This study aimed to produce scientific data on in vitro and in vivo efficacy of extracts of Uvaria chamae, Lantana camara and Phyllantus amarus on multiresistant Salmonella spp isolated in Benin. In vitro anti-salmonella tests were used to assess the activity of the extracts of the three plants and to select the extract to be used for the in vivo tests.
The performance of the in vivo efficacy test was preceded by an experimental infection which made it possible to choose the optimal concentration of the inoculum.
In vitro anti-Salmonella activity of Uvaria chamae, Phyllantus amarus and Lantana camara
The aqueous and ethanolic extracts of the leaves and bark of U. chamae showed an inhibition of Salmonella spp with the exception of the ethanolic extract of U. chamae’s roots. The aqueous extract of Uvaria chamae was active on 90% of the virulent Salmonella spp and on Salmonella Typhimurium ATCC 14028. These results can be compared to those obtained by Ogueke [17]. This author showed that at a concentration varying between 150–250 mg / ml, aqueous and ethanolic extracts of bark and the ethanolic extract of leaves of Uvaria chamae inhibited Salmonella Typhi.
Aqueous and ethanolic extracts of Phyllantus amarus inhibited Salmonella spp with maximal inhibition diameter of 12 mm. The activity of Phyllantus amarus extracts on Salmonella spp were reported in 2008. Using agar cup diffusion method, the authors showed that ethanolic extracts of P. amarus were active on Salmonella Typhi [18]. In our study, only leaves ethanolic extract of Phyllantus amarus inhibit Salmonella Typhimurium ATCC 14028 (9.33±1.53 mm). The inhibitory power of the extract on the reference strain was greater than those obtained in a previous study. For concentrations ranging from 200 to 1000 µg / ml, the inhibition diameters varied between 7 and 9 mm on Salmonella Typhimurium ATCC 6539 [19].
The ethanolic leaves extract of Lantana camara had an inhibition diameter of 8 mm on Salmonella Typhimurium ATCC 14028. The inhibitory power of extracts of Lantana camara on Salmonella spp has already been reported in the literature. Lyumugabe et al. [20] obtained an inhibition diameter of 11 mm on Salmonella Typhimurium.
Experimental infection of three-day-old chicks with Salmonella Typhimurium ATCC 14028: Preliminary test
To achieve an experimental infection requires that certain experimental conditions be met. The most important are the virulence of the strain used, the choice of the appropriate study model, the choice of the dose and the optimal infective concentration. The choice of chicks as a study model was motivated by two main reasons.
Firstly, Salmonella Typhimurium is known for his ability to infect birds, contaminate eggs [21] and be transmitted to humans. It is the main agent of salmonellosis in humans. Secondly, chicks have been used successfully for experimental infections of non-typhoid salmonellosis : for investigation of the invasiveness of Salmonella enterica in chickens [22] ; for investigation of the dynamics of egg contamination over an extended time course [23]; for investigation of pathogenicity of some avian Salmonella Serovars [24]. In this study, we choose three-day old chicks for various reasons. One had to choose an age of susceptibility, an age when the animal’s immune system is not mature enough to prevent infection. Such an age guarantees the establishment of the infection. Also it is known that in poultry, the signs of the disease are rarely observed after the first two weeks of life [25]. These strategic choices appear to be optimal since, 24 hours after the infection, the animals showed signs of salmonellosis, particularly in groups 2 and 3, which received the concentrations of inoculum 2 and 3 respectively. It was observed wet cloaca, diarrhea stool and somnolence. Clinical signs have been associated with the detection of salmonella in feces. The chosen model has therefore made it possible to reproduce the disease. Several studies have focused on the use of chicks at an age of susceptibility to optimize infection. Osman et al. (2010) used 1-day-old SPF White Leghorn chicks for inoculation with Salmonella Typhimurium. Beal et al. (2004) confirmed that Salmonella Typhimurium is a non-pathogenic commensal in chickens greater than three days of age and can colonize the tract sub-clinically for 8–9 weeks after experimental infection. However, it is possible to use older birds and have interesting results. It all depends on the nature of the study, the virulence of the strain, the concentration and the infective dose. For example, Pande et al. (2016) used with success henses of 14 weeks for oral induction with S. Typhimurium PT 9. The virulence of Salmonella Typhimurium ATCC 14028 was a guarantee because these virulence factors have been characterized by PCR by Deguenon et al.[[27]. This study showed that the strain has 5 virulence genes: invA, spvR, SpvC, FimA and Stn. Spv genes are responsible for the systemic infection and multidrug resistance in humans and animals [28]. SpvC gene is able to inhibit the activation of macrophages [29]. Presence of fimA gene indicates the presence of fimbriae which is important for Salmonella spp to adhere to epithelial cells [27]. Stn gene is suspected to contribute to enterotoxigenic potency [30]. The presence of all these genes therefore guarantees the pathogenicity of Salmonella Typhimurium and its ability to infect chicks.
This virulence explains why salmonellosis symptoms were observed in infected animals. Three infective concentrations were chosen because we had no assurance of sufficient bacterial load to induce salmonellosis in chicks of this age. We had to expand the possibilities. The results showed that only concentrations 2 and 3 could trigger the symptoms of salmonellosis. These symptoms were present until the 10th day of observation in the group 3 chicks while they disappeared between the fifth and the 7th day for the group 2. Salmonella spp were investigated in faecal specimens to support clinical observations. Salmonella spp were detected in chicks of group 2 and 3, three days after infection. Chickens infected with concentration 3 of inoculum still host Salmonella.
By relating microbiological data to clinical observations, it seems obvious that only the infective 3 concentration was able to keep the 3-day-old chicks sicked for 10 days. This observation was reinforced by the count at day 9. The disappearance of Salmonella in group 2 could be explained by a positive reaction of the immune system of the birds.
In vivo anti-Salmonella activity of Uvaria chamae using chick model
Leaves aqueous extract of U. chamae inhibit Salmonella Typhimurium ATCC in chicks at 100, 200 and 400 mg/L but the bacterial load was not canceled. This confirms the in vitro anti-Salmonella tests results which showed that this extract has a bacteriostatic effect on Salmonella Typhimurium ATCC 14028. On the other hand, at 200 and 400 mg / L, the extract showed a better in vivo activity than Colistin on virulent Salmonella spp P19. There is no scientific data on the in vivo activity of extracts of Uvaria chamae on Salmonella spp using chicks model. But interesting data exist about other natural substances. In a last study, Five-months-old local chickens, free of antibodies against fowl typhoid were used for challenge with Salmonella Gallinarum. Administration of extract of Aloe secundiflora showedincrease in the levels of interleukin 6 (IL–6) [31]. In a recent study, Piper beetle L. leaves extract when used in supplementation in drinking water helped to decrease the colonies of Salmonella sp. in small intestine of quails [32]. Mice model were also used to assess in vivo anti-Salmonella activity of natural substances. Punica granatum extract had significant effects on mortality and the numbers of viable S. Typhimuriumrecovered from feces after experimental infection [33].