Epidemiology of Non-Typhoidal Salmonella (NTS) in Humans and Animals in the Gambia and Senegal

Non-Typhoidal Salmonella (NTS) species are important food-borne pathogens. Although acute gastroenteritis is the most common clinical symptom, complications can occur resulting in bacteraemia with or without focal infections. Food products, especially food of animal origin such as poultry are associated with the transmission to humans. In Africa, NTS are among the most common cause of bloodstream infections in children younger than 5 years. Epidemiological data on NTS are lacking in Africa both for human and animal infections. Therefore, a study providing a better understanding of the factors that lead to the emergence of NTS is a prerequisite for the design of improved intervention strategies to control these pathogens. The aim of this thesis was to study the epidemiology of NTS pathogens in humans and animals in The Gambia and Senegal. Chapter 1 reviews the current status of knowledge on NTS infections in Africa with focus on The Gambia and Senegal. It also provides the background against which these studies were conducted. Chapter 2 describes the prevalence of NTS along the poultry production chain in Casamance, Senegal. Fifty seven randomly selected broiler farms, 42 street restaurants and 285 chicken carcasses were studied. The following farm prevalences were reported: 35.1, 38.6 and 29.8% in chicken faeces, on carcass skin, and in muscles, respectively. NTS were found in chicken meat servings of 14.3% of the 42 street restaurants and in 40.4% of the 285 chicken carcasses examined. The most prevalent serotypes among the eighteen identified were Salmonella Brancaster (57.9%), Salmonella Goelzau (10.7%), Salmonella Kentucky (8.4%), and Salmonella Hadar (7.3%). The following serotypes were for the first time identified in Senegal: Salmonella Bandia, Salmonella Bessi, Salmonella Brunei, Salmonella Hull, Salmonella Istanbul, Salmonella Javiana, Salmonella Magherafelt, Salmonella Molade, Salmonella oxford, Salmonella Poona, Salmonella Rubislaw, Salmonella Tamale, Salmonella Zanzibar and Salmonella Goelzau. The prevalence of NTS on skin and in muscle was significantly associated with the detection of Salmonella in feces (P≤ 0.001). The high levels of contamination of skin and muscle can be attributed to poor hygiene at the farm level and the non-hygienic handling of chicken carcass meat during and after slaughtering. This conclusion is supported by the fact that some serotypes are present both on the farm (as found in feces) and in carcasses (on skin and meat). Food can also become contaminated through environmental contact, because hygienic measures applied in the restaurants are poor. A large proportion of our isolates (77.7%) were resistant to two or more antibiotics commonly used in Senegalese veterinary practices and in human medicine (trimethoprim-sulfamethoxazole, tetracycline, trimethoprim, streptomycin, sulfonamides, and spectinomycin). The high prevalence of Salmonella in broilers in Casamance and the level of antibiotic resistance are of concern and constitute a real threat to public health. Chapter 3 reports on the molecular characterization of 261 NTS serotypes isolated in poultry and poultry products in Senegal using consecutively RAPD and MLST. These techniques have provided details on the genetic diversity among the serotypes. Twenty distinct profiles were generated by the RAPD assay, the latter corresponding to the eighteen strains obtained after serotyping. Salmonella Kentucky showed two distinct profiles; this distinction was later confirmed by MLST. The MLST assay revealed genetic diversity resulting in 19 clones of which 16 were new and have never been reported anywhere in the world. The three known clones, namely Salmonella Kentucky ST198 previously reported in Senegal, Salmonella Agona ST13 and Salmonella Istanbul ST33 were isolated in many countries from both human and animal sources. This shows that these clones are geographically widely distributed and are circulating in a wide range of hosts. However, one new clone of multi-resistant Salmonella Kentucky was found. This study provided us with new insights into the genetic diversity on NTS in Senegal. Molecular tools remain essential to study the epidemiology of NTS by tracking the sources of infection and/or contamination. These same techniques were used to study the animal to human transmission in The Gambia in the next chapter. In chapter 4, eight diarrheic children with confirmed salmonellosis and 6 healthy carriers were traced back to their compounds and Salmonella identified from the domestic animals (poultry, sheep and goat) living in close contact in the same compound. The most common serotypes identified were Salmonella Colindale in humans (21.42%) and Salmonella Poona in animals (14.28%). Among the animals, poultry carried the highest proportion of Salmonella (66.7%). In fact, poultry are considered as the most common asymptomatic carriers of Salmonella. However, serotypes in humans were different from those in animals except in one case where Salmonella Moualine was simultaneously found in chicken and a diarrheic child but in different compounds. After proceeding MLST on all isolates, we found that those two Salmonella Moualine were distinct but genetically very close because they differed at only one locus sucA. The similarity matrix of the strains revealed close genetic relatedness among Salmonella serotypes. There was at least 80% similarity and the majority varied between 98% and 100%. This showed the stability of Salmonella clones which are not subject to high genetic variability. There was therefore no indication of clonal groups which are adapted to a specific host because the genetic tree did reveal that all lineages contained isolates of mixed origin (human and animal). The association between salmonellosis and other diseases, most often malaria, in our study shows the role of opportunistic infections and malaria in NTS infections. Almost all serotypes were susceptible to all antibiotics tested. This is due to the fact that antibiotics are not yet commonly used by of the rural population in The Gambia for treatment of NTS infections as well in humans as in the animal production system. Our results do not support the hypothesis that humans and animals in close contact in the same household carry genotypically similar Salmonella serotypes. Nevertheless these findings have stirred up the problem of the transmission of NTS in Africa and have highlighted the poultry population as playing a pivotal role of healthy carriers in the epidemiology of NTS. Based on this study, we suggest other areas to be investigated such as the environment and human-to-human transmission. Little is known on the molecular epidemiology of NTS particularly with respect to their virulence genes. Therefore, to assess their occurrence and contribution to disease in humans and animals in The Gambia and Senegal, we screened all serotypes isolated from humans, animals and food in both countries (chapter 5). A total number of 185 NTS was tested by PCR for the presence of 12 virulence genes. Among these genes, 10 belong to the five described Salmonella Pathogenicity islands thought to be implicated in Salmonella pathogenesis; and the other two genes are carried by plasmids. All genes were present at a level of more than 70% except sopE and pefA which were observed in 33% and 44% of the isolates, respectively. The most prevalent gene was invA (95.5%) which is an invasion gene conserved within the Salmonella genus. It has been widely used to diagnose Salmonella in humans and animals. However, the sopE gene associated with outbreaks in human and animals was present in all serotypes isolated in humans with diarrhoea except one. Interestingly, Salmonella Istanbul and Salmonella Javiana isolated from chicken serving restaurants carried all the virulence genes of the five pathogenicity islands. There was a significant association between some virulence genes (sopB, sopE and pipD) and resistance to certain antibiotics namely amoxicillin, ticarcillin, trimethoprim plus sulfamethoxazole, tetracycline, trimethoprim, spectinomycin, streptomycin, sulfonamides and nitrofurantoin. This association shows that resistance might increase the virulence of NTS during infection. These findings showed that all strains of Salmonella isolated from humans and animals are potentially pathogenic. This is very worrying because the most virulent serotypes were also the most frequently detected in food and in animals, and caused diarrhoea in immuno-compromised children. Furthermore, virulence and resistance are intrinsically linked and the presence of the virulence genes sopB and pefA is likely to increase the resistance to a series of antibiotics (trimethoprim plus sulfamethoxazole, tetracycline, trimethoprim and sulfonamides).