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1.0 INTRODUCTION Salmonella is a genus of rod shaped, Gram negative, oxidase negative, non-spore forming, predominantly motile bacteria belonging to the family Enterobacteriacace. Salmonella strains are approximately 0.7 1.5m wide and 2.0 5.0m in length (Giannella et al., 1996). The bacterium ferments glucose and usually with the production of gas. In addition, they are able to grow in minimal media containing glucose as the sole source of carbon and ammonium ion as a nitrogen source. Most serovars are phenotypically identified by urea hydrolysis, the absence of tryptophan deaminase, non-lactose fermentation, the production of hydrogen sulphide (H2S), decarboxylase lysine and ornithine and growth on simmon citrate agar (Grimout et al., 2000). Microbial diseases constitute a major cause of deaths in many parts of the world, particularly in developing countries Salmonella has been identified as an important food and environmental pathogen. Many strains of Salmonella can infect Humans and animals and many cause pathological conditions resulting in morbidity and mortality. In industrialized countries, most of the outbreaks are associated with food borne transmission by contaminated poultry, egg, meat, milk and other dairy products (Kariuki, et al., 2002). In the United States alone, Salmonella accounts for 60 of all bacterial disease outbreaks (Chen et al., 2000). In developing countries like Nigeria, hygiene is often limited and therefore the probability of Salmonella contamination of food at various stages of processing and handling is always very high. In particular there is a tradition of poor hygiene and poverty in Ado- Ekiti, Ekiti-State, South Western, Nigeria. These conditions expose the people to a variety of food related and water- borne diseases. This is best seen in children who frequently suffer from gastroenteritis infections including bacterial diarrhoea that carry a very high mortality. Apart from common problems associated with developing countries, Nigeria has long been involved with Boko Haram insurgence which has resulted in the immigration of people from Northern Part of the country to the south leading to over- population in places like Ado-Ekiti, As a result of this, there is now an over-pressure on the water supply and sanitation infrastructures of the urban and rural areas of Ekiti-State. In many area of Ado Ekiti, Untreated sewage is channeled into rivers and streams which are the major sources of drinking water. The contaminated water is also used to irrigate plots of vegetables which are sold in nearby markets. Despite the wide range of bacterial infections, limited data exist on the bacteria quality of water and food, which are the likely sources of human infections. It is therefore difficult to evaluate the importance of Salmonellosis in Nigeria due to lack of co-ordinate epidemiological surveillance system (Akinyemi et al., 2005). Epidemiological studies to investigate Salmonella infections could assist in identifying the major routes of transmission and, in the development of control measures to interrupt the transmission cycle without the implementation of control measures, these continuous cycles of Salmonella infections and re-infections in human and animals via the environment will not be broken. Epidemiological studies cannot be performed accurately based on phenotypic characteristics alone as this lacks sensitivity the interpretation of these phenotypic results may be highly subjective and they are not suitable for strain differentiation. Sensitive and reliable methods are therefore required to differentiate Salmonella Strains beyond the phenotypic level. At present genotypic typing methods are widely used for epidemiological studies, they typically produce a strain specific finger printing pattern that can be used for bacterial identification and strain differentiation. Using these methods, strains responsible for outbreaks can accurately be differentiated from other strains of species or subspecies (Terletski et al., 2004). In the present study, O H Antisera technique was employed to characterize clinical and environmental Non-typhoidal Salmonella isolate from food, blood, urine and stool samples obtained from defined areas of Ado- Ekiti, Nigeria. This was done in order to know the prevalence of each strain of the isolates, and in doing so to understand the epidemiological links amongst the isolates. 1.1 Taxonomy and Typing Of Salmonella The genus Salmonella belongs to the family of Enterobacteriaceae. Salmonella species are gram negative, facultative anaerobes bacteria that diverged from E. coli approximately 100-160 million years ago and acquired the ability to invade host cell. Based on DNA relatedness, Salmonella is today divided into species S. enterica and S. bongori. S. enterica comprises over 2500 known serovars and each year new serovars are discovered. S. enterica that has diverged from S. bongori approximately 71-100 million years ago is further divided into six subspecies, shown in figure 1.1. The majority of the bacteria causing diseases in humans and warm blooded animals fall into the subspecies enterica (I). The serovars are classified by the Kauffman white scheme on the basis of surface agglutination reactions of their O-H- and Vi-antigen (Vi-antigens are only found in S. typhi S. paratyphi and S. dublin) S. typhimurium and S. enteritidis may be subdivided into phagetypes based on the lysine pattern after infection with a defined panel of bacteriophages. This is still the primary method for sub typing of Salmonella and often used in combination with antimicrobial susceptibility testing. However, this typing method has some limitations. Phagetypes are not always very stable and have been shown to change for example upon introduction of plasmids. Moreover, pure cultures from some serovars have shown variable expressions of minor O- antigen thus apparently unrelated Salmonella strains can have close phylogenetic relationships. Most of the human infections are caused by only few phage types of the two serovars S. enteritidis and S. typhimurium. To distinguish variants with small difference in these phage types, a high discriminatory power is needed which is provided by many molecular typing methods. The most frequently used method are pulsed field gel electrophoresis (PFGE), multi locus sequence typing (MLST) and multi locus variable number tandem repeat analysis (MLVA). It is suggested to use a combination of conventional and appropriate molecular typing methods. To identify the sources of human illnesses and to monitor the spread of successful clones in national or international outbreaks and epidermics. Genus SALMONELLA Species S. bongori S. enterica Subspecies enterica (I) arizonae (iiia) diarizonae (iiib) houtenae (iv) indicia (vi) salamae (ii) Serovars S. typhi S. Newport S. paratyphi S. Santpaul S. enteritidis S. virchow S. dublin S. typhimurium S. infantis S. cholerasis S. hadar Phagotype DT104 U288 DT120 DT170 DT12 DT135 U292 Figure 1.1 Taxonomic Schemes of Salmonella Serovars and phagotypetes 1.2 Stress tolerance of Salmonella Definition of bacteria stress Microbial stress can be defined as sudden environmental changes that alter The behavior of a micro-organism and reduce its growth. Usually stress is referred to as environmental conditions that cause injury to the cells, which results in some loss of functions. Sub- lethal injury damages but does not kill the micro-organism as it is able to respond to the stress and to repair the damage lethal injury eliminates the bacteria. In a bacteria population however, a fraction of the bacterial cells may survive depending on the degree of the injury and time of exposure to the stress Metabolic injury is related to damages of various components of the cells and is frequently distinguished from structural injury which refers more to damages of the cell wall or membranes. Both injures are characterized by the failure of the bacteria to form colonies on selective media where uninjured cells would normally grow. Bacterial stress can be generally classified into three categories physical, chemical, and nutritional. Salmonella encounter various stress conditions in the food chain and during infection. Salmonella species are exposed to numerous stress conditions as they pass through the food chain and also during the course of infection. It is exposed to numerous stress conditions. Like many food borne pathogens, Salmonella can sense and respond effectively to diverse stress signals inside and outside the host. During food production, the food-associated microbes are exposed to food preservation techniques such as freezing, chilling, heating, smoking and curing. Salmonella might also survive on food surfaces where it experiences desiccation, nutrient limitation and when it gets in contacts with disinfectants. Following ingestion, an enteric pathogen, such as Salmonella must cope with low pH in the stomach, ranging from 2.5-4.5 largely depending on the alimentation. When Salmonella species enter the gut, They are exposed to various stress conditions which upon invasion, are taken up by phagocytes where they encounter a new series of stressors, unfortunately Salmonella is known for tolerating a remarkable broad range of stress condition and also to survive rapidly changing environments. Cross-protection and virulence of stress adapted cells studies have shown that the explosive to sub lethal stress can confer protection to the subsequent exposure to the same stress or other stress condition. Dehydrated Salmonella cells can be example acquire high tolerance to heat, ethanol, high salt concentration and UV radiation. This is due to the production of stress proteins of Rpos mediated general stress response system that maintain cell viability and thereby confer protection to different stresses. Thus, certain stress conditions have the potential to increase virulence. Because many of the host defense mechanisms show similarities to conditions that bacteria experience during food processing for example acid or heat tolerant S. enteritidis were observed to be more virulent in mice. Certain stress conditions that resemble host defense mechanism can also serves an signals for the expression of virulence genes, for example the Spv genes located on the Salmonella virulent plasmid. The general stress signal factor Rpos is not only important for the regulation of stress proteins but is also involved in regulation of the Spv virulent gene. The stress conditions that enhance virulent in the Salmonella include glucose starvation, low pH, elevated temperature and iron limitation. Pre -exposed to acids is known to induce resistance to acid and thereby leads to a decrease in the infectious dose. 1.3 Pathogenesis of non-typhoid Salmonella in human Salmonella serovars that causes disease, in humans can be divided into typhoid and non-typhoid according to their disease pattern. Typhoid Salmonella including the human specific serovars S.typhi and S. paratyphi causes enteric fever, a systemic disease. The most common disease outcome of non-typhoid Salmonella (NTS), such as S.typhimurium and S.enteritidis is a self limiting gastroenteritis, a localized infection of the terminal ileum and colon that does not require antibiotic therapy. Depending on the host immune status, the bacteria strain and infection does, a systemic infection can occur in human this can manifest as septicemia or focal infection. A frequent model for studying Salmonellosis is the oral infection of mice with S.typhimurium which establish a systemic infection in the animal model (figure1.2) this is different from the usually pathogeneses in humans and it is important to be aware of the limitations of the model. However, it has provided the opportunity to identify important Salmonella virulence factors and to study the course of a systemic Salmonella infection. Infection does, passage though the stomach and colonization of the gut. In food borne infections, infectious does of a pathogen depends on three factors 1.) On host resistance, which in impaired in young, elderly and immuncompromised 2) on the composition and matrix with fat for example lowering the infection dose and 3) On virulence and physiological state of the ingested bacterium, the infectious does for Salmonellosis is therefore variable. It is thought to be typically between 106- 108 CFU for humans but can be significantly lower for example in outbreaks. After ingestion Salmonella passes the stomach where it is exposed to an acidic pH .The surviving bacteria cells enter the intestines where Salmonella is able to colonize multiple sites of the small intestines, the colon and the cecum, the ileum is however the most frequently invaded site of Salmonella in humans. 1.4 Adhesion and invasion of intestinal epithelial cells The epithelium of the intestine in covered and protected by a mucus layer thus, it has been suggested that Salmonella invades preferably the mucus free m cells situated in the peyers patches and dendritic cells (DCS) that lie within the epithelium. The first cellular contact presumably mediated by frimbriae is followed by the invasion of the epithelial cells. This process is conferred by the type III secretion system (T3SS) I, a protein complex that is associated with at least 20 structural and regulatory protein T3SS-I has a needle like structure and injects protein effectors into the epithelial cell. The translocated protein cause cytoskeletal rearrangements in the host cell which lead to membrane ruffling and bacterial internalization through macropinocytosis. The internalized bacteria cells reside in a membrane bond vacuole (SCU) in which they cross the epithelia layer. Knodler et al suggested that a Subpopulation of Salmonella replicates with in the cytosol of the epithelial cells. These cells are thought to be extruded from the monolayer into the intestinal lumen. This host defense mechanism might serve as a reservoir of disseminated from Salmonella. The extruded bacteria can infect new host cells or can be excreted and eventually infect new host. Upon translocation to the lamina propria, bacteria flagellin functions as the major proinflamatory determinant. It is recognized by toll like receptor (TLR) 5 located on Salmonella is thought to either invade macrophage or once again to enter epithelial cells from the basolateral side. Interaction of Salmonella with macrophage within the lamina propria, Salmonella in immediately taken up by mononuclear phagocytic cells (macrophage or Dcs). Associated with peyers patches. Macrophages recognize pathogen associated molecular pattern (PAMPs) of Salmonella, such as flagellin, leading to an uptake of the bacteria. The contact of Salmonella, with macrophages and other immune cells trigger the production of pro inflammatory cytokine and chemokines However, effector protein of T3 SS-1 early in infection have also been suggested to be directly implicated in eliciting the inflammatory response. Inside the macrophage, Salmonella is located within the phagosome, during the first 2-3h before replication begins, Salmonella modifies the phagosome, Maturation and create a unique niches, the Salmonella containing vacuole (SCV). It has been suggested that Salmonella arrest the degradative pathway of the phagosome by inhabiting its fusion with the lysosome. But contradictory studies exist that propose the survival of Salmonella despite the fusion of these two compartments. Within the phagocytes, Salmonella faces a number of challenges, such as procurement of nutrient, the defense against antimicrobial peptides, the protection against oxidative stress and acidification. Upon phagocytes activation that occurs during up take of Salmonella. The phagocytes NADPH oxidase reduces molecular oxygen to cytoxin superoxide. This constitute the initial bacterial phase called the oxidative burst and it is followed by a prolonged bacteria static phase in the presence of nitric oxide which in synthesized by the nitric oxide synthase (NOS). Microarrays studies have shown that Salmonella regulates up to 919 genes differently when it senses the specific intravacuolar environment in macrophase. It is though that genes needed for the early stages of infection. Such as T3ss 1 and flagellin are repressed. The genes that is unregulated. Including a second Salmonella secretion system, the T3ss-2 it translocates effector protein across the vacuolar membrance into the cytsol. Among other T3ss-2 Is involved in maintaining and positioning of the SCV, in the defense against killing by the NOS and NADPH oxidase and in the delay of the apoptotic cell death. During the course of infection Salmonella can remain with the SCV of macrophage for several hours to several days. It may be spread systemically but Salmonella cannot also cause macrophage apoptosis allowing it to disseminate to new macrophage and to infect the epithelial cells form the basolateral side. Macrophage lysis results in the releases of proflammatory cytokines and inflammation. If this process is beneficial for Salmonella is still controversially discussed. It allows Salmonella to colonize the gut associated tissue but also help the host in cleaning the infection Oral infection Figure1.2 The course of an infection with S. typhimurium in the after oral administration modified after wary and wary 2000. 1.5 AIM To characterize and study the prevalence of Non typhoidal Salmonella (NTS) in Ado- Ekiti metropolis. 1.6 Specific Objective 1 TO Characterize Non- typhoidal (NTS) Salmonella species from clinical and Non-clinical samples 2 To determine the prevalence of Non typhoidal Salmonella from clinical and non clinical samples in Ado-Ekiti 3 To determine the antibiogram (antimicrobial susceptibility pattern) of Non typhoidal Salmonella from Clinical and Non-Clinical Samples. 4 TO determine the serotypes of the Non-typhoidal Salmonella by serotyping technique. CHAPTER TWO 2.0 Literature Review Despite global improvement in public health facilities bacterial infections still remains an important public health problem worldwide. Salmonella is one of the most important food-borne pathogens and is often linked to water-borne transmission as well (weigel et al, 2004). the disease caused by members of Salmonella is called Salmonellosis. S. enterica subspecies enterica (subspecies I) is responsible for 99.3 of the infection in human and animal (pignato et al., 1998). Most of the infections are zoonotic in origin but serotype like S. typhi and S. paratyphi infect only human (Yan et al., 2013). During the past ten years there has been a dramatic increase in Non- typhoidal Salmonellosis. In USA alone there are estimated 1.5 million cases of Non-typhoidal Salmonellosis each year, accounted for nearly one third of deaths associated with food-borne illness (Yan et al., 2003). The predominant serotypes responsible for Non-typhoid Salmonellosis are Salmonella enterica sub enterica serovars enteritidis and typhimurium (Schoeni et al., 1995, Soumet et al., 1999). Molecular methods based on DNA, have become powerful alternatives in identification of species and strains which is essential for effective investigation of both endemic and epidemic microbial disease. Apart from more traditional methods this review will also focus on the identification and typing of Salmonella strains. 2.1 Global Distribution of Serovars and Trends in Human Salmonella serotyping still serves as the predominantly used surveillance tool for detection of outbreaks and corresponding source, to monitor trends overtime, and attribute different food and animal reservoir to human infections. Despite this there is today only limited knowledge of the global distribution of Salmonella serovars in humans in last decade. Some countries have collected annual prevalence data on serovars distribution among humans, but very few publications have summarized the global distribution of the serovars responsible for human infections and further analyzed the data (Herikstad et al., 2002,). An equally important feature for surveillance is quality assurance systems and quality control, which is necessary to ensure reliable data, only a small number of international quality assurance systems exist to evaluate the quality of serotyping conducted worldwide by national reference laboratories (Petersen et al .,2002 Anonymous, 2007) In January 2000, The WHO launched WHO Global food borne Infections Network (GFN) formerly known as Global Salmsuvr (GSS) a global effort to enhance laboratory-based surveillance of Salmonella infections and other food borne disease, and to promote prevention and control activities. Enhancing worldwide serotyping of Salmonella is a key objective of WHO GFN and is facilitated by bench training at international capacity building courses. To ascertain the performance of participating laboratory and to promote enhanced laboratory based surveillance, an External Quality Assurance System (EQAS) was established as a part of WHO GFN in 2000 (Petersen et al., 2002). Each year, EQAS distributes a set of blinded bacterial cultures of identifications, serotyping and antimicrobial susceptibility testing. A key component of this program is the internet based Country Data Bank (CDB) to which member countries are encouraged to annually upload data on the 15 most common Salmonella Serovars ( HYPERLINK http//www.antimicrobialresistance.dk http//www.antimicrobialresistance.dk). The results of the WHO GFN EQAS data from 2000 2011 revealed that a total of 350 laboratories in 96 countries participated in annual EQAS testing at least once during the seven EQAS iterations, a total of 756 reports were received from the participating laboratories. Cumulatively 76 of participating laboratories submitted data for all eight strain and 82 of the strains were correctly serotyped. The goal of EQAS program is for all participating laboratories to perform Salmonella serotyping with maximum of one error. The percentage of laboratories reaching the threshold reporting one or zero error increased significantly (p 0.04), from 48 in 2000 to 68 in 2007. In each EQAS iteration, 84 to 96 of the laboratories correctly serotyped. The S. enteritidis isolate that was included in the panel of test strains as an internal quality control strain. Regional variation in performance were observed, with laboratories in Central Asia and Middle East performing less well overall than other regions errors that resulted in incorrect serovars identification were usually caused by difficulties in the detection of phase II flagella antigen, or differentiation within antigen complexes. Some of the errors likely are related to the quality of the antisera available. The same problem existed in detecting H antigen, was reached by the National Institute for Public Health and Environment (RIVM) which served as the community reference laboratory for Salmonella as designate by the European Commission. They evaluated 26 European national reference laboratories in 2007 and found that 98 and 96 of the laboratories correctly serotyped the isolate O and H antigen respectively (Anonymous 2007). In 2008, the global distribution of Salmonella Serovars per country, based on data from the WHO GFN CDB were summarized and analyzed in search for tends from 2001-2007. All data included were based on reliable data from countries which managed the quality assurance threshold of the WHO GFN EQAS. 2.2 HUMAN EPIDEMILOGY Food-borne diseases have been estimated to infect up to 76million people in the United States annually. This would equal one fourth of the people infected in the developed world per year if these data were extrapolated. The burden of Salmonella is expected to be much greater in the developing parts of the world (Schlundt et al., 2004). Salmonella is overall the most common food borne pathogen in the United State, however in some state campylobacter are more frequent than Salmonella. According to the WHO, human NTS infections constitute a major public health burden in society and represent a huge cost for many countries ( HYPERLINK http//www.who.int www.who.int). In 2000, it was estimated that in the United States, NTS resulted in 14 million infections annually in a population of about 293 million inhabitants with approximately 168,000 visits to general practitioner (GP). A total of 16,430 people were hospitalized resulting in 582 deaths (Mead et al., 1999 Mc Dermott et al., 2006). A similar study was conducted the same year in the United Kingdom, This revealed that 41.51NTS case occurred each year with 15.03 laboratory confirmation among a population of 60million people, resulting in 1,576 hospitalization and 119 death (Adak et al., 2000 Mc Dermott et al., 2006). This was approximately twice as many hospitalization and deaths in the United State compared to the United Kingdom. Recently, the financial burden of NTS was estimated in the United State based on foodnet data, the data revealed the costs of medical care, lost productivity and mortality exceeded more than 3.6 billion annually (Voetsch et al., 2004 Mc Dermott et al., 2006). In comparison the annually cost of NTS in Denmark are estimated to be 15.5million which was approximately four times less that in the United State ( HYPERLINK http//www.who.int www.who.int). A few countries around the world have an established laboratory based surveillance of NTS, or have recently improved the data quality and reporting to implement programmes measuring the burden of Salmonellosis one of the measurements in the surveillance reports are the notification incidence or isolation rate per 100,000 inhabitants all estimating the burden on the society caused by Salmonellosis. Bangtrakulnonth 2006, Ben Aissa et al., 2007. In the United States, The National Salmonella surveillance system reported in 2006 an increase of 12.3 of NTS compared with 2005 due to increased reporting. In general the level decreased compared with data from 1996. The number of human Salmonella case also seems to have declined in Tunisia based on data collected from 1994 to 2004 with the lowest isolation rate monitored in 2004 for the entire surveillance period (Ben Aissa et al., 2007). The 2008 Annual Surveillance Report New Zealand showed, as many other country reports a decreasing notification rate of NTS. Based on data from 29 countries, the number of Salmonellosis cases in Europe decreased by 8 between 2005-2006. In 2006, the National Salmonella and Shigella center in Thailand found 3,758 NTS cases, representing a decrease compared with previously published data showing the number of human cases from 1993 to 2002 (Bangtrakulnonth et al., 2004). The worldwide reported trend of decreasing human caused by NTS Seems to be in agreement with what have been observed elsewhere. A decreasing number of Salmonella isolates were serotype from 2001-2007 when assessing the global distribution of Salmonella serovars in 37 countries worldwide. The data were based on quality data submitted to CDB of the WHOGFN. Despite the decreasing occurrence of NTS infections in human, the problem is Still large and in largely preventable and therefore an unnecessary burden on public health. 2.3 Symptoms and Human Infection The symptoms of Salmonella infections usually appear 12 to 72 hours after infection of the organism and include diarrhea, fever, abdominal cramps, nausea, and sometimes vomiting but asymptomatic infections may also occur. The illness usually lasts from 4 to 7 days, but are in most cases self-limiting ( HYPERLINK http//www.cdc.gov www.cdc.gov, HYPERLINK http//www.who.int www.who.int. Mc Dermotte et al., 2006). NTS gastroenteritis will develop into bacteremia in about 5 of cases. Bacteremia often requires hospitalization with a prolonged course of illness and could potentially result in a fatal outcome (hohmannel 2001). A cohort study with almost. 49,000 participants showed that people with gastrointestinal infections caused by NTS have an excess mortality with a relative risk of 13.13 up to a month after being infected (Helms et al., 2003) Several studies have described certain serovars such as S. dublin and S. choleraesuis often being associated with invasiveness (Hohmanel et al., 2001, Helms et al., 2002, Jones et al., 2008). An observational study based on patient data from 11.656 isolate (2002-2007) estimated the risk factor of the ten most common Salmonella serovars from these patients, The data showed that 87.40/0 of 68.1 S. choleraesuis isolate originated from blood sample with a significant increased odds ratio of 44.00 (95 cl 34.3- 56.5) when compared to other NTS serovars S entertidis 511 45 121-, S typhimunium and S. sopina did also seem to be highly invasive when compared to other NTS Serovars (Vi). These data correspond well with an investigation describing the differences in the outcome of Salmonellosis based on the various serovars (jones et al 2008) 60 and 67 of all S. choleraesuis and S. dublin infections, respectively require hospitalization compared with other serovars, however S. heideibery, S. poona, S. panama, S. virchow, S. paratyphi and S. sandigo also seemed to more frequently cause invasive diseases (Jone et al 2008). Several studies have shown that invasive NTS is endemic in sub-Sahara Africa (morpeth et al., 2009 Vandenberg et al., 2009). In some of these countries the mortality in children caused by NTS bacteremia exceeded the burden of childhood malaria (Morpeth et al., 2009). 2.4 Age, Season and Risk Factor Human Salmonella infections are age specific and affect mostly children, elderly people and immunological compromised patient (Holmannel et al., 2001 Jafari et al., 2009). The reason for the typical ages pattern is believed to be a result of children acquiring immunity to Salmonella and determinating immune status in the elderly. This observation was supported by Hendriksen et al., who showed that 2002 2007 in Thailand, 32.6 of all Salmonella cases were observed among children from 0 to 5 year of ages and peaked again with 14.0 of all cases in people older than 60 years in Thailand. The previously mentioned risk factor analysis from Thailand showing an odds ratio between 1.63 (95 Cl 1.1 2.5 and 1.51 (95 Cl 1.1 2.0) in the age group of 6 20 and 21 40 years for being infected with S. choleraesuis compared to other NTS infection. In the risk factor analysis, additional serovars seemed to be age specific such as S. anatum, S. enteritidis and S. welterreden, which mainly affected people older than 6 years. In constract, S. Stanley, S. panama and S1 (1) 4, (5) 121 predominately infected children less than 6 years of age. Several surveillance reports have illustrated the general infection pattern of human Salmonellosis associated with yearly seasonality with the summer months being the season having the highest incidence of infections (Cho et al., 2008). In Tunisia, however, the general pattern of infection seems not to be consistent with hypothesis that most infections occur in the summer months. S. enteritidis infection, pecked in January followed by S. Livingstone peaking in April, whereas S. Corrallis peaked in October (Ben Aissa et al., 2007). Ben-Aissa et al did not indicate a reason for the skewed seasonal pattern but one possibility could be that this was the same time of the year when the serovars peaked in the animal reservoir. In Thailand, the seasonality of Salmonella infections were in general in agreement with other studies having most infections in the summer period. 2.5 Antimicrobial Treatment and Antimicrobial Resistance Antimicrobial treatment is not routinely recommended for empiric treatment of gastrointestinal infection caused by NTS in healthy people as the infection often in self-limiting. Antimicrobial treatment should be given to patients with severe illness, immunosuppression or patients suffering from bacteremia (Hohmannel et al., 2001). Treatment with first line antimicrobial should include ampicillin, chloramphenicol or trimethoprism sulfamethoxazole (Hohmann et al., 2001). The choice differs by region and chloramphenicol is not used in most developed countries but is common in developing countries. Ampicillin and trimethoprism Salfamethoxazole are good choices but many do not even considering them and choose instead fluoroquinolone or 3rd generation cephalosporin. Unfortunately the recent increased development of resistance to many antimicrobials often leaves the GP with no alternative than to treat the infection with either a fluoroquinolone or 3rd generation cephalosporin. These antimicrobial are routinely used for empiric treatment if the susceptibility of the of the isolates is unknown or if the patient suffers from bacteremia (Hohmannel et al., 2001) for pediatric patients, treatment with a fluoroquinolone contraindicated and practitioners will rely on ceftriaxone or another 3rd generation cephalosporin (Hohmannel et al., 2001). Several studies from the United States, Canada, and Denmark have shown an increased risk of hospitalization or ever death associated with multi-drug resistant NTS compared with susceptible NTS. (Holmberg et al., 1987 lee et al., 1994 Molbal et al., 1999 Helms et al., 2002 ). In a Danish study, an increased risk of invasive illness has been observed with 3.5 of the patient investigated being hospitalized. An increased mortality was recorded in 12 of the patients in up to two year after the infections for both cases the infections were associated with quinolone or multi-drug resistant S. typhimurium (helms et al .,2002 helms et al., 2007). Recently, muiti-drug resistant NTS collard et al., 2007 lee et al., 2009 (vanderbery et al., 2007). Several publication have described the increase occurrence of muiti-drug resistant NTS and isolates resistant both fluoroquinilone and 3rd generation cephalosporin in South East Asia and Africa (Archambault et al., 2006 Aarestrup et al., 2007). Recently described the level of antimicrobial resistance from 2003 to 2005 in seven Southeast Asia countries. 2.6 Main Reservoirs As a zoonotic food-borne bacterium Salmonella has reservoirs in various animals. The most common domesticated animal hosts are chickens, pigs and cattle. But many others domestic animals as well as wide range of wild animals can also harbor this organism. Because of the ability of Salmonella to contaminate meat during slaughter and to survive in fresh meats and meat products that are not thoroughly heated, animal products constitute a main vehicle of transmission. Another important vehicle of transmission is eggs that are contaminated on the surface or in the interior of the egg. Finally, products and other vegetables that are contaminated with animal manure during growing or processing are increasingly recognized as an important source of human Salmonella infections. 2.7 Transmission Local and Global The primary reservoirs of NTS are the intestinal tract of colonized domestics and wild animals. In most industrialized countries food of animal origin is the primary vehicle for human Salmonella, however, multiple routes of transmission has been documented including vector and water borne, animal human and environmental contact as well as many others. In the Netherlands S. typhimurium isolated form a pig, a calf and a child in the farm were identical, indicating animal to human transmission (Hendrilken et al., 2004). Salmonella is passed on from the intestinal tract of the host to meat products during slaughter, where faecal contamination often occurs. Human infections are required from contaminated meats due to inadequate cooking or poor kitchen hygiene, the latter of which can result in gross contamination of uncooked foods such as vegetables. 2.8 Surveillance Methods In the last decade, our ability to distinguish in a rapid and reliable way between epidemiological unrelated isolates from the same bacterial species has increased, thereby enhancing our capacity to detect outbreaks, conduct surveillance and understand or elucidate the epidemiology of certain types or clones. Thus bacteria typing technique have been developed to measure genetic relatedness among emerging pathogenic strains, clone or cluster of bacteria from a single species. In the beginning of the bacteria typing era typing systems were based solely on phenotypic methods such as serotyping (Grimont et al., 2007) phage typing (Smith et al., 1951) and antibiogram typing. These methods are currently in use in many countries as part of national surveillance programmes. The results obtained by these phenotypic tests, particularly serotyping has been the basis for the elucidation of the epidemiology of NTS for decades. In addition, conventional serotyping and antimicrobial susceptibility testing are excellent surveillance tools aid also used as first line methods in outbreak detection caused by NTS. 2.9 DETECTION METHODS Salmonella cells can be injured or stressed as a result of food processing conditions or environment factors. The optimal isolation and cultivation of Salmonella therefore requires enrichment procedures. Including non-selective and selective enrichment for the resuscitation of injured Salmonella cell non-selective (pre-enrichment) medium such as buffered peptone water is usually used. The choice of pre-enrichment medium, can however, differ depends on the nature of the sample examined. The pre-enrichment stage is often followed by two enrichment steps. During the first step selenite cystine broth or rappaport-vassiliadin Broth or both can be used to allow for the growth of Salmonella species but the inhibition of other organisms. Following this enrichment step selective media is used for the confirmation of Salmonella include Salmonella species. The most commonly Salmonella used media selective for xylose lysine deoxychocolate agar (XLD), Salmonella-Shigella (SS) agar, Wilson-Blair bismuth sulfate agar. After overnight incubation colonies can be examined for the required characteristics Salmonella species grown on XLD plate, for example, usually produce H2S, resulting in black centered colonies,(harrigan and park,1991). Supplementation of the pre-enrichment, or enrichment media with ferrioxamine could also improve the isolation of Salmonella quantitatively as well as qualitatively. It can significantly improve the isolation of low numbers of Salmonella from mixed cultures when used as a supplement in buffered peptone water (BPW) or selenite cystine broth (reissbrodt et al., 1996). Ferrioxamine supplies iron to Salmonella via the special update and utilization system of the cell. Other competitor organisms such E. coli have no update and utilization system for ferrioxamine. Presumptive Salmonella cultures are usually confirmed by means of a number of biochemical tests. A rapid screening test in the urease test, which should be negative for Salmonella isolates (Ewing, 1986). Following the urease test other biochemical tests can be performed. These tests could be performed individually or by using commercially available identification systems such as the API 20 E system. 2.10 Pulsed Field Gel Electrophoresis in Salmonella Surveillance of Salmonella outbreaks and illnesses in the US includes not only serotyping of the bacteria, but also further discrimination by molecular serotyping methods. Although serotyping has been a frequently used sub typing method for over 50 years, the method does not allow for discrimination between bacteria strains with the same serotype or fingerprinting of Salmonella serotypes the bacteria. When an increase in food borne outbreaks of certain Such as enteric and S. typhimurium arose in the 1980s in the northeastern US, The need for discrimination between bacteria strains in outbreak investigations became apparent (Salem Imen et al., 2012). The multiple sub typing methods were created. phage Typing and plasmid profiling were two early methods of discrimination however, these methods were limited due to the fact that analyzing was based on entities that could undergo losses or conversion (Salem Imen et al., 2012), more discriminatory DNA based methods such as amplified fragment length polymorphic (AFLP), restriction fragment length polymorphic (RFLP), Ribotyping, multiplex PCR, Multilocus sequence typing (MLST)multi-virulence locus sequence typing (MIST AND PFGE were then established, multiple studies have compared these methods and evaluate their usefulness, discriminatory power and convenience (Foley et al., 2006). However, PFGE is considered the gold standard for molecular sub typing among public health officials and epidemiologists due to its popularity in the 1990s and creation of the CDC, standardized protocol and surveillance reference network called pulse net (swaminathan et al 2006). Pulses field gel electrophoresis has remained the gold standard for lack of a clearer ideal typing method (Ward et al., 1987). The PGFE method works by digesting genetic bacteria DNA into large fragment using restriction enzymes and subsequently comparing these fragment binding patterns on an electrophoresis gel. Due to the large size of these genomic fragments (up to 12mbp) a novel mode of electrophoresis was created to allow the DNA to unravel and separate in the agarose gels. This method relies in the orientation change and migration of DNA through agarose, based on the response to periodic directional change in electric field pulses (Hostetter et al., 1991). The most popular PFGE apparatus used among laboratories is the contour-clamped homogenous electric field (CHEFMAPPER) system that uses electrode clamped to specific voltages and arranged to a hexagonal orientation allowing for a uniform electric field switch of 1200 angle between pulses (Foxman,2005). Sample preparation is also unique in that DNA is encapsulated in low-melting point agarose gel plug, then protecting the shearing susceptible restricted DNA fragments. Bacterial cell plugs undergo multiple lysis and buffer washes to remove an unwanted protein. Once plugs undergo an incubation step with restricted enzyme, restricted DNA fragments can be fractionated using the PFGE CHEF map per system, gel strained for visualization of bands and band analysis completed using comparisons software. The PFGE system allows for high reproducibility between national health laboratories due to the use of standardized protocol of the CDC, and has been extremely useful in Salmonella outbreak surveillance and characterization of Salmonella serotype in humans, domestic animals and wildlife (swaminathan et al., 2006). 2.11 Immunological Method The Tradition method for the detection of Salmonella is time consuming, and great efforts have therefore been made to develop rapid methods for the detection of Salmonellosis. Many of these rapid method have been based on immunological techniques in the test antibodies produced by experimental animals are used to detect antigenic difference between micro-organisms. Two classes of antibodies are used, polyclonal and monoclonal antibodies in order to use antibodies to identify specific organisms, they must not cross react with other organisms (Towner and cockayne 1993). The speed and sensitively of these immunological technique or argue vary, and highly affected by the duration of the enrichment period required to get detectable amount of targets. Cells as well as the complexity of the sample and the specifiable of the reporting mechanism. 2.12 Microscopy Fluorescent antibodies can be used to detect bacteria under a fluorescent microscope that detect the light transmitted after excitation of the specific fluorochrome bound to the antibody (guy et al., 2000). In this procedure organisms are fixed directly onto the slide, and are then incubated with the specific antibody. The antibodies can be labeled directly with a fluorochrome to allow visualization of the antibody (direct Immunofluorence assay) or a fluorochrome- labeled antibody conjugal can be added to detect the unlabelled antibodies. (Indirect Immunofluorence assay) (tower and cockayne 1993). Immunofluorence with polyclonal antiserum has been used for the detection of Salmonella species. O antisera are mostly applied but H antisera can also be used (mortener et al., 1981). A modification of the Immunoflorescent technique has been developed to different between dead and live Salmonella cells (Duffy et al., 2000) The method is based on combining anti-Salmonella anti bodies labeled with Texas Red with a viability Strain sytox green. The technique included pre-enrichment enrichment and selective enrichment steps. The technique has been applied for the detection of S. enteritidis. The main advantage of this technique in that it helps to distinguish between viable and non-viable cells especially in processed and preserved food products in order to make an accurate assessment of the number of Salmonella in the product (Duffy et al., 2000). 2.13 Polymerase Chain Reaction Polymerase chain reaction (PCR) use, a thermostable polymerase to produce multiple copies of specific nucleic acid region (Towner and cockayne, 1993). The DNA fragment is amplified in a three step process. During the first step the target DNA in denatured at a high temperature, this is followed by the annealing of the primers to the opposite strand of DNA at a temperature that only allows for hybridization with the correct target sequence. During the third step extension of the oligonucleotide primers occurs using the target DNA as a template for the new strand that in formed. When this process is repeated for several cycles an exponential amplification of the targeted DNA fragment is obtained (Towner and cockayne. 1993 olsen et al., 1995). The use of PCR is one of the most promised approaches for the detection of Salmonella species. PCR has advantages in terms of speed, sensitivity and specificity and has been widely used to detect Salmonella species in food clinical and environmental samples. Combining the PCR with an enrichment step has improved the sensitivity of the assay and assisted in diluting out PCR- inhibitory substances (Olsen et al., 1995). For the detection and identification of Salmonella species, several primers have been designed. Some of the primers are used for the identification of all isolate belonging to the genus. These primers sets target genus such as INVA ST and hin/H2 which are specific for all salmonella SPP. Others primers are only specific for some individual Salmonella serotypes. These targets include sef A that encodes then SER 14 fambrial antigen. A virulence plasmid for S. enteritidis, S. pullurum and S. gallinerum, and the flic gene, that encodes Flagelin H1 present in S. typhimurium (awany et al., 2003). 2.14 Salmonella in Humans and Animals Salmonella enterica is recognized as a major zoonotic bacteria pathogen worldwide. These are over 2,500 Salmonella serovars all are regarded as capable of causing salmonellosis in human. Non-typhoidal Salmonellosis (NTS) in one of the most frequently foodborne disease reported worldwide in developing as well as developed countries. The spread of Salmonella infection in humans is favored by a wide range of animal reservoir and by a wide commercial distribution of both animal and food products. NTS constitutes a major public burden and represents a significant cost and economic impact. Salmonella enterica infection is one of the leading causes of food borne illness in united state and has been estimated to cause 1.4 million cases of human Salmonellosis resulting in 16,430 hospitalizations with almost 600 death each year( hohmannel et al 2001) in addition, the degree. Of under reporting has been estimated at 38fold of the number of each reported to the center for disease control and prevention (CDC) Salmonella account for an estimated 27 of all food borne illnesses caused by known bacterial agents with the majority of human Salmonellosis Cases related to the consumption of the contaminated food products. The CDCs food borne disease active surveillance network (foodnet) estimated that the annual cost due to loss of work, Medical care and lost productivity caused by food borne Salmonellosis in the United States ranges 12.3 billion to 13.6 billion. Salmonellae are facultatively anaerobic, gram negative rod shape bacteria belonging to the family Enterobacteriaceae. Although members of this genus are motile by peritrichous flagella non- flagellated variant, such S. pullorum and S.gallinarum and non-motile strain resulting from dysfunctional flagella do occur. Salmonellae are chemoorganotrophic, with an ability to metabolize nutrients by the respiratory and fermentative pathways. The bacteria grows within a wide temperature range between 80C and 450C and PH range of 4 to 8 with an optimum temperature of 370C and pH of 6.5 to 7.5. They catabolize D- glucose and other carbohydrates with the production of acid and gas. Typical Salmonella identifications include a production of acid and hydrogen sulfide gas from glucose fermentation in triple sugar iron agar, reduction of nitrate into nitrile, the oxidase test negative and failure to hydrolyze urea. The genus Salmonella contains two species Salmonella enterica and Salmonella bongori (formerly subspecies V). Salmonella enterica is divided into six subspecies which is referred by a Roman numeral and subspecies name (enterica) salamae (II) azizonar (IIIa), diarizona (IIIb) houtenae (IV) indica VI. Over 99 of the serovars belong to Salmonella enterica species consisting of the major sarovars causing human salmonellosis. A serological confirmation of Salmonella surface antigens with specific antibodies comprise somatic antigen (0) lipopolysaccharides on the external outer membrane filamentous portion of the flagella associated with the peritrichous flagella, and the capsular antigens (VI) associated with the Salmonella capsule in serovar typhi, paratyphi and dublin. The white-kauffmann scheme is practical summary of the antigenic structure of different Salmonella serovar. The top ten most frequently reported serovars to the centre for disease control and prevention from human and various Kinds of animal sources with the most common serovars comprising typhimurium, newport, enteritidis and heidelbery. There surveillance data demonstrated. The persistence of Salmonella serovars in animals which can account for their introduction into the food chain and some Salmonella serovar in food animal are epidemiological disseminated into the derived food products more successful than others. Since there are number of common characteristics among Salmonella serovars isolated from humans animals, a spread of Salmonella into food supply can likely to be occurred. A wide range of food especially foods of animal origin such as poultry products, beef, pork and seafood products have been implicated in food borne illness attributed to human Salmonellosis associated with the consumption of contaminated fruit and vegetables. Others sources of exposure include water, handling of farm animals and pets, and human-to human contact may be serving as a source of Salmonella infection. There has been an approximately estimated 1-6 of the prevalence of Salmonella from farm animal in the United States. Salmonella shedding in feces of livestock and poultry are mainly responsible for persistence in these reservoir in addition, Salmonella spreads horizontally between animal via the fecal-oral route through fecal contamination of the environment airborne transmission and contamination of feed, in opposite, appear to play less important roles for Salmonella disseminating among farm animals An intermittent feeding, stress during transport duration of lairage (stay in holding pens at slaughter house) increase the spread of Salmonella by the fecal-oral route. Therefore, the prevalence of intestinal carriage among livestock increases in excess of 10 before slaughter. 2.15 Salmonella in Environment Salmonella are disseminated in the natural environment such as water, soil and sometimes in food plants. Animal are considered as the major reservoir of Salmonella but significant dissemination into the environment has resulted from the activities of humans and animals. Both humans and animals can excrete Salmonella when clinically diseased or after if they remain carriers. Salmonella organisms do not multiply significantly in the natural environment however, they can survive several weeks in water and several years in soil if conditions of temperature, humidity, and pH are favorable. A number of studies have reported of the incidence of Salmonella in animals and environmental sites including studies of waste disposal. Salmonella serovars are disseminated into the aquatics environment from a diverse range of source including effluent discharges, agriculture run-off and excretion by wild animals. Salmonella are able to survive in natural water for a period. Study by Pedersen (1995) demonstrated that Salmonella and Escherichia could survive for approximately 56 day in fresh water systems. Salmonella can be detected in fresh water pools in forest area however, Thomason and his colleagues reported that Salmonella could not be detected from the pool sites. In addition, the authors concluded that consumption of contaminated water by wild life and other animals completes the cyclic movement of Salmonella in nature. In freshwater, Salmonella appeared to die off in 2-3days however, when nutrient was supplementary to water Salmonella began to grow In marine water, sediments are important for Salmonella to attach and settle into for increased Survival, open seas are crucial for eliminating Salmonella from human activities. Elimination of Salmonella and other bacteria in the seawater has been demonstrated to be predominantly due to protozoa. Also in area of high bacteria load, higher numbers of protozoa were presented Salmonella which resulted in the higher elimination rate of Salmonella, May be present in any waste from human or animal activities. The risk associated with sewage sludge could lead to both human and animal infections and aquatic flora and selfish contamination. Thousands of acres nationwide are permanently closed to harvesting due to high levels of bacteria. Many stream in NC and nationally are listed as impaired due to fecal coliform contamination. The source of bacterial contamination are rarely known, and fecal coliform levels determined using traditional monitoring approaches do not differentiate relative contributions of human and animal sources. Bacterial source appeared to be primarily urban wildlife, Such as raccoons, beaver, geese and more than 100 pets, with high concentrations located close to tributaries several potentially useful fecal bacterial indicator organisms have also been proposed as useful tools in the quantification of indicators of pollution sources that are a threat to public health including E.coli and Salmonella enterica, multidrug resistance (MDR) analysis has proven as a useful screening technique for generally differentiating animal from human sources of fecal coliform contamination and could assist resource managers in developing strategies to more effectively manage water sheds impacted by multiple pollution source including shellfish harvesting. In previous work, we were able to detect similar as well as distinct strain of Salmonella from environmental specimens using various types of antimicrobials as described by Galanis 2002. Salmonella are ubiquitous and found in any phase of natural and man-made environments. Salmonella incidence and distribution in the environment has been increased by human activities as a result of modern agriculture. It has been accepted that Salmonella could not be easily eliminated and thus effort should address to controlling its introduction and spread into agriculture and food chains. To date, a number to control measures have been introduced, achieved and influenced on general food borne pathogens Salmonella, however, will continue to be a feature of humans, animals and the general environment. 2.16 Control, Intervention and Prevention Surveillance of NTS is not only about collecting data, but also analyzing the data to identify critical point to intervention many types of analyses are possible case control studies to identify risk factors can provide detailed information about the nature and magnitude of different reservoir contributing to infection. Source attribution is partitioning of the human disease burden of one or more foodborne infection to specific sources, guiding authorities to factors need to known to attribute burden of illness determined. In addition the proportion of food borne disease due to international travel should be estimated and food items categorized. In 2004, a Danish mathematical model was published for quantifying the number of domestic and sporadic cases caused by different serovars and phage types as a function of the prevalence of the same Serovars in each major animals or food sources (hald et al., 2004) in the following years, the model was enhanced to include information on antimicrobial susceptibility. Using this model, researchers in Denmark were able to quantity the contribution of various animals, food and international travel to human infection with resistant NTS (Hald et al., 2007). In the United States, the foodborne Disease Active surveillance Network (food Net) adopted the same model to attribute the human Salmonella. Cases to various sources (Gerner-Smidt et al., 2006). Denmark has one of the best national programs in the world for controlling and preventing Salmonella and other nation look to Denmark for leadership in this area. In the 1980 the incidence of Salmonellosis in Denmark steadily increase and was linked to the consumption of boiler chicken and pork. This led to a targeted national control programmes (Wegener et al. 2003 mousing et al., 1997). Based on the general prevention strategy known as Hazard Analysis and Critical Control point (HACCP) in a systematic preventive approach to food safety that addresses physical, chemical and biological hazard as a means of prevention, rather than finished product inspections. The HACCP strategies is to identify key step (critical control points) in chain from farm to fork (Busani et al., 2006).where interventions will have the most impact in reducing or eliminating food safety hazards. In 1995, the most extensive nation-wide control programme ever attempted was launched in Danish finishing swine herds. All swine herbs were tested and categorized based on their Salmonella prevalence. Herds were assigned to three categories. Herds with low and acceptable prevalence, moderate prevalence, and clearly unsatisfactory prevalence, moderate prevalence and clearly unsatisfactory prevalence (Wegener et al., 2003) swine herds belonging to the unsatisfactory categories were slaughter using specific hygienic precaution according to the inherent risk. In 2001, the classification scheme was extended to include a fourth category, namely herds being negative in serological tests (Adak et al., 2002). A similar approach was implemented for poultry. All shell eggs from layer flocks should be free from S.enteritidis and S.typhimurium and suspected or confirmed positive eggs should be pasteurized prior to marketing. In addition, infected flocks were eliminated or slaughtered ad separately or late in the day to avoid cross- contamination (Wegener 2003 Hald. et al., 2005) There control programmes and targeted intervention resulted in major reduction in the incidence were reduced by 95 (1993 to 2001) 85 (1993 to 2001) and 75 (1997 to 2001) respectively (Wegener et al., 2003). The benefits of implementing the control programme by eliminating infected animals and diversifying slaughter was estimated to save the tax payers in Denmark s 25.5 million annually. In 2003, the European parliament and council issued, in combination with the harmonized surveillance system regulation 2160 on the control of Salmonella and other specified food borne zoonotic agents. This regulation was intended to control Salmonella and other specified foodborne zoonotic agents by reducing transmission from poultry and pigs (Anonymous 2003b) The regulation enforced strict rules for all member states to reduce the prevalence of Salmonella in primary production. They control programmes target breading flocks of Galus gallu in 2004, followed by laying hens broilers, turkeys, slaughter pigs and breeding pigs over the subsequent four year (Anonymous, 2003b). The increased number of human infection caused by international travel is in many countries a major concern. Currently there is no vaccine to prevent infections caused by NTS for travellers. Thus, in many counties, public health authorities have initiated campaigns to educate the public on steps to reduce the risk of Salmonellosis. Consumer are advised not to eat raw or under cooked eggs poultry or meat, not to eat raw and unpasteurized dairy products and to clean vegetables before consumption. The WHO instituted a similar communication programme as part of their global strategy to decrease the burden of food borne diseases. This lead to the WHO report the five keys to safer food which was published in 2001, and included and associated training materials develop to provide countries with materials that are easy to use, reproduce and adapt to different target audience ( HYPERLINK http//www.who.int/ www.who.int/ foods safety /consumer/5key/en/). Several publications have shown that adopted children carrying Salmonella is an over looked problem (Saimen et al., 2001 Weill et al., 2004 fabre et al., 2009 ) .The American Academy of paediatrics recommends that a stool specimen be collected from all adopted that a stool specimen be collected from all adopted children entering the united states and cultured for the presence of bacterial pathogen such as Salmonella Hostetler et al., 1991 Nicholson et al., 1992 stauffer et al., 2002) the utility of the recommendation was highlighted to all countries in a study where family members were infected with S. concord which was introduced by adopted children . Another approach in preventing travel associated Salmonellosis is by publishing data on the burden of Salmonellosis and routes of transmission, in different countries. Thus, urging the country to take action to limit the source of infections among the general population and traveler by improving the food safety. Aarestrup et al 2007 however, this might be a challenge due to the limited knowledge of the epidemiology of Salmonella. In developing countries and the circumstances associated with the production systems, which often are many small operations where animals are reared with minimal oversight such as free range. To date, only a few studies describe effective serovars specific control programmes that do not rely on vaccination. Multidrug resistant S.typhimurium DTI04 appeared in the 1980s and became a major cause of Salmonellosis around the world. In Demark, a component to control and prevent S. typhimurium DT104 to spread and cause infections in humans was incorporated into the existing control program for pig herds (Adak et al., 2002). Farmer with S. typhimurium DT104 infected herds had to follow a hard intervention plan which included restrictions on livestocks trade and special slurry management carcasses of infected pigs had to be heat treated or decontaminated before leaving the abattoir (Nielsen et al., 2001). Likewise, a Danish control programme for S. dublin was launched in 2002 attempting to reduce the increasing number of human infections and the economic losses for cattle industry, where this serovars was a problem. In constract to the control programme in pigs, this programme for sought to identify cattle herds free of infection by periodic measurement of S. dublin antibody titres in bulk milk and by modeling the spread between herds (Hard et al .,2005 Jordan et al., 2008). The outcome of the modeled control programme showed that restricting the movement of herds between regions was more important than attempting control within herds. However, a combination seems be more effective but needs to further explored (Jordan et al., 2008). To date, all attempts to develop a comprehensive vaccine for humans and animals that will cover all important serovars of Salmonella have failed. Today, only vaccines for human against S. typhi exist. One study revealed that a Vis typhi vaccine did not protect against S. paratyphi A or B since these serovars do not express the vi polysaccharide. However, a Ty 21as typhi vaccine conferred substantial cross protection against S. paratyphi B but not S. paratyphi A (Levine et al., 2009) this example highlights the difficulties in developing Salmonella vaccines. A major problem is the both serovar-dependent and host-dependent factors as well as the attributes influencing serovar host specificity, are known among the move than 2,500 serovars (barrow et al., 2007). Some success has been seen with S. enteritidis, which is associated with chickens and is mainly transmitted in eggs. In the United Kingdom, a widespread vaccination program was implemented for lay-laying hens to reduce transmission of S. enteritidis .The pragramme was successful in achieving a significant decrease of human infections caused solely by S.enteritidis (Gast et al., 2007) 2.17 Future Prediction and Perspective Zoonotic infection is responsible for a large and growing proportion of the mortality and morbidity throughout the world. Foodborne Zoonoses will likely continue to be important in the future as the global population move more toward meat products as a source of protein (Merianos et al., 2007 Murphy et al., 2008) .In general, the human infections caused by NTS will most likely not significantly decrease in the future unless the global intervention towards eggs succeeds. However, the serovar distribution will probably on a global scale be influenced by increased trade and consumption of exotic food or food produced to low costs. Most of the research conducted in the last decades has been focused on S. enteritidis and S. typhimurium, while relatively little is known about the epidemiology of rare serovars with the potential is increased globally. Recently focus on NTS in the African region revealed that serovars causing gastrointestinal infections also are invasive (morpeth et al., 2009) .We predict to see stronger evidence of NTS causing bacteremia in human from Africa in time due to the effort by WHO to implement and enhance Salmonella Surveillance and burden to illness, studies in this area, Another alarming development which lay head in the increasing frequency of antimicrobial resistance in NTS. In the last decade the western world has tried to minimize the usage of fluoroquinolones and third and fourth generation cephalosporins in both the human and veterinary medicine. These two drug classes are paramount in treating Salmonellosis caused by multi-drug resistant strains recently, the WHO has stated that antimicrobial resistance in a global public health concern and has top priority. Evidence shows extensive usage and resistance to fluoroquinolone and third and fourth generation cephalosporins from developing countries driven in part by low price and weak or absent use restriction (Arachambault et al., 2006 Aarestrap et al., 2007 fabre et al., 2009 Lee 2009) in the past two decades, our understanding of Salmonella biology and epidemiology has grown tremendously Advances in technology are moving toward rapid, high throughput, comprehensive analytical methods. It is highly likely that many, if not all, phenotyping technique will be supplanted by platform based in DNA sequence and gene expression tools. To make this transition, it will be necessary to validate the sequenced-based method to the older phenotypic methods in order to avoid the loss of knowledge and the ability to compare with historical data (Hyytia et al., 2007). It is expected that within the next decade, microbial genomic sequencing will become inexpensive and routine worldwide. Currently, the technique in faced with limiting factors of how to assemble, process and handle the large amount of data full genome sequencing will create. Despite of this limiting software to resolve the problem will most likely be developed in the future making rapid tools for analysis available for extraction of biological and epidemiological data. The data could be applied in multiple ways for typing genetic comparison or ever non-specific vaccines. CHAPTER THREE 3.0 Materials and Methods 3.1 Study Areas The minimum samples size for this study was calculated as fellow with the confidence level of 95 and confidence interval of 5, the minimum sample size is calculated using the formula below NZ2(P)(1-P) C2 Where Z1.96 for confidence level Cconfidence interval of 5 Ppercentage picking of choice NMinimum number of sample required 3.4 Study Design This study was done in order to find the prevalence and antimicrobial resistance of Salmonella in clinical samples (blood, urine and stool) and non- clinical samples (egg, vegetable, tomatoes, intestine sample, meat and yam powder).since the study was a laboratory based research, the isolation and identification of Salmonella contamination was done on all clinical and non-clinical samples presented for routine tests. The convenient sampling method was used whereby all samples collected were regarded as samples for the study. Isolates of confirmed Salmonella were also tested for antimicrobial resistance in order to establish the resistance of the organism against antimicrobial. 3.5 Sample Collection Food samples that were collected included meat, yam powder, egg, intestine sample, tomatoes and vegetables, which were purchased from retail outlets, and were placed in sterile plastic bags. The samples were taken to the laboratory and were analyzed within 6hours of collection. Samples were purchased directly from farmer in Ado-Ekiti as well as from market located in the center of the city. Samples were aseptically cut, placed in separate plastic bags and were transported on ice to the laboratory, where they were analyzed within 6hrs of collection. With the addition of BPW at a 1 6 ratio to 6.5 g sample with 58.5ml BPW. Bend until clumps were dispersed. Incubate at 37oc for 18-24 hours. Fresh blood, urine and stool samples that were reactive for Salmonella using test kits were collected from Ekiti State University Teaching Hospital Ado-Ekiti and were placed in transport medium. The samples were transported to the Microbiology Laboratory, where they were analyzed within 6hrs of collections, followed by the Isolation and identification of Salmonella strain. 3.6 Cultural Method Isolation of Salmonella strain from blood, urine, stool and food samples were performed using standard culturing techniques. During cultivation pre-enrichment for the resuscitation and multiplication of damaged Salmonella isolates were followed by selective enrichment and plating. The isolation procedures were performed as follows 6.5 g of the foods were weighted and blended and were added to 58.5ml of buffered peptone water (LABM 046) and appropriate dilutions were performed. The samples were incubated at 37oC for 24hrs for enrichment. Following incubation, a loopful of inoculum from the enrichment culture was streaked onto xylose lysine deoxychocolate (XLD) agar (LAB M032) plates and incubated at 37oC for 24 hrs. 3.7 Identification using API 20 E The identity of all presumed Salmonella isolates was confirmed using the API20E strips. A single isolated colony of the Organism is suspended in sterile distilled water and inoculated in API20E Biochemical Test strip which contains dehydrated bacterial media/biochemical reagents in 20 separate compartments. Bacteria react with them sterile oil is added into amino acid Arginine, amino acid ornithine, H2 S and Urease Compartment. The tray is incubated at 37C For 18-24hours the colours were read after 24hrs while reagents were added to other to know whether they are positive or negative. 3.8 Biochemical Tests Tartrate utilization and gelatin hydrolyzing tests were employed to differentiate between the different Salmonella subspecies. The tartrate utilization test was carried out according to the methods described by Ewing (1986) .the organic acid medium consisted of 10 g peptone 12ml of 0.12 bromothymol blue solutions and 10 g sodium potassium tartrate which were added to 1L distilled water. The pH was there after adjusted to 7.4 with 10ml sodium hydroxide. The tubes, each containing 3ml of the medium were inoculated with a loopfull of overnight culture and were incubated at 37oC for 24hrs. A volume of 0.5ml of a 50 neutral lead acetate solution was added to each of the tubes (experiment and control) tartrate utilization was indicated by the decrease in the volume of precipitate formed when compared with the control tube. Tartrate utilization was also confirmed using phenol red tartrate agar as described by Ewing (1986). The agar deeps were inoculated by stabbing colonies of the organisms into the medium with a straight wire, using overnight culture of the Salmonella isolates. Then tubes were incubated at 37oC for 48hrs. 3.10 Antibiotic Susceptibility testing of the isolates Antibiograms were obtained for the positive Salmonella isolates by collecting antibiotic susceptibility data using the disk diffusion method, Bacterial cultures used for the susceptibility tests were obtained from iron triple agar. Adjusted to 0.5 Macfarlands standard. Sterile cotton swab was dipped into the suspension, rotated several times, pressing firmly on the inside wall of the tube above fluid level to remove excess inoculums and swabbed uniformly over the surface of Muller Hinton agar plate. The plates were held at room temperature for 30min to allow drying. Plates were incubated for 19hrs and zones of inhibition were read automatically by meter-ruler. Minimal inhibitory concentration (MIC) values and breakpoint concentration used were based on the Clinical Laboratory Standard Institute (CLSI) former (NCCLS) m100 (27) and m31 (28) reference (CLSI, 2016). The plates included susceptibility testing to the following antibiotics Chloramphenicol (30g), Ofloxacin (10 g) Co-trimoxazole (30g) ciprofloxacin (30g), Amoxicillin (30g), Augmentin (30g), Gentamycin (10g,) Pefloxacin (30g) and Streptomycin (30g). CHAPTER FOUR 4.0 Results Table 4.1 shows the prevalence of non-typhoidal Salmonella in clinical Samples. Out of 55 stool samples 9 were positive, from 60 blood samples 6 were positive and from 35 urine samples 4 tested positive for strains of Salmonella. A total of 19 samples were positive for non-typhoidal Salmonella among clinical samples. in tables 4.2 depicts the prevalence of non-typhoidal Salmonella in non-clinical samples, from 27 raw eggs,9 were found positive for strains of Salmonella, among 25 groups of vegetables 11 were positive for strains of Salmonella, 4 out of 8 tomatoes were positive,5 out of 12 intestinal samples were also positive for strains of Salmonella. From 21 different sources of meat, 10 were positive and 1 out of 7 sources of yam powder was positive for non-typhoidal Salmonella. A total of 40 samples were positive among non-clinical samples. The results from non-clinical samples showed that Salmonella was isolated from nearly all the food items used as non-clinical Samples. Gender distributions of clinical samples are shown in table 4.3. 80 were males while 70 were females, 9 males cases were positive for strains of Salmonella and 10 female cases were positive for strains of Salmonella. The age distribution showed that children from ages 1-10 have the mostly affected cases of non typhoidal Salmonella with 6 cases the highest frequency, while elderly from ages 61-70 was next with 4 cases, Table 4.4 showed the percentage ages distribution of patients with positive isolates. Out of 150 patients from which Samples were collected 106 were diagnosed of typhoid fever while 44 were diagnosed of malaria, 15 patients were positive to strains of Salmonella from the typhoid fever cases, while only 4 cases were positive to Salmonella from the malaria cases.78.9 of the clinical isolates were from typhoid fever patients while 21.1 of the positive isolates were from the malaria patients as showed in Table 4.5. 4.1 The result of API20E The results of API20E showed that Organisms are positive to decarboxylation of Amino acid lysine, decarboxylation of Amino acid Ornithine, Hydrogen Sulphide,glucose,Mannose,fermentation of Sorbitol, fermentation of rhamnose, fermentation of arabinose while the organisms is negative to beta-galactosidase, decarboxylation of Amino acid Amine, Urease, Tryptophan deaminase, indole, Voges proskauer test, gelatinase liquefaction, fermentation of inositol, fermentation of Sucrose and fermentation of amygdalin. Table 4. 6 shown the biochemical reactions involved in API20E Test kits and typical Salmonella reaction. 4.2 Biochemical test Tartrate Utilization and gelatin hydrolysis were further tested to determine which subspecies the isolates could further belong, all isolates collected from both Clinical and Non-Clinical Samples gave a positive reaction compared to control tube, except for few Samples, positive tests for tartrate utilization were indicated by the development of an acid reaction(yellow colour)culture that tested positive for tartrate utilization were considered to belong to Salmonella enterica Subspecies enterica or Subspecies 1 and negative isolate were considered to belong to the environmental isolate or Salmonalla Subspecies however, most of the Clinical Samples didnt show positive reaction. Tartrate Utilization was also confirmed using phenol 1 red tartrate agar ,positive test for gelatin hydrolysis were indicated by the liquefaction are considered to be possible for the human origin(Salmonella enterica Subspecies 1) all isolates obtained from Clinical and non-Clinical Samples were negative for the gelatin hydrolysis. 4.3 The Antibiotic Sensitivity profiles of the isolates All Isolates were Susceptible to Ciprofloxacin 55, Amoxicillin 53 Streptomycin 50, Gentamycin 48, Chloramphenicol 32, Co-trimoxazole 30 Augmentin 28.Ciprofloxacin was the most effective agent against all the NTS Isolates. High resistance was recorded against Pefloxacin 78 and Ofloxacin 81 as shown in Table 4.7. 4.4 Serotyping of the Salmonella isolates Of the Salmonella Isolates collected most strains were serologically identified. Majority of the serotypes were Salmonella enterica which was determined by antigen H and O Antisera. Serogroup A,( 9 )Isolates ,Serogroup B,( 15) Isolates Serogroup C1, (12) Isolates, Serogroup C2, (10) Isolates, Serogroup D ,(8) Isolates and Serogroup E,( 5)Isolates. Ever though these Strains belong to Salmonella enterica Subspecies enterica the serotypes cannot be determined further because there were over expression in the H phase. Table 4.8 showed the percentage distribution of Salmonella Serogroups isolated from Clinical and non-Clinical Samples. Table 4.1 Prevalence of Non-typhoidal Salmonella in Clinical Samples Samples Number Of Samples Number Positive Samples Total Percentage P valueStool 55947.40.366Blood 60631.6Urine 35421.0Total 15019100 Table 4.2 Prevalence of Non-typhoidal Salmonella in Non-Clinical Samples Samples Number Of Samples Number Positive Samples Total Percentage P valueEgg27922.50.0415Vegetable 251127.5Tomatoes 8410Intestine sample 12512.5Meat 211025Yam powder 712.5Total10040100, Table 4.3 Gender distribution from Clinical cases SexNumber Of patients Number of Positive patients Total Percentage Male 80947.4Female 701052.6Total 15019100 Table 4.4 Percentage Age Distribution of Positive Patients Age distribution No of positive isolatePercentage 1-10 11-20 21-30 31-40 41-50 51-60 61-70 TOTAL 6 2 0 3 3 1 4 1931.6 10.5 0 15.8 15.8 5.3 21.0 100 Table 4.5 Percentage Diagnosis distribution of the isolates in clinical Sample Diagnosis No of positive isolate to NTS No of positive isolate to NTS Typhoid fever 106 Malaria 44 Total 15015 4 1978.9 21.1 100 Table 4.6 The Biochemical reactions involved in API20E test kits and typical Salmonella reaction Test Substrate ReactionResult (-)Result ()Salmonella SPPONPG ADH LDC ODC CIT H2S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARAONPG Arginine Lysine Ornithine Citrate Na thiosulfate Urea Tryptophan Tryptophan Na pyruvate Gelatin Glucose Manitol Inositol Sorbitol Rhamnose Sucrose Melibiose Amygdalin ArabinoseBetagalactosidase Arginine dihydrolase Lysine Decarboxylase Citrate utilization H2S production Urea hydrolysis Deaminase Indole production Acetone Gelatinase Fermentation Fermentation Fermentation Fermentation Fermentation Fermentation Fermentation Fermentation FermentationColorless Yellow Yellow Yellow Pale Colorless Yellow Yellow Yellow Colorless No diffusion Blue green Blue green Blue green Blue green Blue green Blue green Blue green Blue green Blue green Yellow Red Red Red Blue Black Red Brown Red Red Black diffusion Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow – – – – – – – – – – – Table 4.8 Distribution of Salmonella Serogroups isolated from clinical and non-clinical samples Serogroups No of isolates in each Serogroup of Isolates in each SerogroupSerogroup A 9 15.3 Serogroup B 15 25.4 Serogroup C1 12 20.3 Serogroup C2 1 16.9 Serogroup D 8 13.6 Serogroup E 5 8.5 Total 59 100 Table 4.7 Antibiotic susceptibility pattern of non-typhoidal salmonella isolated from clinical and non-clinical sources. Name of Antibiotics Number Of Sensitivity ()Number of resistant () Number of intermediate () Ciprofloxacin (10g) 32(54) 27(46) 0(0.0)Amoxacillin (30g) 31(53) 28(48) 0(0.0)Streptomycin (30g) 30(51) 29(49) 0(0.0)Gentamycin (10g) 28(47) 31(53) 0(0.0)Chloramphenicol (30g) 19(32) 40(68) 0(0.0)Co-trimoxazole (30g) 18(30) 41(70) 0(0.0)Augmentin (30g) 17(29) 42(71) 0(0.0)Pefloxacin (30g) 12(20) 46(78) 1(2.0)Ofloxacin (10g) 11(19) 48(81) 0(0.0) CHAPTER FIVE DISCUSSION Although it is known that non-typhoidal Salmonella infections are widespread in Nigeria, no data exist on bacterial quality of clinical and non-clinical sources. This study has therefore, been conducted to assess the clinical and non-clinical Salmonella infection transmission. The incidence of Salmonella in non-clinical sources was relatively higher than in the clinical samples. The prevalence rates in both clinical and non-clinical cases were 23.6. Most of the non-clinical samples were found positive for the organisms. This indicates contamination of Salmonella in the foods used as non-clinical sources. The isolation of Salmonella isolates from non-clinical samples also indicates high incidence of Salmonella in certain food samples e.g. eggs, vegetable and meat. It has been reported in many studies that Salmonella is usually isolated from food of animal origin especially poultry meat, beef and raw eggs (fratamico 2012). In this study eggs, vegetable, tomatoes, intestine sample, meat and yam powder samples were tested since they are common source of foods in Ado-Ekiti, Ekiti- State, Nigeria. The study also showed that, most of the vegetable may be contaminated while growing, during harvest, handling and distribution. In many areas of Ado-Ekiti untreated waste water flow into rivers and streams. This contaminated water is used to irrigate some plots of vegetable that are sold in the markets. The use of Animal wastes or human wastes as fertilizer could also be an important source of contamination. Following the isolates, Salmonella species were differentiated phenotypically using biochemical test included API20E, tartrate utilization and gelatin hydrolysis. Based on the result of biochemical tests and serotyping all the Salmonella isolates were identified as Salmonella enterica sub species which are commonly associated with humans and warm-blooded animals. Based on the result of the serotyping Serogroup B and C were the most frequently isolated .Many serotypes of sub species are pathogenic and are able to cause infections such as gastroenteritis, septicemia and other serious conditions in human and animals. The antimicrobial susceptibility studies of all the isolates showed that Ciprofloxacin was the most active antibiotic showing 55 Susceptibility in all the isolated strain of NTS Majority of the isolates were however resistant to pefloxacin and Ofloxacin. In Nigeria Resistance to these antibiotics has also been reported (Olowe et al., 2007) Antimicrobial-resistant Salmonella are increasing due to the use of antimicrobial agents in food animals at sub-therapeutic level or prophylactic doses which may promote on the farm selection of antimicrobial resistant strains and markedly increase the human health risks associated with contaminated meat products. Molla et al (2003) reported that the isolates of Salmonella from food items and workers from Addis Ababa were resistant to the commonly used antibiotics including streptomycin, ampicillin and tetracycline. Furthermore, Zelatin et al., (2001) also reported resistance of Salmonella isolates to commonly used antimicrobials including ampicillin, streptomycin, nitrofuratoine, kanamycin and tetracycline with 100, 66.7, 58.3 and 33.3 respectively. Similarly, in previous reports by Akinyemi et al., (2005) a high resistance was seen for ampicillin. The results of the current research also indicated resistance of majority Strains Salmonella isolates to antimicrobials such as Pefloxacin and Sparfloxacin. The difference could be due to the increasing rate of inappropriate utilization of antibiotic which favors selection pressure that increased the advantage of maintaining resistance genes in bacteria (Akinyemi et al 2005).Moreover, this increase antibiotic resistance, in addition to public health problems may lead to economic loss of exporting foods and animal products and cost of drug of choice to treat human and animals due to resistance development. , Ciprofloxacin, Amoxicillin and Streptomycin showed good antimicrobials activities against Salmonella isolates it was found that most isolates were susceptible to Ciprofloxacin Amoxicillin and Streptomycin. This result was comparable with previous reports among isolates of sheep and goat meat. Akinyemi et al( 2005) from Nigeria also reported that the effectiveness of such drugs like ciprofloxacin could be because this drug is not well distributed in the society and not readily prescribed rather than it is used as drug of choice in antibiotic resistant person. In addition to this, Ciprofloxacin is not commonly used to treat animals in Nigeria. CONCLUSION In this study, the isolation of Salmonella from clinical and Non-clinical samples, indicated the wide spread distribution of Salmonella in Ado- Ekiti Ekiti State, Nigeria. The result of the study also showed that the occurrence of Salmonella was not confined to a particular location as Strains of Salmonella was recovered from most of the sampled. Due to logistical reasons the sample collection is over a period of Eight month. Based on the results of the present study it can be concluded that clinical and non-clinical source have an important role in Salmonella transmission in Ado-Ekiti, Ekiti -State Nigeria. 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A perspective on the role of pulsed-field gel electrophoresis for surveillance in Mexico in Molecular biology and molecular epidemiology of infections.(Juan J. Calva and EdmundoCalva editors). Mexico, 2009, 90-107 Xia S, Hendriksen RS, Xie Z, Huang L, Zhang J, Guo W, Xu B, Ran L, Aarestrup FM. Molecular Characterization and Antimicrobial Susceptibility of Isolates from Infections in Humans in Henan Province, China J. Clin.Microbiol., 2009 47 401 – 409. Yan S. Pendrak, M., Abel Riddir, B Pundirson, J., Fehorki D., and Foley. F 2003 An overview of salmonella Typing Public Health Perspectives. Clin and Spp in Raw 4, 189 204. PREVALENCE AND CHARACTERIZATION OF NON- TYPHOIDAL SALMONELLA (NTS) FROM CLINICAL AND NON CLINICAL SOURCES IN ADO-EKITI, NIGERIA. BY ADENIJI NURUDEEN (MATRIC NO AAA012543) A PROJECT SUMMITED TO THE DEPARTMENT OF MEDICAL MICROBIOLOGY AND PARASITOLOGY FACULTY OF BASIC MEDICAL SCIENCES LADOKE AKINTOLA UNIVERSITY OF TECHNOLOGY OGBOMOSO, OYO-STATE NIGERIA. IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR AWARD OF MASTER OF SCIENCE M.SC 2017 Abstract Non Typhoidal Salmonella Strains (NTS) are an important cause of infectious diarrhoea world-wide. In the absence of immune deficiency, gastroenteritis caused by Non typhoidal Salmonella is usually mild, self-limiting and rarely requires intervention. Nevertheless, non typhoidal Salmonella are also an important cause of invasive diseases, particularly in developing countries. This study was conducted to determine the occurrence and phenotypic characterization of non typhoidal Salmonella in Ado-Ekiti. In this study, blood, urine, stool and foods samples were collected. Samples were examined macroscopically, cultured, screened for NTS and antibiotic susceptibility test was also performed using standard procedures. Biochemical tests were performed using API20E, gelatin hydrolysis and tartrate utilization was employed to differentiate Salmonellae into subspecies. All isolates were identified and then subjected to serotyping with antigens H and O antisera. A total of 250 samples (150 clinical and100 non-clinical) were tested, 40/100 (40) non typhoidal Salmonella was obtained from non-clinical sources and 19/150 (22.66) from clinical sources. Out of 150 patients from which clinical samples were collected 106 were diagnosed of typhoid fever while 44 were diagnosed of Malaria,15(78.9) patients tested positive for typhoid fever were also positive for non-typhoidal Salmonella. While only 4(21.1) Malaria patients were positive for non- typhoidal Salmonella. The age distribution showed that children from ages 1-10 have the highest frequency of non-typhoidal Salmonella with 6(31.6) cases while elderly from ages 61-70 were next with 4(21.0) cases. Most of the isolates utilized tartrate while none of the isolates hydrolysed gelatin. Results of antimicrobial sensitivity showed that the majority of the organisms were sensitive to Ciprofloxacin (55), Amoxicillin (53) and Streptomycin (50)while the isolates are Susceptible to Pefloxacin (20) and Tarivid (18). Serotyping results showed that SerogroupB, 15(25.4) and Serogroup C1, 12(20.3) were the most common. The occurrence of non typhoidal Salmonella coexisting with typhoid and malaria is a threat to human health It is therefore recommended that continuous studies should be done with non typhoidal Salmonella presence in foods, animals and humans and there is also a need to improve hygiene practice. ACKNOWLEDGEMENT I express my sincere gratitude to God for blessing and helping me throughout this research project. I also wish to express my sincere gratitude to my supervisor, Professor Samuel Sunday Taiwo, for his very valuable support and suggestions from the early stage of the project to the end. Working with him was a privilege and his words of encouragement were instrumental to the success of this research. I also thank my friends for their moral and academic support. I am grateful to my lecturer in the Department of Medical Microbiology and Parasitology, Ladoke Akintola University of Technology, Osogbo Dr Ojorongbe, Dr Olowe, Dr Opaleye, Dr Adesiji, Dr Bolaji and Dr Adekunle for the opportunity given to me to further my study. I am sincerely grateful to all the staff of Department of Medical Microbiology and Parasitology, Ekiti State University Teaching Hospital, Ado-Ekiti, Nigeria for allowing me to use their laboratory and all the member of Staff of the Department of Medical Microbiology and parasitology Obafemi Awolowo University Teaching Hospital, Ile -Ife for their support and contribution toward the success of this research. I also wish to thank Dr. Adebayo Makinde and Semi Akintayo of the General Hospital Omuo-Ekiti, Ekiti-State for their support. I thank you all. DEDICATION This project is dedicated to Almighty God. CERTIFICATION This is to certify that this project was carried out by ADENIJI NURUDEEN (Matric Number AAA012543) of the Department of Medical Microbiology and Parasitology and was supervised and approved in fulfillment of the requirement for the award of M.SC. . Prof. S.S. Taiwo Date (Supervisor) . Dr.O.O Opaleye Date (Head of Department) Abbreviations API Analytical profile index system BPW Buffered peptone water CDB Country Data Bank CDC Center for Disease Control EQAS External Quality Assurance System HACCP Hazard Analysis and Critical Control Point H2S Hydrogen Sulphide MIC Minimal Inhibitory Concentration NTS Non Typhoidal salmonella PCR Polymerase Chain Reaction PFGE Pulsed field Gel Electrophoresis PT phage Type RFLP Restriction Fragment Length Polymorphism SS Salmonella-Shigella Agar WHO World Health Organization XLD Xylose Lysine Deoxychocolate Agar List of Tables S/N Title Page Table 4.1 Salmonella Isolates from Non Clinical Source 48 Table 4.2 Sex Distribution from Clinical Source 49 Table 4.3 Gender Distribution from Clinical Cases 50 Table 4.4 Percentage Age Distribution of Positive Patients 51 Table 4.5 Percentage Diagnosis Distribution of Isolate 52 Table 4.6 The Biochemical Reactions Involved in API20E Test Kit and Typical Salmonella Reaction 55 Table 4.7 Distribution of salmonella serogroups Isolated form Clinical and Non- Clinical Samples 56 Table 4.8 Antibiotic Susceptible Testing of the Isolate 57 TABLE OF CONTENTS Abstract ii Acknowledgement iv Dedication v Certification vi Abbreviations vii List of Tables viii CHAPTER ONE Introduction 1 Taxonomy and typing of Salmonella 3 Stress tolerance of Salmonella 6 Pathogenesis of non typhoidal Salmonella in human 7 Adhesion and invasion of intestinal epithelial cells 8 Aims and objectives 12 CHAPTER TWO Literature Review 13 Human epidemiology 15 Symptoms and human infections 17 Age Season and Risk Factors 19 Antimicrobial Treatment and antimicrobial Resistance 20 Main Reservoir 21 Transmission Local and Global 21 Surveillance Methods 22 Detection Methods 22 Pulsed Field Gel Electrophoresis in Salmonella 24 Immunological Method 25 Microscopy 26 Polymerase Chain Reaction 27 Salmonella in Humans and Animals 28 Salmonella in Environment 30 Control, Intervention and Prevention 33 Future Prediction and Perspective 37 CHAPTER THREE Materials and Methods 40 Study Area 40 Study Population 40 Sample Size Determination 40 Study Design 41 Cultural Methods 42 Identification Using Api20E 43 Biochemical Tests 43 Serotyping of the Salmonella Isolates 44 Antibiotic Susceptibility Testing of the Isolates 44 CHAPTER FOUR Results 46 Biochemical Tests 53 Antibiotic Sensitivity Profile of the Isolates 54 Serotyping of the Salmonella Isolates 54 CHAPTER FIVE Discussion 58 Conclusion 61 References 62 PAGE MERGEFORMAT 56 Passage through the stomach Colonization of the small intestines Invasion of m cell and entry into peyers patches Drainage to mesenteric lymph node Survival and growth within macrophages of liver and spleen Spread to the blood stream Y, dXiJ(x(I_TS1EZBmU/xYy5g/GMGeD3Vqq8K)fw9
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