 Infected and treated with 0.5 ml Augmentin (12 hourly) RA, EL, UF, PM, SS RA, EL, UF, PM, SS RA, EL, UF, PM, SS RA, EL, UF, PM, SS A, EW, FS, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF 6 Infected and treated with 1 ml Honey - ORS (12 hourly) RA, EL, UF, PM, SS RA, EL, UF, PM, SS RA, EL, UF, PM, SS A, EW, FS, NM, SF A, EW, FS, NM A, EW, FS, NM, SF A, EW, FS, NM, SF 7 Infected and treated with 1 ml ORS (12 hourly) RA, EL, UF, PM, SS RA, EL, UF, PM, SS RA, EL, UF, PM, SS RA, EL, UF, *PM, SS A, EW, FS, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF 8 Infected and treated with 1 ml homemade (12 hourly) RA, EL, UF, PM, SS RA, EL, UF, PM, SS RA, EL, UF, PM, SS A, EW, FS, *PM, SS A, EW, FS, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF 9 Not infected and not treated A, EW, FS, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF A, EW, FS, NM, SF Key: A = Activeness, RA = Reduced activity, EL = Eating little, EW = Eating well, UF = informed stool, FS = Formed stool, SF = Smooth fur, SS = Scattered fur, PM = Presence of mucous, *PM = High presence of mucous, NM = No mucous John-Isa et al.; AJI, 2(1): 18-25, 2019; Article no.AJI.52667 22 Fig. 1. Total counts of S. typhimurium in the faeces of wistar faeces after infection and treatment Key: A = Infected and not treated, B = Infected and treated with 1 ml honey, C = Infected and treated with 2 ml honey, D = Infected and treated with 3 ml honey, E = Infected and treated with 0.5 ml augmentin, F = Infected and administered with 1 ml honey – ORS, G = Infected and administered with 1ml commercial ORS, H = Infected and administered with 1 ml homemade ORS, TM1 = Treatment day 1, TM2 = Treatment day 2. TM3 = Treatment day 3, TM4 = Treatment day 4 and TM7 = Treatment after day 7 The infected rats lost weight as a result of the infection with S. typhimurium however, administration of honey to the infected rats caused a significant increase (p<0.05) in their weight but the infected and not treated rats recorded weight loss for a longer duration than those that were administered different volumes of honey (Fig. 2A-E). The observation that the infected and not treated rats did not gain back their body weight throughout the duration of the research is in agreement with the work of Momoh et al. [13]. A B 0 1 2 3 4 5 6 7 24 h/TM1 48 h/TM2 72 h/TM3 96 h / TM4 168 h / TM7 lo g cf u /m l Infection period (h)/Treatment A B C D E F G H 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) Period (Day) Infected and not treated with honey Not infected and not treated 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) Period (Day) Infected and treated with 1ml honey Not infected and not treated John-Isa et al.; AJI, 2(1): 18-25, 2019; Article no.AJI.52667 23 C D E Fig. 2 A–E. Average weight of wistar rats before infection with S. typhimurium, at the onset of infection and after treatment with honey and augmentin Infection of rats with S. typhimurium caused a decrease in their PCV, HB and RBC and increase in their neutrophil counts, showing a sign of infection but after treatment with honey (between 2ml and 3ml), there was no significant difference in the PCV, WBC of the group of rats treated with honey and the group not infected, not treated (control) (Tables 2a and b). Administration of honey to apparently healthy rats (control) caused a significant (p<.05) increase in the PCV and lymphocytes of the rats. This shows that the honey has both haematinic and immunomodulatory potentials. 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) Period (Day) Infected and treated with 2ml honey Not infected and not treated 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) Period (Day) Infected and treated with 3ml honey Not infected and not treated 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) Period (Day) Infected and treated with 0.5ml Augmentin Not infected and not treated John-Isa et al.; AJI, 2(1): 18-25, 2019; Article no.AJI.52667 24 Table 2a. Effect of honey on the haematological parameters of wistar rats infected with S. typhimurium Group PCV (%) HB (g/L) WBC (10 9 /L) RBC (10 12 g/L) 1 32.50 ± 4.95 b 10.85 ± 1.66 b 12.70 ± 0.28 a 3.00 ± 0.82 b 2 40.00 ± 2.83 ab 13.50 ± 0.71 ab 11.70 ± 0.28 ab 3.20 ± 0.25 ab 3 42.50 ± 2.12 a 14.14 ± 0.66 a 8.77 ± 0.49 c 4.04 ± 0.87 ab 4 43.00 ± 4.24 a 14.30 ± 1.36 a 8.94 ± 0.42 c 4.29 ± 0.07 a 5 40.33 ± 4.51 ab 13.50 ± 1.51 ab 10.86 ± 0.17 abc 4.06 ± 0.30 ab 6 35.50 ± 0.71 ab 11.90 ± 0.28 ab 10.88 ± 0.05 abc 3.66 ± 0.03 ab 7 39.67 ± 4.51 ab 13.19 ± 1.56 ab 9.56 ± 1.92 bc 4.07 ± 0.34 ab 8 35.00 ± 6.00 ab 11.64 ± 2.00 ab 9.83 ± 1.47 bc 3.66 ± 0.53 ab 9 42.33 ± 2.08 a 13.66 ± 0.35 ab 8.82 ± 1.06 c 4.07 ± 0.58 ab Key: 1 = Infected and not treated, 2 = Infected and treated with 1ml honey, 3 = Infected and treated with 2ml honey, 4 = Infected and treated with 3ml honey, 5 = Infected and treated with 0.5ml augmentin, 6 = Infected and administered with 1ml honey – ORS, 7 = Infected and administered with 1ml commercial ORS, 8 = Infected and administered with 1ml homemade ORS, 9 = Not Infected, not treated, PCV = Packed cell volume, HB = Haemoglobin concentration, WBC = White blood cell and RBC = Red blood cell Table 2b. Effect of honey on the haematological parameters of wistar rats infected with S. typhimurium contd Group Lymphocytes (%) Neutrophils (%) Monocytes (%) Eosinophils (%) Basophil (%) 1 31.50 ± 0.71 f 64.50 ± 0.71 a 3.00 ± 0.00 a 1.50 ± 0.71 a 1.00 ± 0.00 a 2 37.50 ± 0.71 bc 61.00 ± 1.41 ab 2.50 ± 0.71 a 2.00 ±1.41 a 1.00 ± 0.00 a 3 40.00 ± 2.83 ab 56.50 ± 0.71 b 1.50 ± 0.71 a 1.00 ± 0.00 a 0.00 ± 0.00 4 40.50 ± 0.71 a 57.00 ± 1.41 b 1.50 ± 0.71 a 1.00 ± 0.00 a 0.00 ± 0.00 5 39.33 ± 150 abc 56.00 ± 4.00 b 1.67 ± 1.15 a 1.00 ± 0.00 a 0.00 ± 0.00 6 37.00 ± 1.41 cd 60.00 ± 2.83 ab 2.00 ± 1.41 a 1.50 ± 0.71 a 0.00 ± 0.00 7 32.67 ± 1.15 ef 60.33 ± 3.79 ab 2.00 ± 1.00 a 1.67 ± 0.58 a 0.00 ± 0.00 8 34.67 ± 0.58 de 60.67 ± 1.15 ab 2.33 ± 0.58 a 1.67 ± 0.58 a 1.00 ± 0.00 a 9 40.00 ± 1.00 ab 57.33 ± 1.15 b 1.33 ± 0.58 a 1.33 ± 0.58 a 0.00 ± 0.00 Key: 1 = Infected and not treated, 2 = Infected and treated with 1ml honey, 3 = Infected and treated with 2ml honey, 4 = Infected and treated with 3ml honey, 5 = Infected and treated with 0.5ml augmentin, 6 = Infected and administered with 1ml honey – ORS, 7 = Infected and administered with 1ml commercial ORS, 8 = Infected and administered with 1ml homemade ORS and 9 = Not infected, not treated 4. CONCLUSION This study has shown that honey sample from FUNAAB (HF) caused enhanced immune response in the rats against S. typhimurium. This honey also has haematinic, immunomodulatory, and immunostimulatory potentials in rats infected with S. typhimurium. These findings therefore could be exploited in boosting the immune system and in the treatment of diarrhoeal diseases caused by this bacterium. CONSENT It is not applicable ETHICAL APPROVAL As per international standard written ethical approval has been collected and preserved by the author(s). ACKNOWLEDGEMENTS The authors appreciate the effort made by Mr Jimoh, Kabiru Ayobami for cross checking the statistical analysis. COMPETING INTERESTS Authors have declared that no competing interests exist. REFERENCES 1. Torpdahl M, Tsai-Ling L, Shiu-Yun L, Ishien L, Sung-Hsi W, and Chien- Shun C. 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