Leaves of Lantana camara (Verbenaceae), Leaves and roots of Uvaria chamae (Annonaceae), Leaves of Phyllanthus amarus Schumach.&.Thonn were collected from the wild. In Benin, no permission was necessary to collect these samples.
Identification was done at the Beninese National Herbarium (University of Abomey Calavi) by Professor Hounnankpon YEDOMONHAN (https://chercheurs.inrab.org/details/173).
Reference numbers in the Herbarium are AA6686/HNB for Phyllantus amarus, AA6687/HNB for Uvaria chamae, AA6688/HNB for Lantana camara. This experimental research has been done in compliance with our Institution (University of Abomey-Calavi), national and international guidelines. All the plants collected in this study have been replaced by young ones in order to maintain the species survival.
210 three-day old and 90 three-week old Isa Brown male chicks were used for the experimentation. The birds were taken from a commercial hatchery ‘’Terre et Associés’’, Abomey-Calavi (Benin). The birds were kept in an enclosure carefully cleaned and disinfected. During the experiment, the animals took water and feed. All experiments were conducted according to the protocol approved by Ethical committee of Research Unit of Applied Microbiology and Pharmacology of natural substances. After the procedure, animals were killed in compliance with the Beninese code for the care and use of animals for scientific purposes. All animal restraint for killing was ethically carried out carefully to avoid fear, distress or pain. In order to limit fear, distress or pain related to restraint, ketamine was administered to animals.
Eleven bacterial strains were used:
- Salmonella Typhimurium ATCC 14028 were acquired from Research Unit in Applied Microbiology and Pharmacology of natural substances, University of Abomey-Calavi, Benin.
- Ten multiresistant strains of Salmonella spp: they were isolated by Deguenon et al. [27]. The strains were multidrug-resistant to penicillins, first generation cephalosporins and some aminoglycosides.
Production of aqueous and ethanolic extracts
Method described by Legba et al. (2018) has been used. The organs were dried in the laboratory at a temperature of 16 ° C for. The dried material was then powdered using a Retsch SM 2000/1430 / Upm / Smf type mill. Fifty grams of powder were macerated in 500 ml of distilled water or ethanol on a Stuart Bioblock Scientific Fisher stirrer for 72 hours at room temperature. Homogenate was filtered with hydrophilic cotton and Wattman No. 1 paper. This filtrate was then dried at 40 ° C for ethanolic extract and 50°C for aqueous extract in the Pasteur oven.
In vitro anti-Salmonella assessment of aqueous and ethanolic extract of Uvaria chamae, Phyllantus amarus and Lantana camara
Salmonella Typhimurium 14028 and the ten multiresistant Salmonella spp were used. A 24-hour pure colony of portion from the Mueller Hinton medium of each strain was emulsified in 5 ml of physiological water to give a turbidity of 0.5 Mc Farland [34].
Each inoculum was seeded by swab on Petri dishes containing Mueller Hinton agar. Wells of 6 mm diameter were hollowed out using sterile Pasteur pipette tip. 50 μl of each extract was deposited in the wells. A well containing sterile distilled water served as a negative control. After 1 hour pre-diffusion at room temperature, the petri dish was incubated at 37 ° C in an oven for 24 hours. After incubation period, the dishes were examined for the measurements of the zones of inhibition. [34].
Method used by Legba et al. (2018) were used. 100 μl of the stock solution of each extract prepared at 200 mg / ml were added to 100 μl of Mueller-Hinton Broth. A series of two-fold dilution from well to well was made then 100 μl of different bacterial suspensions were added. Positive and negative controls were prepared respectively by adding 100 μl of MH broth to 100 μl of bacterial suspension and 100 μl of MH broth to 100 μl of the extracts. The plates were incubated at 37°C for 24 hours. The MIC was estimated using Tetrazolium. The content of each well was cultured on Agar MH Agar and incubated at 37 ° C for 24 hours for the determination of CMB. CMB is the lowest concentration of extract to which no colony of bacteria can be observed. The antibiotic potency (a.p) of each extract was then calculated with the formula CMB/CMI.
Experimental infection of three-day-old Isa Brown male chicks with Salmonella Typhimurium ATCC 14028
A preliminary microbiological examination of the cloaca of the chicks helped to check if chicks are exempt from Salmonella spp. A cloaca swab was performed on all chicks and Salmonella spp were searched according to the method described by Deguenon et al.[27].
Salmonella inoculums were prepared from a pure isolate of the bacterial strain in distilled water at three selected concentrations: 3 x 108 CFU (Concentration 1), 6 x 108 CFU (Concentration 2), 9 x 108 CFU (Concentration 3).
- Groups 1 (n=3): 2 ml of Inoculum concentration 1
- Groups 2 (n=3): 2 ml of Inoculum concentration 2
- Groups 3 (n=3): 2 ml of Inoculum concentration 3
- Groups 4 (n=3): 2 ml of Distilled water
Oral inoculation was performed using 20-gauge feeding needle and disposal syringe. To confirm the viability of the strain used for inoculation, a sample of each inoculum (about 100 microliters) was taken before and after oral administration for culture at 37 ° C for 24-45 hours.
The birds were observed for 10 days and the symptoms of salmonellosis were recorded. On days 3, 6, and 9, the feces from each lots of chicks were collected. Salmonella was sought using method described par Deguenon et al.[27]. Five (5) grams of fecal samples were transferred immediately following collection in 45 ml of buffered peptone water. Samples were homogenized in the broth either by vortex mixer and then incubated for 18-24 h at 37 °C. 1 ml of pre-enrichment was then inoculated in 9 ml of selenite cystine broth for 24 h. Isolation was done on petri-dishes containing Xylose Lysine Decarboxylase (XLD) and incubated for 24h. API 20E Gallery was used for positive identification of all suspicious colonies from fecal material. Salmonella counts were performed on Day 9 samples to assess bacterial load. The method described by Pande et al. [23].
In vivo anti-salmonella assessment of Leaves aqueous extract of Uvaria chamae
Salmonella Typhimurium 14028 and Salmonella spp (P19) were used for this step. After inoculation, chicks were treated with leaves aqueous extract of Uvaria chamae and colistin.
For each strain, 90 three-day-old chicks were randomly assorted into six groups:
- Group 1: non-infected and non-treated (G1, n=18)
- Group 2: infected and untreated (G2, n=18)
- Group 3: infected and treated with 200 mg/l of Colistin (G3, n=18)
- Group 4: infected and treated with 100 mg/l of Uvaria chamae leaves aqueous extract (G4, n=18)
- Group 5: infected and treated with 200 mg/l of Uvaria chamae leaves aqueous extract (G5, n=18)
- Group 6: infected and treated with 400 mg/l of Uvaria chamae leaves aqueous extract (G6, n=18).
Preliminary examination and inoculation were performed as previously but a single concentration of inoculum were used: 9.0 108 UFC/ml. The birds were observed for 9 days and the symptoms of salmonellosis were recorded. From the third day after infection, the chicks are subjected to oral treatment with the aqueous leaf extract of Uvaria chamae and colistin as reference antibiotic. The treatment was done for 7 days. On days 3, 6 and 9 after infection, faeces from each group were collected and Salmonella counts were performed [35].
Effects of Leaves aqueous extract of Uvaria chamae on biochemical and hematological parameters
In order to evaluate the toxicity of Uvaria chamae leaves aqueous extracts for chicks, the different concentrations of extracts tested and Colistin were administered for 7 days to three-week-old chicks.
90 three-week-old Isa Brown male chicks were divided in five groups:
- Group 1: oral administration of 100 mg/L of Leaves aqueous extract of chamae (G1, n=18)
- Group 2: oral administration of 200 mg/L of Leaves aqueous extract of chamae (G2, n=18)
- Group 3: oral administration of 400 mg/L of Leaves aqueous extract of chamae (G3, n=18)
- Group 4: oral administration of 200 mg/L of Leaves aqueous extract of chamae (G4, n=18) of Colistin (G4, n=18)
- Group 5: water
The hematological (white cell Number (NB); Blood red Cell (NR), Hemoglobin (Hb), Hematocrit (The)) and biochemical (Uremia, creatinine, AST and ALT) data were recorded on Days 0, 4 and 7.
Statistical analysis
Microbiological data were analyzed using ANOVA test with Graph Pad Prism 7.0 Software. Hematological and Biochemical data were analyzed by the SPSS 17.0 Software.