Excerpt
TABLE OF CONTENTS
DEDICATION
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF APPENDICES
LIST OF ABBREVATIONS AND ACRONYM
ABSTRACT
1. INTRODUCTION
1.1. Back Ground of the Study
1.2. Statement of Problem
1.3. Significance of the Study
1.4. Limitation of the Study
1.5. Objectives of the Study
1.5.1. General objective
1.5.2. Specific objectives
2. LITERATURE REVIEW
2.1.Salmonellosis
2 .2.Taxonomy and Nomenclature
2.3. Etiology and characteristics
2.4. Epidemiology
2.4.1. Mode of Transmissions
2.4.2. Fish as Source of Salmonella
2.4.3. Potential Risk Factor of Occurrence of Salmonella in Fish
2.5. Pathogenesis
2.6. Clinical Signs and Symptoms
2.7. Diagnosis
2.7.1. Culture method
2.7.2. Rapid detection method
2.8. Treatment
2.9. Prevention and Control
2.10. Salmonella in Humans
2.11. Salmonella in Fish
2.12. Antimicrobial Resistance
2.12.1. Antibiotics Resistant Salmonella Isolated from Fish
2.13. Public Health Importance of Salmonellosis
3. MATERIALS AND METHODS
3.1. Descrption of the Study Area
3.2.Study Animals
3.3. Sample Size Determination
3.4.Study design
3.5. Methods of Data Collection
3.5.1. Questionnaire survey 23
3.5.2. Sample collection and transportation 23
3.5.3. Laboratory analysis 23
3.5.4. Confirmation of Salmonella Isolates 24
3.6. Antimicrobial Susceptibility Testing
3.7. Ethical clearance
3.8. Data Management and Analysis
4. RESULTS
4.1. Occurrence of Salmonella
4.2. Antimicrobial susceptibility of the isolated Salmonella
4.3. Socio-Demographic Characteristics of the Respondents
4.4. Risk Factors Associated with Occurrence of Salmonella
5. DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5. 1. Discussion
5.2. Conclusions
5.3. Recommendations
6. REFERENCES
7. APPENDICES
DEDICATION
This work is dedicated to my father Bane Bodena, my mother Alemitu Beyene, and the fishermen of Lake Fincha.
ACKNOWLEDGEMENTS
First and foremost, I would like to express my heartfelt gratitude to almighty God for His unending mercy in allowing me to exist in this dynamic world, overcoming all odds during my studies, and remaining in my and my family's lives forever.
My heartfelt gratitude goes to the Guduru District Administrative Office for granting the study leave and securing my salary during the study period.
My heartfelt gratitude goes to my advisors, Dr. Bizunesh Mideksa and Mr. Abebe Olani, for their unwavering encouragement, constructive criticism, and advice.
My heartfelt gratitude also goes to the National Animal Health Diagnostic and Investigation Center (NAHDIC) staffs for their unwavering laboratory assistance and counseling service. It is also my pleasure to thank my friends Getahun Deresse for hosting me to conduct molecular characterization, Misgana Tefera and Amare Tesfaye, as well as NAHDIC staff members, for their technical, moral, and material support in the completion of this study .
My heartfelt gratitude goes to the people who work in the livestock and fishery office in the study area for their enthusiastic cooperation and field guidance during sample collection.
I would like to take this opportunity to express my heartfelt gratitude and love to my wife, Habib Yadeta, and our children (Anatoly and Monenus), for their patience, care, love, encouragement, and understanding during my absences and in all aspects of our family life. Finally, I want to thank everyone who is involved in this study, whether directly or indirectly.
LIST OF TABLES
Table 1: Occurrence of Salmonella from different fish species samples and fishermen hand's swabs
Table 2: Results of antimicrobial susceptibility testing of S. Typhimurum isolates from Fish
Table 3: Socio -demographic characteristics of the respondents
Table 4: Knowledge of respondents on Salmonella and its prevention
Table 5: Practice of fishermen on prevention of occurrence of Salmonella
Table 6: Result of Univariable logistic regression analysis of risk factors of occurrence of Salmonella
Table 7: Multivariable logistic regression analysis for the predictors of occurrence Salmonella in fish at Fincha Lake
LIST OF FIGURES
Figure 1: Map of the study area
Figure 2: Salmonella Typhimurim isolated from fish by Conventional multiplex PCR
LIST OF APPENDICES
Appendix 1: Data collection tool for questionnaire survey
Appendix 2: Type and preparation of microbiological media used for isolation and antimicrobial susceptibility test of Salmonella
Appendix 3: Procedures of salmonella species identification by Conventional multiplex PCR for Salmonella Enteriditis, Salmonella Typhi ,and Salmonella Typhimurum Identity test procedure
Appendix 4: The Susceptibility of each antimicrobial was determined depending on the following the measure of zone inhibition diameter
Appendix 5 : Plating and biochemical tests record format used for Salmonella isolation.
LIST OF ABBREVATIONS AND ACRONYM
Abbildung in dieser Leseprobe nicht enthalten
ABSTRACT
Salmonella is one of the most common foodborne and important zoonotic pathogens worldwide. Because of the difficulties in ensuring optimal hygienic food handling practices, the problem is even more severe in developing countries. A cross-sectional study with a simple random sampling approach was conducted to estimate the occurrence, identifying risk factors, and assessing the antimicrobial susceptibility profile of Salmonella isolated from fishermen and fish species from Fincha Lake. Salmonella was isolated, phenotypically identified by Omni Log and the antimicrobial susceptibility test was performed using the disk diffusion method and interpreted following the Clinical Laboratory Standard Institute recommendations. Tilapia (220), common carp (85), and catfish (79) were the most known fish species in Lake Fincha. Samples were collected from skins ( 65 ),Gills ( 96 ), Intestines ( 110 ) , and Muscles ( 113 ) parts of these three species. Muscles 3 (2.65%) samples were found to be the most Salmonella positive, followed by gills 2 (2.08 %). No Salmonella was isolated from fishermen's hand swabs ( 0% ).The prevalence of salmonella in fish was 1.30 % ( 95% confidence interval (CI ) : 0.42-3.01 ).The overall prevalence of Salmonella in the study area was 1.21% ( 95% CI: 0.39-2.80 ) . All of the isolated Salmonella (n=5) belongs to S. Typhimurium Serovars. The antimicrobial susceptibility test revealed that the isolates were resistant to Cefotaxime, Tetracycline, and Ampicillin disks. However, Chloramphenicol, Sulfamethaxazole+Trimethoprim, Amoxicillin + Clavulanic acid, Gentamicin and Cefoxitin were found effective in inhibiting the growth of all of the isolates. Isolation of Salmonella from fish was associated with contamination of the harvesting areas, contamination of the Lake, collecting and processing materials and equipment. Factors independently predicted Salmonella occurrence were the presence of contamination (AOR: 2.84, 95%CI: 1.45-1.83, P=0.041), fish that was not iced after landing (AOR: 8 95% CI: 0.06-5.91, P=0.032) and poor handling practice (AOR: 29.3, 95% CI: 0.06-0.37 P=0.0.035). In conclusion, Salmonella has been isolated from fish collected from Lake Fincha, so appropriate Measures should be taken to prevent contamination and infection.
Keyword s: Antimicrobial Resistance, Risk Factors, fish, Lake Fincha, Salmonella
1. INTRODUCTION
1.1. Back Ground of the Study
Salmonella infections are a major public health concern around the world. Annually, it is estimated that 93.8 million cases and 155,000 deaths are associated with gastroenteritis Salmonella species worldwide. Based on the current evidence, 85.6 % of pathogens were food-borne, and infection was linked to a variety of food types (Majowicz et al., 2010). Salmonella in animals is a major concern because animals can serve as latent carriers of Salmonella serotypes and shed the organism into the environment without any obvious clinical signs, posing a risk of human infection (Kiflu et al., 2017).
To date, more than 2,700 Salmonella serotypes have been detected. Many have been linked to human illness (Jones et al., 2008). Non typhoidal Salmonella (NTS) serotypes are one of the most common causes of newborn diseases, causing diarrhea, bacteremia, and focal suppurative infections. S. Enterica subsp. enterica, especial serotypes like S.Enteritidis and S.Typhimurium, are responsible for a great number of infections in humans and other mammals (Dunkley et al., 2009). Aquatic foods are abundant in a world where water covers more than 70% of the land surface. It could provide an interesting aspect of global food to improve human nutrition, health, and well-being (Tacon and Metian, 2013). Despite the low mass consumption of fish in developing nations, it contributes 180 kcal per capita per day, in a few countries where people have developed a taste for fish (Lokuruka, 2009).
According to the Food and Agriculture Organization (FAO) (2020), fish production has increased dramatically in the last 60 years, representing approximately 179 million tons in 2018 with a market value of $401 billion. Global fish consumption increased as well, increasing from 9.0 kg per capita in 1961 to 20.5 kg in 2018. Aquaculture production accounts for 46% of total production and 62% of the total sale value. Global aquaculture production is expected to double by 2050 as a result of rising demand for high-quality protein, reduced wild fish catch, and progressions in fish farming technologies (Ibrahim et al., 2020). The aquaculture potential of the capture fishery from major Lakes, reservoirs, small water bodies, and rivers, on the other hand, was recently reported to be 94500 tons/year (Gashaw and Wolff, 2014). Fish is a source of unsaturated fats, cal led omega 3 fatty acids, which affect cardiac functions including hemodynamics and a rterial endothelial function (Wolfe, 2010). Fish is an excellent source of high- quality protein, contains the essential amino acids that are necessary for human health (Hoyle and Merritt, 1994). Fish skin surface, intestine ,and gills, however, carries a high microbial load (1.72 ± 0.68 x 108 to 7.00 ± 3.39 x 108)
(Mhango et al., 2010). Aquaculture food safety hazards include contaminants such as environmental pollutants, fish disease, and hygienic aspects (microbial agents) (Hastein et al., 2006). Fish handling hazards may include catching, slaughtering, and processing for consumption (Hastein et al., 2006). Fish's natural habitat is highly vulnerable to pollution from domestic, industrial, and agricultural discharges. As a result, fish and other aquatic life forms are vulnerable to all potential hazards (Raufu et al., 2014).
Microorganisms are commonly found on the skin and gills of fish, as well as inside the fish, in areas such as the digestive tract and internal organs such as the kidney, liver, and spleen. Fish and fish products, particularly raw or undercooked products, have been linked to outbreaks of bacterial pathogens, bio toxins, histamine, viruses, and/or parasites (Galaviz-Silva et al., 2009). Salmonella has been isolated from a variety of fish and seafood (Elhadi, 2014). It may be transferred to seafood as a result of poor sanitary conditions during transportation and marketing (Sarter, 2007). Salmonella prevalence in sea foods has been studied in various parts of the world, and health risks have been assessed. Salmonella distribution in seafood was found to be highest in the central Pacific and Africa (12%) and lowest in Europe/Russia and North America (1.6%) (Dh Al-Khayat and Khammas, 2016). Salmonella prevalence in fish food has been reported in Khartoum, Sudan (9.2 %) (El Hussein et al., 2010), Coimbatore, India (14.7 %) (Lakshmanaperumalsamy, 2014). Despite constant surveillance and intensive efforts, Salmonellosis-related food poisoning outbreaks are increasing in Western countries, with fishery products accounting for a significant portion of the outbreaks reported (Gandra et al., 2014). In developing countries such as Ethiopia, there is no such continuous monitoring system, and the exact number of cases is unknown (Grace, 2015).
Antimicrobial resistance is not a new phenomenon, but the number of resistant organisms, geographic areas affected by drug resistance, and breadth of resistance in single organisms are unprecedented and increasing (Bujjamma, 2015). Nonetheless, some, most notably those in the developed world, saw the resistance problem as a curiosity of minor health concerns confined to gastrointestinal organisms in distant countries (Pouokam, 2017). Previous studies on Salmonella infection and drug resistance patterns in fish were not adequately addressed and this leads to fish not being considered and prioritized as one of the challenging industries for food poising and drug-resistant development. There are over 15 classes of antibiotics, all of which target essential physiological or metabolic functions of the bacterial cell (Malorny et al., 2004), and none have escaped a resistance mechanism. Every year, millions of kilograms of antimicrobials are used in the prevention and treatment of diseases in humans, animals, and agriculture around the world (Malorny, 2004). Antimicrobial resistance mechanisms are as diverse as the antimicrobials themselves (Antonelli et al., 2019).
1.2. Statement of Problem
Salmonella has become a threat to the global food system, from production to processing and consumption (Ao et al., 2015). Salmonella is not a biological contaminant that was first identified in fish, where it was introduced through contaminated water or improper handling (Santana, 2012). As a result, understanding Salmonella is critical for ensuring food safety and quality. The fact that this bacterium survives in soil and water and can be transferred to fish is important in providing information on the nature of the contamination and the possible route of dissemination of this bacterium, allowing traceability of the microbial source in fish slaughterhouses (Setti et al., 2009).
Poor post-harvesting practices such as irresponsible behavior and attitude toward hygiene practices of fish fillets, transporters, and fish traders/retailers) in their workplace; poor personal hygiene practices such as: dirty clothing and hands used to handle fish; exposed cuts and wounds; long dirty hands washed with dirty water without soap (Cunha-Neto et al., 2019). According to the best of our knowledge, only a few studies have been conducted in Ethiopia to assess the occurrence, risk factors and antimicrobial resistance profiles of Salmonella isolated from personnel and fish, but none has been conducted in Lake Fincha, our study area. A preliminary survey of GramNegative bacterial pathogens from commonly caught fish Species was conducted at Lake Hayiq with a prevalence of 1.6 % Tesfaye et al. (2018). They isolated Salmonella from fish but did not mention the associated risk factors or the pathogen's antimicrobial resistance.
1.3. Significance of the Study
This study provided new information about the occurrence of Salmonella in fish and helped to fill a knowledge gap about associated risk factors. Finally, this work was forwarded to the appropriate body to serve as a source of information for future investigations and corrective actions.
1.4. Limitation of the Study
There was a lack of funds, a problem with transportation access, a lack of related literature sources conducted at the study site, and the availability of secondary data on the most recent five years of actual duty reports.
1.5. Objectives of the Study
1.5.1. General objective
The general objective of this study was to evaluate the occurrence, identify risk factors, and assess the antimicrobial susceptibility status of Salmonella isolated from fish and fishermen Lake Fincha.
1.5.2. Specific objectives
- To estimate the occurrence of Salmonella in fish species harvested from Lake Fincha
- To estimate the occurrence of Salmonella in fishermen working in this study area
- To determine the antimicrobial susceptibility profile of Salmonella isolated from fishermen and fish species of Lake Fincha
- To identify the associated risk factors for the occurrence of Salmonella in fish species of Lake Fincha
- To identify the serotype of Salmonella
2. LITERATURE REVIEW
2.1.Salmonellosis
Foodborne Salmonellosis often follows consumption of contaminated animal products, which usually results from infected animals used in food production or from contamination of the carcasses or edible organs (Alemayehu et al., 2003). Salmonella remains among the main causes of foodborne illness in developing as well as in developed countries. Salmonella causes 31% of food-related deaths followed by Listeria (28%), Campylobacter (5%), and Escherichia coli O157: H7 (3%) (Michael and Samuel, 2001). Salmonella spp. is mainly transmitted by the fecal-oral route. They are carried asymptomatically in the intestines or gall bladder of many animals, and are continuously or intermittently shed in the feces (OIE, 2005).
2 .2.Taxonomy and Nomenclature
The current nomenclature of the genus Salmonella, which is used by the CDC, is based on recommendations from the WHO collaborating center and adequately addresses the concerns and needs of clinical and public health microbiologists (Deb and Kapoor ,2005). Salmonella is classified scientifically as follows: Domain: Bacteria, Kingdom: Monera, Phylum: Proteobacteria, Class: Gamma Protobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae, Genus: Salmonella, and Salmonella enteric and Salmonella bongori are the two species (Hafez and H.M, 2005). Salmonella enterica is divided into six subspecies (I-VI); S. enterica subsp. enterica is the most common (I), S. enterica subsp. salamae (II), S. enteric subsp. arizonae (IIIa), S. enterica subsp. diarizonae (IIIb), S. enterica subsp. houtenae (IV) and S. enterica subsp. indica (VI) (Brenner et al.,2000),based on biochemical properties (biotype), differences observed in multilocus enzyme electrophoresis (MLEE), phylogenetic analysis using 16S rRNA or other sequences, or analyses using other molecular techniques such as amplified-fragment length polymorphism (AFLP). Salmonella enterica subspecies I is responsible for 99 % of all human infections (DE and MS, 2006).
2.3. Etiology and characteristics
Salmonella strains are straight rods usually motile with peritrichous flagella (except S. Pullorum and S. Gallinarum), facultative anaerobes, ferment glucose usually with gas production (except S. Typhi and S. Dublin), but fail to ferment. Salmonella multiplies best at temperatures ranging from 35 OC to 37 OC, pH levels ranging from 6.5-7.5, and water activity ranging from 0.94-0.84. They are classified as chemo-organotrophic organisms (organisms that obtain energy by the oxidation of electron donors in their environments) (Food Research International, 2010). They can also multiply in environments with little or no oxygen (European Commission, 2000). The bacteria are heat sensitive and will not survive at temperatures above 70 OC; thus, they are susceptible to pasteurization but resistant to drying for years. Particularly in dried feces, dust, and other dry materials like feed and certain foods (Radostitis et al., 2007).
2.4. Epidemiology
Salmonellosis epidemiology is complicated, because there are over 2,700 distinct serotypes (serovars) with different reservoirs and geographical incidences. Food consumption, production, and distribution changes have increased in the frequency of multistate outbreaks associated with fresh and processed foods. Salmonellosis can affect all livestock species, with young, debilitated, and parturient animals being the most susceptible to clinical disease, and all age groups of humans being affected (Rounds et al., 2010).
Unlike Salmonella Typhi and Salmonella Paratyphi, which have a human host specificity, NTS can be acquired from both animals and humans (Braden, 2006). Salmonella is one of the leading causes of bacterial foodborne diseases in both developed and developing countries, though the incidence appears to vary by country (Chiu et al., 2004). Significant outbreaks of Salmonellosis occurred around the world at different times.
For instance, in the United States, 164,044 (approximately 32,000 annually) during 1998 - 2002 (Lynch et al., 2006); in China approximately 70% 80% and during 1992 - 2005 (Liu et al., 2008), in Germany, a total of 42,851 (Robert Koch Institute, 2008) (EFSA, 2009). In 2006, a total of 160,649 confirmed cases of human salmonellosis were reported in the EU (Liu, 2010). In many countries, the incidence of human Salmonella infection has increased drastically over the years. Salmonellosis is an important global public health problem causing substantial morbidity and mortality ( CDC, 2009) .
2.4.1. Mode of Transmissions
Salmonella is found in high concentrations in the rumen (Anderson et al., 2000), rectum (Ransom et al., 2002), cecum, and colon (Galland et al., 2001). People are frequently infected when they consume contaminated animal-derived foods such as meat, eggs and milk. They can also become infected by ingesting organisms in animal feces, either directly or indirectly through contaminated food or water (OIE, 2005). Salmonella spreads cyclically between humans, animals, food, and environmental sources. Non- typhoidal Salmonellae typically spread through the food chain. Animals can become infected by eating contaminated feed, drinking contaminated water, or coming into close contact with infected animals. Livestock, animal feed, and high levels of fecal shedding from infected animals have all been identified as important entry points into the food chain on a farm. Another source of contamination is the slaughtering of animals (Liu, 2010).
2.4.2. Fish as Source of Salmonella
Fish may be exposed to Salmonella s pp. through consumption of contaminated feed or by residing in contaminated water. Relatively little has been reported about the persistence, or possible dissemination, of Salmonella in fish exposed to these bacteria via feed. A few experiments in fresh water fish, e.g. Rainbow trout (Oncorhynchus mykiss), Israeli 8 mirror carp (Cyprinus carpio) and Tilapia (Tilapia area), have shown that high doses of orally-administered Salmonella could result in the persistence of Salmonella in the gastrointestinal tract, and sometimes also the entry of Salmonella into the internal organs and muscle tissue (Nwiyi and Onyeabor, 2012). Studies have pointed to a 10.4% (19/384) prevalence of Salmonella in fresh fish in Iran, where five different serotypes were detected, namely S. Typhimirium, S. Enteretidis, S. Typh i, S. Paratyphi B and S. Newport (Rahimi et al., 2013).
2.4.3. Potential Risk Factor of Occurrence of Salmonella in Fish
Fish habitat is extremely vulnerable to pollution from domestic, industrial, and agricultural discharges. As a result, fish and other aquatic life forms are vulnerable to all environmental threats (Raufu et al., 2014). Contaminants, including environmental pollutants, fish disease, and hygienic aspects (microbial agents), are all potential food safety hazards in aquaculture (Hastein et al., 2006). Salmonella spp. (enteric bacteria) may be present in fish and fishery products as a result of biological/microbial hazards during storage, processing, and preparation for consumption, to fecal and bacterial contamination (David et al., 2012). Bacterial disease epidemics are common in dense populations of cultured food, aquarium fish, or fish farms. The organic load of the aquatic environment and poor water quality are commonly associated with susceptibility to such outbreaks (Francis-Floyd, 2011).
Other factors may include fish handling, processing, and transport, hypoxia, sudden temperature changes, or other stressful conditions (Olsvik et al., 2013). Most bacterial pathogens in fish can be identified by isolating the organism in pure culture from infected tissues and identifying the bacterial agent (Toranzo et al., 2005). Multiplex PCR was used to detect food-borne bacterial pathogens. Among the risk factors associated with fish handling are catching, filleting, and processing for consumption (Hastein et al., 2006). Poor fish handling refers to inappropriate practices used by fishermen after harvesting their fish products. Poor handling exposes fish to physical, chemical, and biological hazards, resulting in increased microbial contamination and hastening the spoilage rate (Miller and Benansio, 2010). Fish placed on a dirty surface; inappropriate washing of fish in dirty water; fish thrown/displayed on the ground; beating fish with a stick after harvest, or stepping on fish; fish left to insect, bird, and animal predation; delay in gutting the fish after harvest fish in contact with other fish use of filthy boxes, baskets, and ice cubes (Rana, 2015).
2.5. Pathogenesis
Salmonella infections are a leading cause of diarrhea and mucosal inflammation, and they can lead to severe systemic disease. In most cases, the infection begins with the consumption of tainted food (Dougan et al., 2011). Salmonella infections in humans vary greatly in nature and severity, and are influenced by the infecting Salmonella serovar, strain virulence, infecting dose, host age, and immune status. Salmonellae typically colonize the intestine through the adhesion of the bacteria to epithelial cells via fimbrial antigens. The cells infiltrate the intestinal mucosa and proliferate in the gut-associated lymphoid tissue (GALT) (Gondwea et al., 2010).
Pathogens spread from infected tissues to regional lymph nodes, where macrophages form the first effective barrier to prevent further spread (Radostits et al., 2007). Systemic disease can occur if the macrophages are unable to prevent the spread (Liu, 2010). During systemic disease, bacteria spread from the GALT into the venacava via the efferent lymphatic and thoracic ducts, from which they spread throughout the body (Gondwea et al., 2010). The bacteria multiply in the spleen and liver before being released into the bloodstream and infecting other organs. Salmonellae can live and multiply inside host cells (Liu, 2010). Salmonella contains large clusters of virulence genes that work together in a complex virulence function of different Salmonella infection outcomes (Radostits et al., 2007).
Pathogenicity islands are chromosomal regions that have been acquired through horizontal gene transfer. These islands contain genes involved in gastrointestinal and systemic pathogenesis (Bäumler et al., 2000).Some pathogenicity islands encode type III secretion systems (TTSS) that are responsible for the contact-dependent translocation of substrate proteins into eukaryotic host cells or for Salmonellae survival in macrophages (Kingsley and Bäumler, 2002). Salmonella also has adherence fimbriae, which allows it to easily attach and adhere to cell surfaces, particularly mucous membranes (Chees brough, 2006).
2.6. Clinical Signs and Symptoms
Bacteremia is associated with 2-8% of NTS infections and is not always preceded by gastroenteritis, Immune compromise (including HIV, malignancy, chemotherapy, and steroid therapy) and extreme age (3 months and older) are risk factors for NTS bacteremia. In up to one-third of cases of NTS bacteremia, risk factors are not apparent. Extra intestinal focal infections (for example, arthritis, meningitis, and pneumonia) affect 5-10% of those with bacteremia (Matheson, 2010). The most common manifestation of NTS infection is acute gastroenteritis. Nausea, and/or vomiting are common symptoms. Fever, abdominal cramps, and bloody diarrhea are also possible symptoms. The asymptomatic carriage can be found in up to 4.7% of healthy hosts (Sirinavin et al., 2004). These gastroenteritis symptoms appear 6 to 72 hours after ingesting the bacteria and are usually self-limiting, resolving within two to seven days (Pegues et al., 2010).
2.7. Diagnosis
Although a tentative diagnosis of Salmonellosis may be made, the clinical signs and findings at postmortem examination are not unique to Salmonellosis. They should confirm this in a diseased animal or at necropsy by isolating organisms in their feces and counting the viable counts. Fecal samples, rather than swabs, should be collected, and these should be obtained before the administration of antibiotics. Oral secretion and blood culture may also be used to isolate the organism, though these methods are less reliable than feces culture and must be used with caution to avoid contamination. Salmonella is commonly found in the tissue of animals that have died from Salmonellosis, and a sample of spleen, liver, hepatic, mediastinal, and bronchial lymph nodes can yield a count of 106 organisms/gram excreting a similar concentration can be found in the ileum's wall and content, as well as the cecum, colon, and lymph nodes. Internal organs should be sampled to distinguish between animals that died of enteritis but did not have septicemia (.Jones et al., 2007).
2.7.1. Culture method
The traditional Salmonella culture method involves pre-enrichment, selective enrichment, isolation of pure culture, biochemical screening and serological confirmation, which requires 5-7 days to complete. The USDA and FDA recommended method involves a 6-24 hours, pre- enrichment step in a nonselective broth such as lactose broth, tryptic soy broth, nutrient broth, skim milk, or buffered peptone water with a recommended incubation temperature of 37 °C. The selective enrichment step requires additional 24 hours incubation in Rappaport Vassiliadis (RV) broth, selenite cysteine (SC) broth, or Muller Kauffmann tetrathionate broth. The incubation temperature of 41.5°C ± 1°C for RV broth and 37°C ±1°C for SC and MKTT broth is, used. Bacterial cells are isolated on selective agar plates such as Hektoen enteric agar (HEA), xylose lysine deoxy-cholate (XLD), and/or brilliant green agar (BGA). Biochemical testing is done using triple sugar iron agar, urea, citrate, indole and lysine iron agar (LIA), which requires an additional 4-24 hours (ISO, 2002).
2.7.2. Rapid detection method
Detection of Salmonella antibodies using enzyme immunoassay (EIA): The detection of Salmonella antibodies by EIA provides a sensitive and cost-effective method for mass screening of animal flocks/herds for signs of a past/current Salmonella infection. The method's limitation is that the immune response of the individual animal is not elicited until 1-2 weeks after infection. There are a variety of commercial kits available for testing poultry, cattle, and pigs. This method has the obvious advantage of being automated, as no incubation is required to increase the number of bacterial cells (Zamora et al, 2002).
The EIA is a well-established antigen-assaying technique. Antibodies labeled with an enzyme bind to Salmonella antigens, and the amount of antigen present is determined by enzymatic conversion of a substrate, which typically results in a color change that can be read visually or using a spectrophotometer. To provide enough Salmonella cells for detection, the EIAs rely on standard cultural procedures for pre-enrichment and selective enrichment. EIA technology that allows detection earlier in the resuscitation and/or culture process can provide even faster results. For retrospective diagnosis, serological tests such as ELISA, serum agglutination, and complement fixation can be used.For the retrospective diagnosis of Salmonellosis or the detection of carriers (Zamora et al., 2002). Real-time quantitative polymerase chain reaction (QPCR), reverse transcriptase PCR (RTPCR), and nucleic acid sequence-based amplification (NASBA) have all been used to detect Salmonella from various food matrices. Salmonella enterica was detected at 1 cfu ml-1 after 8-12 hours cultural enrichment in the TaqMan-based Q-PCR with the invA gene as the target. The NASBA method has been used to detect viable Salmonella cells and is more sensitive than RT-PCR, as well as requiring fewer amplification cycles than conventional PCR methods (Pal et al., 2020).
2.8. Treatment
Salmonella gastroenteritis is typically a self-limiting disease, with diarrhea resolving within three to seven days and fever resolving within 72 hours (Fuaci and Jameson, 2005). As a result, therapy should focus primarily on replacing fluid and electrolyte losses. As a result, antimicrobials should not be used routinely to treat nontyphoidal Salmonella gastroenteritis or to reduce convalescent stool excretion. Antimicrobial therapy, on the other hand, should be considered for any systemic infection (Parry et al., 2002). Antibiotic treatment is usually not advised, and in some studies, it has been shown to prolong Salmonella carriage. Neonates, the elderly, and the immune- compromised (e.g., HIV patients) with non typhoidal Salmonella gastroenteritis are especially vulnerable to dehydration and dissemination, and may require hospitalization and antibiotic therapy (Fuaci and Jameson, 2005).
Because of the rising prevalence of antimicrobial resistance, empirical treatment for life-threatening bacteremia or local infection caused by non-typhoidal Salmonella should include a third- generation cephalosporin and a quinolone until susceptibility patternsaredetermined. Amoxicillin and trimethoprim-sulfamethoxazole are effective in the eradication of long- term carriage (WHO, 2012).The high concentration of amo xicillin and quinolone in bile and the superior intracellular penetration of quinolone are theoretical advantages over trimethoprim- sulfamethoxazole (WHO, 2012).
2.9. Prevention and Control
To control the spread of the pathogen and human infection, the level of Salmonella contamination in various food animals, food products, and the environment must be monitored regularly (Norrung and Buncic, 2007). The most serious meat safety concerns affecting consumer health and causing product recalls involve microbial and, in particular, bacterial pathogens (Sofos, 2005). Controlling these pathogens at all stages of the farm-to-fork chain is critical to reducing the occurrence of food-borne disease in humans (Norrung and Buncic, 2007). Salmonellosis is the most common foodborne and zoonotic disease in the world. Reduced Salmonella prevalence necessitates a comprehensive control strategy for animals and animal foodstuffs, as well as restrictions on infected flocks until they have been eradicated (Breytenbach, 2004).
In addition, mandatory testing before slaughter should be conducted like the one being implemented in Sweden (Boqvist and Vagsholm, 2005).Non-typhoidial Salmonella organisms are found in a variety of domestic and wild animals, including cattle, poultry, swine, rodents, and pets such as iguanas, turtles, dogs, cats, chicks, and ducklings. Bacterial excretion in humans infected with Salmonella can last for the duration of the infection and as a temporary carrier state for months. Ingestion of the organisms in food derived from infected animals or contaminated by the feces of an infected animal or person may be the mode of transmission. Contaminated meat, poultry, eggs, milk, and their products, as well as water, fruits, and vegetables, could be the source. Preventive measures should therefore include educating food handlers about hand hygiene, refrigerating foods in small portions, thoroughly cooking all foodstuffs, avoiding recontamination of cooked food, and maintaining a sanitary kitchen to prevent rodent, and insect contamination (Varma et al., 2005).
Safe food production necessitates knowledge of the nature and origin of the animals, animal feed, and the health status of farm animals. It also requires knowledge of the use of veterinary medicinal products, the results of any analysis of farm samples, slaughter data on ante-mortem and post-mortem findings, and the risks associated with postharvest production stages (Snijders and Knapen, 2002). No part of the food chain can be considered in isolation, but must be viewed as a component of the whole. It must also include customers. Additional measures to control secondary contamination could include contamination prevention through cleaning and disinfection, personnel hygiene, and proper processing (Nowak et al., 2006). The growth of microorganisms in meat and poultry products can be controlled by keeping them at a cold temperature of 100 °C during transport and storage, especially for Salmonella (Coleman et al., 2003).
2.10. Salmonella in Humans
Human Salmonella infections are a major public health concern around the world (Lee et al., 2015). Salmonella is a highly infectious and contagious pathogen. The risk of illness increases with the amount consumed, and outbreaks are generally associated with high infectious doses. Based on human challenge experiments, dose-responses for different common Salmonella serotypes linked to different food matrixes causing infection in humans are not available (Bollaerts et al., 2008).
Nonetheless, this information is critical for quantifying the risk of infection and illness in humans. Based on data from all types of outbreaks, the available information on dose-response relationships indicated an ID50 of 7 CFUs for infection and an ID50 of 36 CFUs for illness (Teunis et al., 2010 ). Salmonella infections can affect anyone, but certain groups, such as adults 65 and older, children under the age of five, and those with compromised immune systems, are more likely to develop the disease (CDC, 2020). A susceptible population is more likely to become ill at low dose levels when the pathogen-food matrix combination is extremely virulent, and at high dose levels when the combination is less virulent (Bollaerts et al., 2008).
The three most prevalent Salmonella serovars associated with foodborne illness were S. Enteritidis, S. Typhimurium and S. Newport in the USA (Luvsansharav et al., 2020) and S. Enteritidis, S. Typhimurium and monophasic S. Typhimurium in the EU (EFSA and ECDC, 2018). Information on the dominant serotypes is not readily available for Africa which could be due to the lack of an integrated surveillance and reporting system. According to the meta-analysis study by Tadesse (2014), S. Concord (34%), S. Typhi (32.5%), S. Typhimurium (9.4%) and S. Paratyphi (6.1%) are the dominant serotypes reported from humans in Ethiopia.
2.11. Salmonella in Fish
Salmonella in freshwater fish has been usually related to the fecal contamination of water from where fish were harvested (Mhango et al., 2010). Fishes work as a passive carrier of Salmonella that may excrete Salmonella spp. without apparent symptoms and represent no clinical disease. The high prevalence (60%) of Salmonella in catfish was reported by (Elhadi, 2014).The high prevalence rate was attributed to the high temperature in pond water because high temperature promotes the organism's growth rate (Misganaw and Getu, 2016). Pal and Gupta reported microbial contamination of fish grown in ponds in and around Calcutta (Malorny , 2008 ). Different serovars (S.Tyhpimurium, S. Anatum, S.Newport) of potentially human pathogenic. Salmonella was isolated from largemouth bass, channel catfish, common carp, and sucker mouth catfish in the natural river system. In a freshwater dam fish, the contents of the blood, tissues, intestine, and skin surface were compared. Salmonella spp. were found in every part of the fish (Heinitz et al., 2000).
The bacterial load in fresh and smoked fish was determined using the pouring plate method (Ibrahim and Sheshi, 2014). The prevalence of Salmonella in various fish body parts was investigated (Lakshmanaperumalsamy and Mohamed Hatha, 2014). Salmonella isolates from freshwater Lakes, farmed fish, and market fish were found in 31%, 5%, and 10% to 28% of the samples, respectively (Issue, 2015). Mailoa and Sabahannur (2013) isolated pathogenic Salmonella from smoked fish, which can cause food poisoning. The factors that may have contributed to the contamination of fish with Salmonella came from terrestrial sources, such as the unorthodox use of cattle and poultry feces as fertilizer or manure on farmland near a canal, river, or pond Bibi et al. (2015). As a result, topsoil washed into the water reservoir during the rainy season increases fish and environmental contamination. These have primarily aided in the growth of human and fish microbes. When untreated sewage water enters lakes or fish farms via runoff or storm water, it contaminates the fish. Transporting fish in contaminated fishing boats or containers, as well as washing fish in pond/lake water, may also pollute the aquatic environment and cause food contamination (Raufu et al., 2014).
2.12. Antimicrobial Resistance
Salmonella's main clinical picture is self-limiting gastroenteritis, which in severe cases may necessitate fluid and electrolyte replacement. Antibiotics are only given to patients who have serious diseases or are at high risk of contracting invasive diseases (WHO, 2014). Third-generation cephalosporin antibiotics, quinolones, and macrolides are among the antibiotics used to treat typhoid fever. However, there has recently been an increase in the number of typhoid Salmonellas and non-typhoid strains with high levels of resistance to quinolones and cephalosporins (Cosby et al., 2015).
The emergence of multiple drug- resistant Salmonella (MDR) is a global concern, and the presence of Salmonella MDR in food is a risky condition, representing an increase in the severity of foodborne disease, leading to increased hospitalization rates and the possibility of death (Crump et al., 2015). In contrast, the epidemiology of Salmonella spp. antimicrobial resistance is complex and may be influenced by factors such as antibiotic consumption, human travel, transmission between patients in hospitals, importation and trade of food of animal origin, trade in live animals in the country or between countries, and exposure through an animal or human environment (ECDC/EFSA/EMA, 2017).
2.12.1. Antibiotics Resistant Salmonella Isolated from Fish
Contribution of aquaculture to the global supply of fish, crustaceans, molluscs and other aquatic animals has increased considerably over the past four decades, also becoming a risk factor for consumers, due to the widespread and inappropriate use of antimicrob ial drugs in aquaculture and risks associated with the spread of resistance between pat hogens to human. Antibiotic residues in food can generate allergies and toxicity that a re difficult to diagnose, due to the lack of knowledge on the source of their intake, which can cause exposure in sub inhibitory concentrations and lead to the appearance of resi stance in both commensal bacteria of the human intestine and fish bacteria, with possi ble dissemination of resistance genes to various bacterial populations (Abakpa, 2015). In Morocco, 28 (49.1%) Salmonella isolates showed resistance to ampicillin (22 isolat es), nalidixic acid (9 isolates), sulfonamide compounds (2 isolates) and tetracycline (1 isolates). Six isolates showed resistance to two antimicrobial substances (Setti et al., 2009).
In a study carried out by Elhadi, (2014), a total of 140 Salmonella isolates from six different types of frozen freshwater fish: catfish (Clarias gariepinus), carfu (Corfu toothcarp), mrigal (Cirrhinus cirrhosus), tilapia (Oreochromis mossambicus), rohu (Labeo rohita) were detected in samples imported from five different countries ( Thailand, Vietnam, Bahrain, Myanmar and India). Isolates were tested for susceptibili ty to 18 selected antimicrobial agents, with most isolates showing resistance to tetracy cline (90.7%) followed by ampicillin (70%) and amoxicillin and clavulanic acid (45%).
2.13. Public Health Importance of Salmonellosis
Salmonellosis is a major global public health problem that causes significant morbidity and thus has a significant economic impact. Although the majority of infections cause mild to moderate self-limiting disease, serious infections that result in death do occur (De Jong and Ekdahl, 2006). Despite improvements in hygiene, food processing, food handler education, and consumer information, foodborne diseases continue to be the most serious public health problem in most countries (Dominguez et al., 2002). Salmonellosis incidence is defined as the detection of Salmonella in animals or groups of animal products or surroundings that can be specifically linked to identifiable animals or animal feed. On the human side, a registered medical practitioner in the United States is required by the Public Health (Control of Disease) act to notify the local authorities if a patient has or is suspected of having a foodborne disease (Kemal et al., 2014). Infection can occur if fishery food contaminated with Salmonella is consumed raw or undercooked. Salmonellosis transmitted through food from fisheries is a major cause of human morbidity worldwide (Helmi et al., 2020).
Studies show that the occurrence of resistant microorganisms has a negative impact on human health. The use of antimicrobial agents in humans and animals affects the intestinal tract, putting those who are affected at a higher risk of infection. This is the percentage of Salmonella that would not have occurred if the Salmonella were not resistant .Furthermore, antimicrobial agents used in animals can result in increased transmission of resistant microorganisms between animals, which could lead to transmission of such microorganisms to humans via food. Prolonged illness may indicate an increase in the frequency of treatment failure and the severity of infection. Salmonella Dublin is mostly but not entirely specific to cattle, with an average of 10 human cases reported in Ireland each year. Apart from its pathogenicity two other characteristics of S.Dublin make it particularly important for Ireland from a public health viewpoints (Jones et al., 2007).
3. MATERIALS AND METHODS
3.1. Descrption of the Study Area
The study was carried out at Lake Fincha. Lake Fincha is located in central Oromia, Horo Guduru Wollega Zone, in the Abay Chomen District. Abay Chomen District is a district in Ethiopia's Oromia region, part of the Horo Guduru Wollega Zone. Abay Chomen District is located 289 kilometers northwest of Addis Abeba ,and is bounded on the south by Lake Fincha, which was formed when the Fincha dam flooded the Chomen swamp, on the south west by Horo district, on the north west by Amuru Jarte, on the North by Abay River which separates it from the Amhara Region on the East and by Guduru district (Figure 2). This District's elevation ranges from 880 meters to 2,400 meters above sea level. The district's rivers include the Nedi, Fincha, Agemsa, Korke, Gogoldas, Boyi, and Bedessa. The total area of the District is estimated to be 801.7 km[2]; approximately 45, 37, 4, 3 and 11% of the total area are cultivated land, non-cultivated, water bodies, settlements, and woodlands and forests, respectively.
According to a survey of the land in this district, 11.4 % is arable or cultivable, 2.2 % is pasture, 1.4 % is forest, and the remaining 83.8 % is mountainous, unusable, or part of the Fincha Sugar Project. Niger seed is a significant local cash crop. Fincha town is the district capital. Lake Fincha is a man-made Lake formed as a reservoir by a hydroelectric dam on the Fincha River. The Lake has a surface area of 293km2 and is located 2302 meters above sea level. Various fish species were introduced to Fincha reservoir for various purposes, including Nile Tilapia (Oreochromis niloticus), Common carp (Cyprinuscarpio), and Catfish (Celarias gariepinus) in the 1980s. In 1998, the former two species, as well as Tilapia zillii, were reported to be well established in the reservoir (AChDAO, 2020).
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Figure 1: Map of the study area
3.2.Study Animals
The study was conducted on Fish species from Lake Fincha like Nile Tilapia, Common carp, and Catfish harvested from Lake Fincha in Fincha and Amerti Nashe Landing sites.
3.3. Sample Size Determination
The sample size required for the study was determined using an expected prevalence of 50% as there is no previous research work on the occurrence of Salmonella i n fish species found in Lake Fincha. The required sample number was obtained by using the formula suggested by Thursfield (Thursfield, 2005) with a 95% confidence interval and 5% absolute precision.
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Based on this, the required sample size was 384 Fish Samples. Additionally, Thirty (30) swab samples from fishermen's hands were taken based on the willingness and the presence of fishermen's by purposive sampling techniques. Overall, 414 samples were subjected to the microbiological test. The sample size for the questionnaire survey was calculated based on the formula recommended by Dr. Hossein Arsham (Arsham, 2020). N= 0.25/SE[2], SE= 5%, N=100, Where N= Sample size, SE= Standard error, to which assuming the standard error is 5% and the confidence interval of 95%.
3.4.Study design
From January 2020 to June 2021 a cross-sectional study with a simple random sampling approach was conducted at Lake Fincha to assess the occurrence, antimicrobial susceptibility, and associated risk factors of Salmonella in 384 randomly selected fresh fish samples and Thirty (30) swab samples from fishermen hands were taken by purposive sampling techniques. To assess perceptions of food hygiene and risk factors for Salmonellosis, a questionnaire survey and focus group discussion with key informants were conducted with focus groups such as fishermen, fish meat filletors, and fish meat sellers. At the start of the study, a brief description and discussion of the study were conducted with the fishermen, fish processors, and fish sellers.
3.5. Methods of Data Collection
3.5.1. Questionnaire survey
Based on the landing sites, fish meat sellers, and the voluntariness of fishermen, a total of 30 individuals were interviewed. To assess perception and risk factors for Salmonellosis, a questionnaire survey and focus group discussion with key informants were conducted. To collect data from the selected fishermen, fish meat sellers, and fish filletors, a face-to-face interview using a semi-structured questionnaire was employed. The information collected using the questionnaire includes their experience, goals for fish harvesting, the harvesting area and equipment, the types of fish species, questions to monitor handling and hygiene, the habit of hand washing before fish processing, landing infrastructure, equipment, and materials (Appendix 1).
3.5.2. Sample collection and transportation
With gloved hands and sterilized scalpel blade, each sampled fish was severed into parts (skin, muscle, Intestine, and gills). A total of 384 samples (65 skins, 113 muscles, 96 gills, and 110 intestines) were collected 6 months period (January 2020 to June 2021). A total of 220 samples from Tilapia which is preferred and largely harvested by fishermen, 85 common carp and 79 catfish as well as 30 hand swabs of volunteer fisherman before handling fish were collected from Fincha and Amerti Nashe landing sites. All samples from each study area were collected aseptically in sterile polythene bags, coded, and transported in refrigerated conditions to the National Animal Health Diagnostic and Investigation Center (NAHDIC) microbiology laboratory for processing and Salmonella isolation.
3.5.3. Laboratory analysis
384 samples from the three fish groups (220 Tilapia, 85 Common carp, and 79 Catfish) and 30 fishermen hand swabs were collected from two landing sites between January 2020 to June 2021.All samples were processed at the National Animal Health Diagnostic and Investigation Center (NAHDIC) Microbiology Laboratory. Salmonella was isolated from fish samples with the method described in the previous study (Andrews et al., 2018). 25 grams of muscles, and 12.5grams of the intestines, skins, and ,gills each part was ground (stomacher 400) with 225ml and 112.5 Buffered Peptone Water (MH 14941-500G, HIMEDIA, and Mumbai, India) for 3min respectively. Pellet was obtained by centrifugation at 20 [0]C, 10,000 x RPM, for 15 minutes for fish sample. The pellet was then dissolved into 10ml of BPW and incubated at 37 [0]C for 24hours. 1ml of BPW later transferred onto Salmonella enrichment Broth to Rappaport and Vassiliadis (M1491-500G, HIMEDIA, Mumbai, India) and incubated at 41.5 [0]C for 24hrs. The inoculums were then later streaked onto Xylose Lysine Deoxycholate (CM 0469, OXOID, and Basingstoke, England) and incubated at 37 [0]C for 48 hours. Due to the color change of the media, typical colonies of Salmonella grown on XLD-agar have a black center and a lightly transparent zone of reddish color, whereas H2S negative variants grown on XLD agar are pink with a darker pink center Lactose-positive Salmonella grew on XLD agar and was yellow with or without blackening. Five colonies were chosen from the selective plating media, streaked onto the surface of pre-dried nutrient agar plates, and incubated at 37 OC for 24 hours (ISO, 2002b).
3.5.4. Confirmation of Salmonella Isolates
All suspected Salmonella isolates were subjected to the following biochemical tests for confirmation: triple sugar iron (TSI) agar, lysine iron agar (LIA), Simmon's citrate agar, urea broth, indole production test, motility test, MR-VP broth and incubated for 24 or 48 hours at 37 OC. Colonies producing an alkaline slant with acid (yellow color) butt on TSI with hydrogen sulphide production, positive for lysine decarboxylase (purple color), negative for urea hydrolysis (no color change ), negative for tryptophan utilization (indole test) (yellow-brown ring), negative for Voges-Proskauer, and positive for citrate utilization were considered to be Salmonella -positive (Quinn et al., 1994).
Presumed Salmonella isolates that passed all biochemical tests for Salmonella characteristics were transferred and cultured on Nutrient Agar (NA) for antimicrobial sensitivity test. Biochemically confirmed Salmonella isolates were subjected to the Biolog Omni Log test. The Biolog Omni Log test was performed by cultivating Salmonella isolates on Biolog Universal Growth Agar (Microbial ID/Characterization product literature, Biolg, USA). The cell suspensions were prepared and pipetted into 96 well Biolog Plates before being incubated at 37 [0]C for 24 to 48 hours (Wragg et al., 2014). The microplates that had been incubated were placed in the Biolog Omni Log reader. The outcome was obtained using computer software (Osielska and Jagodzinski, 2018). The isolates identified as Salmonella by the Biolog Omnilog test were then sub cultured on brain-heart infusion agar and sent to the National Veterinary Institute (NVI) for serotype identification, using the Organization for Animal Health (OIE) Reference Laboratory for Salmonellosis (Tadesse et al., 2016). For serotyping, A fragment of 284 base pairs (bp) of the invA gene was used for genus identification. This gene encodes an important function that allows Salmonella to enter intestinal epithelial cells. A 304 bp Sdf I gene, 401 bp Spy gene, and 738 bp ViaB gene fragments were selected, respectively, for S. Enteritidis, S. Typhimurium and S. Typhi ( Can et al., 2016 ).
3.6. Antimicrobial Susceptibility Testing
The isolates' antimicrobial susceptibility tests were carried out using the Kirby Bauer disk diffusion method following the Clinical and Laboratory Standards Institute of the United States of America (CLSI, 2019) and the Kirby Bauer Disk Diffusion Susceptibility Test Protocol on Muller Hinton agar medium. A sterile loop was used to transfer a loop full of well-grown colonies on nutrient agar from each biochemically and Biolog confirmed isolate into sterile tubes containing 5ml of normal saline solution (0.85% NaCl).The inoculated colonies were vortexed with saline solution until a smooth suspension was formed. Saline solution (if the suspension was more turbid) or colonies (if the suspension was less turbid) were added to the suspension until it met the 0.5 McFarland turbidity standards. The bacteria were then swabbed uniformly over the entire surface of the Muller Hilton Agar plate using a sterile cotton swab dipped in the suspension. To allow drying, the plates were kept at room temperature for 3 minutes in a bio safety cabinet (CLSI, 2019).
Ten antimicrobial disks with the known concentration of antimicrobial were placed on the MullerHinton Agar plate and the plates were incubated for 22 hrs at 37 °C.Each isolate was tested for a series of ten antimicrobials: Tetracycline (TE) (30g g), Cefoxitin (FOX) (30gg), Cefotaxime- 30gg (CTA), Ciprofloxacin (CIP) (5gg), Gentamycin (CN) (10gg), Amocacillin+Clavalunicacid (20+10)30pg (AMC), Sulfamethaxazole+Trimethoprim ((23.75+1.25)Z5pg (SXT), Ampicillin 10gg (AMP), Chloramphenicol 30gg and Meropenem-10gg (ME). Using a caliper, the diameters of the clear zones of inhibition produced by diffused antimicrobial on lawn inoculated bacterial colonies were measured to the nearest mm. According to the published interpretive chart, all ten zones of inhibition against ten antimicrobial agents for each isolate were recorded, compared to standards, and classified as resistant, intermediate, or susceptible (CLSI, 2019). Drug selection was based on the availability of the drugs during the research work and habitual uses in human and animal medications. The isolates showing resistance to three or more antimicrobial classes were considered as multiple drug- resistant (MDR) (Beyene et al., 2017).
3.7. Ethical clearance
During sample collection in this study, the National Animal Health Diagnostic and Investigation Center's (NAHDIC) ethical standards were followed. During sample collection, official permission was obtained from the study area's livestock and fishery office. In addition, participants in the study were treated following the National Animal Health Diagnostic and Investigation Center's ethical standards.
3.8. Data Management and Analysis
The raw data from the study was arranged, organized, coded, and entered into an Excel spreadsheet (Microsoft ®office Excel 2010) before being analyzed with the statistical software STATA 14.0. The frequency of the respondent's perception was described, and the prevalence of Salmonella was calculated by dividing the number of positive samples by the total number of fish tested multiplied by 100.
Salmonella isolates' antimicrobial resistance and molecular profiles were expressed descriptively using frequency distributions and percentages. Logistic regression was used to assess the association of risk factors with the prevalence of Salmonella. Dummy variables were created for those explanatory variables with more than two categories. For all risk factors, the level with the lowest prevalence was used as a reference category. Those variables with a p-value of less than 0.25 in the Univariable analysis were further analyzed by multivariable logistic regression after checking for confounders. In all tested cases, 95% confidence intervals and p < 0.05 were set for significance.
Because there were insufficient positive observations in some variables, the data was not suitable for chi-square analysis whereas Fisher's exact test was used .Thus, logistic regression was used. However, due to a few positive results, some of the variable levels were omitted from the analysis. Following a Univariable logistic regression analysis, the crude odds ratio with P-value and 95 % confidence interval was extracted and displayed.
4. RESULTS
4.1. Occurrence of Salmonella
The overall prevalence of Salmonella in fish samples and fishermen's hand swabs from 414 total samples in the study area is shown in Table (1). Out of 414 samples tested, the overall prevalence of Salmonella was found to be 1.21%. No Salmonella was detected from the swab samples from fishermen's hands in both landing sites in the current study.
Table 1: Occurrence of Salmonella from different fish species samples and fishermen hand's swabs
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CI=Confidence Interval Pos=Positive Pre=Prevalence
All the Salmonella isolated from positive samples (five in numbers) belongs to the Salmonella Typhimurium serotype. According to Figure (2), the Spy (S. Typhimurium) positive result is around 401bp, while the S. Typhi and S. Enteritidis positive results are 738bp and 304bp, respectively.
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Figure 2: Salmonella Typhimmim isolated from fish by Conventional multiplex PCR
4.2. Antimicrobial susceptibility of the isolated Salmonella
The table (2) shows the results of antimicrobial susceptibility testing of Salmonella (N=5) isolated from fish samples using ten different antimicrobial dings. Drug resistance for Cefotaxime-3Og (CTA) (100%) Tetracycline-30g (TE) (100%) Ampicillin-10g (AMP) (100%), Ciprofloxacin-5g (CIP) (60%), and Meropenem-10g (ME) (40 %) were recorded for Salmonella isolates from fish samples of the study areas.
On the otherhand,the greatest number of isolates were susceptible to Chloramphenicol 30g(C)(100%),Cefoxitin30g(FOX)(100%),Sulfamethaxazole+Trimethoprim(23.75+1. 25) g, —25 g (SXT) (100%), Amoxicillin + Clavulanic acid (20+10)-30g (AMC) (100%), Gentamycin-10g(CN) (100 % ) , Meropenem-10pg(ME)(60%),Ciprofloxacin (CIP) - 5 g (40 % ).
Table 2: Results of antimicrobial susceptibility testing of S. Typhimurum isolates from Fish
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Number of isolates (N); Susceptible(S); Intermediate (I); and Resistance(R) As shown in Table (2), 4(80 %) of isolates of Salmonella Typhimurium developed resistance to three or more classes of antimicrobial drugs, implying multidrug resistance.
4.3. Socio-Demographic Characteristics of the Respondents
The sample size proposed was 100. However, due to the refusal of fishermen to participate in this study, the interview was administered to only 30 questionnaires to volunteer fishermen. A questionnaire survey was administered only to a total of 30 people that is 30% response rate was recorded. The majority of respondents 60 % had no formal education, 30 % had primary education, and 10% of them followed secondary school and above. All the Fishermen were male and the majority of them primarily process for commercial purposes 25(83.33%). They mostly received adult fish from collectors 26(86.67 %), and the most common form of fish they presented to consumers was whole fish 28(93.33%). The majority of processors 28 (93.33%) had 3-10 years of experience. Tilapia 21 (70 %) of Fishermen prefer the most common species types that they collect.
Table 3: Socio -demographic characteristics of the respondents
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Assessment of the knowledge of the study participants on Salmonella and its prevention showed that the majority of the fish respondents know that fish collected from contaminated Lake can be contaminated by Salmonella, (66.67%), consuming contaminated fish can cause health problems (66.33%). However, the majority of them didn't know that the use of personal protective equipment can reduce the chance of contamination (90%) (Table 4).
Table 4: Knowledge of respondents on Salmonella and its prevention
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The assessment of fishermen's practices for the prevention of Salmonella occurrence revealed that the majority of fishermen keep the fish properly soon after landing (76.67%), do not practice hygienic handling and storage (80 %), do have proper storage and transportation containers (86.67 %), and do not handle and process the fish without cutting their nails short (73.33 %) (Table 5).
Table 5: Practice of fishermen on prevention of occurrence of Salmonella
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4.4. Risk Factors Associated with Occurrence of Salmonella
The prevalence of Salmonella based on the type of sample in this study area was 3/113 (2.65 %) and 2/96 (2.08 %) from muscles, and gills, respectively. The prevalence of Salmonella in the three species in this study were common carp 2/85 (2.35 %), catfish 1/79 (1.27 %), and tilapia 2/220 (0.91%).In terms of landing sites, the prevalence at Amerti Nashe was numerically higher 2/134 (1.49%) than Fincha 3/250 (1.20 %). According to the Univariable logistic regression analysis, Fish muscles have a higher probability of being contaminated by Salmonella (OR=3.24; 95 % CI: 1.99-6.29; P=0.030) than Fish gills. There was, however, no statistically significant relationship between Fincha and Amerti Nashe landing sites (OR =1.25, 95 % CI: 2.10-75.60; P=0.810). In the logistic regression, contamination, fish iced after landing, and fish handling were significant (P<0.05) and selected for multivariable logistic regression (Table 6). Salmonella contamination of fish was found to be 12.67 times higher than in a contaminated environment than in an uncontaminated environment (OR=12.67; 95 % CI: 1.18-13.63.; P=0.036). Salmonella Occurrence was also significantly higher in fish that were not iced soon after landing compared to fish that were iced soon after landing (OR=16; 95 % CI: 1.45-17.64; P=0.024). Similarly, Salmonella in a fish in poor handling practices is 29.3 times higher than in good handling practices (OR=29.3; 95 % CI=2.40-35.78; p=0.008).
Table 6: Result of Univariable logistic regression analysis of risk factors of occurrence of Salmonella
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Fish protected from contamination
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Those variables with collinearity <50 % and p-value <0.25 were included for the multivariable analysis. The three Variables: Contamination, Iced soon after landing and Good Fish handling were included in multivariable logistic regression analysis. The presence of Salmonella in a fish in a contaminated environment was approximately 2.84 (AOR= 2.84; 95 % CI=1.45-1.83; P=0.041) times more likely than in an uncontaminated environment. Aside from contamination, fish iced after landing was one of the predictors of Salmonella occurrence in a fish. Salmonella in a fish was 6.91 (AOR= 6.91; 95 % CI= 0.06-5.91 P=0.032) times more likely in not iced fish than in iced fish. Furthermore, Salmonella in a fish was about 8 (AOR=8; 95 % CI=0.06- 0.37;P=0.035) times more likely in poor handling practices than in good handling practices .Contamination, the absence of immediate icing, and poor fish handling were computed to be predictor variables for the occurrence of Salmonella (Table 7).
Table 7: Multivariable logistic regression analysis for the predictors of occurrence
Salmonella in fish at Fincha Lake
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AOR (adjusted odds ratio); CI (Confidence interval)
5. DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5. 1. Discussion
In Ethiopia, research on the occurrence, risk factors, and antimicrobial susceptibility profiles of Salmonella isolates from personnel and fish is limited. The current study looked at risk factors and antimicrobial resistance profiles of Salmonella species from personnel and fish. The overall prevalence of Salmonella in this study area was 1.21%.The presence of Salmonella in this study was consistent with a previous study conducted in Ethiopia by Tesfaye et al. (2018), which found a prevalence of 1.6 %. The presence of Salmonella discovered in this study was in contrast to a previous study conducted in Vietnam, which found a prevalence of 36.6 % (Ava et al., 2020). This disparity could be attributed to the previous study area's lack of concern about fish contamination in the processing chain.
Salmonella was found in 2/220 (0.91 %) of the tilapia, 2/85 (2.35 %) of the common carp, and 1/79 (1.27 %) of the catfish in this study. The prevalence in tilapia was found to be 26.6 % in a study conducted in Saudi Arabia (Elhadi, 2014). The occurrence was lower in this study than in the previous one. This disparity could be attributed to poor hygienic practices in the previous study area. Previous research found that Salmonella occurrence in catfish was 35% in Saudi Arabia (Elhadi, 2014) and 11.5 % in Nigeria (Raufu et al., 2014). In this study, the prevalence of Salmonella in catfish was found to be 35% lower than in Saudi Arabia. This difference could be due to differences in the sampled organs, the source of water, hygienic practices, and the temperature of the water in the previous study. It was also discovered to be lower than in previous studies in Nigeria, which could be attributed to poor fish handling and processing practices in the previous study area.
In this study, samples from muscles 3/113 (2.65%) were found to be more Salmonella positive followed by gills 2/96 (2.08 %), but no isolate was detected from the intestine and skin samples. The occurrence of Salmonella in different body parts of fish was studied. Organs of fish found Salmonella positive (skin, intestine, liver and muscle) (Lofty et al., 2011).The Study conducted in Pakistan found Salmonella spp. in various parts of catfish, most notably the entire catfish (80 % ) Following that, 40% of the gills and 20% of the intestines were contaminated (Budiati et al., 2011).
The presence of Salmonella in the gills was found to be lower in this study (2.08 %) than in the previous study in Pakistan (40 %) (Budiati et al., 2011).This disparity could be explained by untreated sewage water, more contaminated processing areas, and the previous study's practice of transporting fish in contaminated fishing boats. The highest occurrence was found in Amerti Nashe 2/134 (1.49 %) and the lowest in Fincha 3/250 (1.20 %) Table (6). Salmonella prevalence varies significantly (p<0.05) by sample type. Common carp was found to be more contaminated than tilapia and catfish among the species tested. Salmonella was found in only two of the four organs tested (muscles and gills).
Antimicrobial resistance develops as a result of improper antimicrobial use in animals and humans, as well as the subsequent transmission of resistance genes and bacteria among animals, humans, animal products, and the environment (Argudn et al., 2017), which can pose serious health risks to humans (Poole, 2015). Consumption of multidrug- Salmonella in conjunction with a raw meat dish is directly related to the global public health crisis caused by antimicrobial resistance (Wabeto et al., 2017). Determining the scope of the problem is critical for developing and monitoring an effective antimicrobial resistance response (WHO, 2014). According to the current study, the greatest number of S.Typhiruim isolates were susceptible to Chloramphenicol (100%), Sulfamethaxazole+Trimethoprim (100%), Gentamycin (100), Amoxicillin + Clavulanic (100%), Cefoxitin (100%), and Meropem (60%).The current study indicated that S.Typhimurium isolates were completely resistant to Cefotaxime 100%), ampicillin(100), Tetracycline(100), and Ciprofloxacillin ( 60%). In contrast to the current study in Nigeria, Raufu et al. (2014) isolated 23 Salmonella strains from African catfish (Clarias gariepinus), with the two most frequent serovars identified as S. Hadar and S. Eko. Overall antimicrobial resistance patterns indicated that most isolates were resistant to streptomycin 10 (43.5%), sulfamethoxazole 8 (34.8%), and trimethoprim 5 (21.7%), which constitutes a serious health risk for humans.
In contrast to the current study Martinez-Urtaza and Liebana (2005) identified 106 Salmonella serovars, S. Senftenberg isolates from 8 Spanish mussel processing plants (from mussels and environmental samples) that were antimicrobial- resistant to 16 antibiotics.
The researchers discovered nine antibiotic-resistant strains, including apramycin, chloramphenicol, nalidixic acid, neomycin, streptomycin, sulfamethoxazoletrimethoprim, sulfonamide, tetracycline, and amoxicillin acid. Yang et al. (2015) investigated the antimicrobial resistance profile of 554 samples, 86 of which found positive for Salmonella. The highest contamination rates were found in oysters (23%), freshwater fish (18%), and shrimp (3%). S. Typhimurium and S. Wandsworth were the most common. Antimicrobial resistance to tetracycline (35.9 %), ampicillin (28.2%), nalidixic acid (26.2 %), trimethoprim- sulfamethoxazole (25.2%), and streptomycin (18.4%), and chloramphenicol (25.2%) was found in the isolates.
Few studies have been conducted in Brazil to characterize the occurrence of bacterial resistance in aquaculture environments, particularly in freshwater fish. Cunha-Neto et al. (2019) isolated Salmonella strains from Native fish samples and they found two unusual serovars in fish samples: S. Abony and S. Schwarzengrund. Antibiograms with ten antimicrobials were performed on isolates, and Sulphamethoxazole + trimethoprim resistance was found in all strains. This highlighted the risks of antibiotic use by farmers, as well as the fact that their discharge into rivers near commercial points poses a risk for the selection of antimicrobial- resistant bacteria. The antimicrobial susceptibility of 21 Salmonella strains obtained from Tilapia (Oreochromis spp.), both whole and in fillets, in the state of Sao Paulo was determined in a review conducted by Fernandes et al. (2018). Isolates were sensitive to Gentamicin (95%), amikacin (66%), and ciprofloxacin (66%) but resistant to florfenicol (80%), which is surprising given its recent use in veterinary medicine in Brazil. Antibiotic-resistant strains of Salmonella have been isolated in fish in Brazil and around the world, demonstrating the transfer of resistance genes among the aquatic microbial population, which can lead to more severe and difficult-to-treat food-borne illnesses.
The high AMR frequencies observed in this study for several drugs could be attributed to sellers' unrestricted access to antimicrobial agents, which leads to abuse and increased selection pressure for resistant strains of bacteria. It could also be due to inadequate or non-existent antimicrobial resistance monitoring programs (Ibrahim et al., 2019).
Fish quality is a complex concept that includes safety, nutritional value, availability, integrity, freshness, eating quality, product size, and type (Abbas et al., 2008). Contamination by microbial pathogens is the most serious issue concerning the safety of fish products. Because of their high water content, neutral pH, and high amounts of amino acids and naturally present autolytic enzymes, fish products are highly susceptible to spoilage (Jeyasekaran et al., 2006). In the current study the presence of Salmonella in a fish in a contaminated environment was approximately 2.84 (AOR=2.84; 95 % CI=1.45, 1.83; P=0.041) times more likely than in an uncontaminated environment.
Aside from contamination, fish iced after landing was one of the predictors of Salmonella in a fish. Salmonella occurrence in a fish was 6.91 (AOR=6.91; 95 % CI= 0.06-5.91P=0.032) times more likely in not iced fish than in iced fish. In contrary to this study Mol (2011) investigated the quality of fish purchased in Istanbul retail markets, which is free of Salmonella species contamination. Furthermore, in this study Salmonella in a fish was found to be approximately 8 (AOR=8; 95 % CI=0.06-0.37; P=0.035) times more likely in poor handling practices than in good handling practices. In this study, from the computed variables contamination, absence of iced soon after landing, and poor Fish handling were found predictor variables for the occurrence of Salmonella.
5.2. Conclusions
Our study showed a prevalence of Salmonella in fish of 1.30%. When compared to other studies conducted in different countries, the current study revealed the lowest prevalence of Salmonella from fish. Although Salmonella does not exist in natural fish micro biota, fish can become asymptomatic hosts with the bacteria primarily residing on the body surface and organs, resulting in cross- contamination during the processing and marketing stages. Water quality and the aquaculture environment have a direct impact on microbiological fish contamination. Salmonella was found in 2.65 % of the muscle samples and 2.08 %of the gill samples in this study. A large percentage of Salmonella isolates were also resistant to various antimicrobial agents, particularly Tetracycline, Ampicillin and Cefotaxime. Meanwhile, the majority of the isolates tested were resistant to three or more antimicrobials, asserting the presence of MDR, which may impede ultimate control of Salmonella infection in fish and suggests a health hazard from consuming raw fish meat. Among the associated risk factors considered for contamination, poor handling practice and a low habit of icing soon after landing were independent predictors of the occurrence of Salmonella species from fish . Salmonella species isolation from fish suggests that hygiene protocols for fish harvesting and processing from farm to mouth should be used to prevent Salmonella contamination of fish by Contaminants. So far, little has been done, but this finding could help in determining the associated risk factors of Salmonella from Personnel and Lake Fincha fish species.
5.3. Recommendations
Therefore, based on the findings the following recommendations were forwarded:
- Fishermen, Fish meat processors and sellers should be made aware of the public health significance of fish diseases caused by contamination especially by Salmonella.
- Each landing site should have its processing plant and facilities.
- Collaborative training should be given for fish harvesters, processors, and sellers on proper Fish handling and hygienic practices.
- It is critical to conduct regular surveillance of fish sold for human consumption to determine the level and type of antimicrobial resistance.
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7. APPENDICES
Introduction
Questionnaire for A Research on Occurrence and Associated Risk Factors of Salmonella In Fish Harvested at Lake Fnchaa,Horo Guduru Wollega Zone,Ethiopia
Dear participants, my name is Ebisa Bnae, I am working a research For MSc at Ambo University. You have been identified as the most knowledgeable respondent about fish and fish processing activities in your cooperatives.
I would like to interview you. This survey is conducted by Ambo university masters graduating class student and will take place in selected fishermen cooperatives at Lake Finchaa.
The interview will take approximately 30 minutes.I will ask you questions about,Cares about fish processing,Personal,Material and equipment hygiene's.Standards and checklists for fish processing Confidentiality and Consent .The information you provide will only be used to assess the Hygiene and associated risk factors for the occurrence of Salmonella in fish. The information you provide is confidential and will not be shared with anyone. It will only be used for this research. Your name and other personal information will not be mentioned on the questionnaire. Only a code will be used. Your participation is voluntary, and you can stop the survey at any time. You are free to refuse to answer any question. But the information that you give us would be quite useful for the study.
Are you willing to participate in the interview of this study?
1. Yes, precede 2. No, stop here
Thank you for your participation in this study!
Appendix 1: Data collection tool for questionnaire survey
Part 1: Address
Woreda: Kebele: Questionnaire code:
Level of education A) No education B) Primary Education C) Secondary education and above
Part 2. Experience and Objective of Fish Harvesting
I. How long you start harvesting fish?
A.3years B.3-10 years C.>10years
2. What is the age of fish you are harvesting?
A. 4-6 months B. >6 months
3. The primary objective of fishing is
A. For commercial purpose B. for self-consumption C. any other
4. Do you have another source of income in addition to fishing?
A. No B. Trade C. other
5. In which form you present the fish?
A. gutted whole B. dried C. filleted D. all type
6. During transport do you encounter the death of fish? A. Yes B. No
7. If your answer is yes for question 7, what is your measure for dead ones?
A. Selling for consumers throwing to the lake C. selling for animal feed processors D. any other? Specify
8. How do you keep fish not presented to the domestic market? (Encircle)
A. Stored in cold store B. stored deep freeze C. other
9. How long does it take to arrive collecting boats to landing sites? (Encircle)
A.30min B.1hr C.3hrs D. 2 hrs. E. any other, specify
10. How long does it takes to transport fish from landing site to processing unit? (Encircle)
A.30min B.1 hr. C.3hrs D. 2 hrs. E. any other? Specify
Part3.Source of Contamination of the Lake and knowledge about salmonella.
II. Have you ever heard of Salmonellosis? (Encircle ) A. Yes B. No
12. Do you know Salmonellosis is a zoonotic disease? (Encircle ) A. Yes B. No
13. Do you believe the lake you are harvesting is contaminated? (Encircle ) A. Yes B. No
14. If your answer is yes to the question, what is the source of contamination? (Encircle)
A. Animal manures B. Poultry feces C. Flood D. Toilet sewages E. Any other specify
Part 4. Harvesting area and Equipment
15. Are the use of finger rings and ear jewelers? (Encircle) A. yes B. No
16. Do you have any medical certification? (Encircle) A, yes B, no
Part 5. Types of Fish Species
17. Which species of fish you are harvesting? ( Encircle)
A. Nile Tilapia B. Common carp C. Catfish
Part 6. Questions to Monitor Handling and Hygiene
Put the mark (V) in the box of your response
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Part 7. Landing Infrastructure, Equipment and Materials
Put the mark (V) in the box of your response
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Part 8. Collector boat, Equipment and Material
Put the mark (V) in the box of your response
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This completes the questionnaire. Do you have any questions? Thank you, I really appreciate your participation in this important study.
Appendix 2: Type and preparation of microbiological media used for isolation and antimicrobial susceptibility test of Salmonella
1. Buffered Peptone Water (MH 14941-500G, HIMEDIA, and Mumbai, India) Preparation: suspend 20.07 grams (the equivalent weight of dehydrated medium per litre) in 1000ml of distilled water. Heat if necessary to dissolve the medium completely. Distribute in tubes or flasks as desired. Sterilize by autoclaving at 121 for (15lbs pressure) 15 minutes. The Final PH is 7.0 ± 0.2 at 25.
Composition (g/l): tryptone10.0gm; Sodium chloride 5.0gm; disodium hydrogen phosphate 9gm; potassium hydrogen phosphate 1.5gm.
2. Rappaport Vassiliadis soya broth (M1491-500G, HIMEDIA, Mumbai, India) Preparation: suspend 27.11 gm in 1000ml distilled water. Heat if necessary to dissolve the medium completely. Dispense as desired in to tubes and sterilize by autoclaving at 115 (10lbs pressure) for 15 minutes. Final PH is 5.2 ± 0.2 at 25.
Composition (g/l): papaic digest of soya bean 4.5gm; sodium chloride 7.2gm; potassium dihydrogen phosphate 1.44gm; dipotasium phosphate 0.4, magnesium chloride hexahydrate 29.00; malachite green 0.036gm.
4. Xylose Lysine Desoxycholate Agar (XLD) (CM 0469, OXOID, Basingstoke, England)
Preparation: Suspend 53grams in one liter of distilled water. Heat with frequent agitation until the medium boils. Do not over heat. Transfer immediately to a water bath at 50. Pour in to plates as soon as the medium has cooled. It is important to prepare large volumes which will cause prolonged heating. PH: 7.4±0.2 at 25
Composition (g/l): yeast extracts 3.0; l-lysine hydrochloric acid 5.0; xylose 3.75; lactose 7.5; sucrose 7.5; sodium desoxycholate 1.0; sodium chloride 5.0; sodium thiosulphate 6.8; ferric ammonium citrate 0.8; phenol red 0.08; agar 15.0.
5. Nutrient Agar (AM5074, Accumix, Malaga, Spain)
Preparation: suspend 28 grams in 100ml distilled water. Mix thoroughly. Boil with frequent agitation to dissolve the powder completely. Sterilize by autoclaving at 15 lbs pressure (121) for 15 minutes. Mix well and pour in to sterile Petri dishes. Final PH (at 25): 7.4 ± 0.2.
Composition (g/l): peptone 5gm; sodium chloride 5gm; beef extract 1.5gm; yeast extract 1.5gm; agar 15gm.
6. Triple sugar agar (M021I-500g, HIMEDIA, Mumbai, India)
Preparation: Suspend 64.62 grams in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Mix well and distribute into test tubes. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Allow the medium to set in sloped form with a butt the of depth of about 2.5cm-5cm. Final PH: 7.4±0.2 at 25.
Composition (g/l): peptone 20gm; beef extract 3gm; yeast extract 3gm; sodium chloride 5.0; glucose1gm; lactose 10gm; sucrose 10gm; iron (48) citrate 0.3gm; sodium thiosulfate 0.3gm; phenol red 0.024gm; agar 12.0gm
7. Simmons Citrate Agar (M 099-500g, HIMEDIA, Mumbai, India)
Preparation: suspend 24.28 grams in 1000ml distilled water. Heat on boiling to dissolve the medium completely. Dispense as desired in tubes or flasks sterilize by autoclaving at 15 Ibs pressure (121) for 15 minutes.
Composition (g/l): magnesium sulphate0.20; ammonium dihydrogen phosphate 1.0; dipotasium phosphate 1.00; sodium citrate 2.00; sodium chloride 5.00; bromothymol blue 0.08; agar 15.00. Final PH: 6.5-7 ± 0.2 at 25
8. MR-VP Medium (M 070-500g, HIMEDIA, Mumbai, India)
Preparation: suspend 17.0 grams in 1000ml distilled water. Heat if necessary to dissolve the medium completely. Distribute in to test tubes 10ml amounts and sterilize by autoclaving at 15 Ibs pressure (121) for 15 minutes.
Composition (g/l): buffered peptone 7.00; dextrose 5.00; dipotassium phosphate 5.00 Reagent required for voges- proskauer reaction 1 a-Naphtanol, ethanolic solution Preparation: dissolve a-Naphtanol in ethanol Composition (g/l): a-Naphtanol 6 grams; ethanol 96 % (volume fraction) 100ml. 1 Potassium hydroxide solution Preparation: dissolve potassium hydroxide in distilled water Composition (g/l): potassium hydroxide 40 grams; distilled water 100ml
9. Mueller-Hinton Agar (CM 0337, OXOID, Basingstoke, England)
Preparation: suspend 38 grams in 1litre of distilled water. Bring to boil to dissolve the medium completely. Sterilize by autoclaving at 121 for 15 minutes. Final PH: 7.3 ± 0.1 at 25.
Composition (g/l): beef, dehydrated infusion 300.00; casein hydrolysate 17.5; starch 1.5; agar 17.00
10. 0.5 McFarland standards
Composition: 1.17% BaCl.2H2O solution and 0.36N of 1% sulfuric acid (H2SO4). Preparation: Add approximately 85Ml of 1% H2SO4 to a 100ml of the volumetric flask, using a 0.5ml pipette add 0.5ml of 1.17% BaCl.2H2O drop wise to the H2SO4 while constantly swirling the flask. Bring to 100ml with 1% H2SO4.place a magnetic stirring in the flask and place on the magnetic stirrer for approximately three to five minutes. Examine solution visually to make certain it appears homogeneous and free of visible clumps. Dispense three to seven ml, cub tube tightly and seal with paraffin and keep at dark and room temperature.
11. Urea broth base (M112S-500g, HIMEDIA, Mumbai, India)
Preparation: Suspend 24.51 grams in 950 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 50°C and aseptically add 50 ml of sterile 40% Urea Solution (FD048) and mix well. Dispense into sterile tubes and allow setting in the slanting position. Do not overheat or reheat the medium as urea decomposes very easily. Final PH: 6.5-7 ± 0.2 at 25
Composition (g/l): Dextrose 1.000, Peptic digest of animal tissue 1.500, Sodium chloride 5.000, Monopotassium, phosphate 2.000, Phenol red 0.012, Agar 15.000 12.
Lysine Iron Agar (M377-500g, HIMEDIA, Mumbai, India)
Preparation: Suspend 34.56 grams in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Dispense into tubes and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool the tubes in a slanted position to form slants with deep butts.
Composition (g/l): Peptone 5.000, Yeast extract 3.000, Dextrose (Glucose) 1.000, L- Lysine 10.000, Ferric ammonium citrate 0.500, Sodium thiosulphate 0.040, Bromocresol purple 0.020, Agar 15.000 Final pH ( at 25°C) 6.7±0.2
Appendix 3: Procedures of salmonella species identification by Conventional multiplex PCR for Salmonella Enteriditis, Salmonella Typhi and Salmonella Typhimurum Identity test procedure
Principle of DNA Extraction
he extraction of DNA consists of four major steps.
1. Preparation of a cell extract:
To extract DNA from tissue/cells of interest:
- The cells have to be separated and the cell membranes have to be disrupted by using "Extraction buffer". Included in the buffer: EDTA and SDS.
- EDTA (Ethylene diamine tetra acetate) removes Mg2+ ions that are essential for preserving the overall structure of the cell membrane
- SDS (Sodium Dodecyl Sulphate) aids in disrupting the cell membranes by removing the lipids of the cell membranes.
Having lysed the cells, the final step in the preparation of a cell extract is the removal of insoluble cell debris and partially digested organelles by centrifugation, leaving the cell extract as a reasonably clear supernatant.
2. Purification of DNA from cell extract
In addition to DNA the cell extract will contain significant quantities of protein and RNA. A variety of procedures can be used to remove these contaminants, leaving the DNA in a pure form.
The standard way to deproteinize a cell extract is to add phenol or a 1:1 mixture of phenol: chloroform. These organic solvents precipitate proteins but leave the nucleic acids in aqueous solutions.
The aqueous solution containing nucleic acid is removed carefully with a pipette. For RNA, however, the effective way to remove it by using ribonuclease enzyme, which will rapidly degrade these molecules into ribonucleotide subunits.
3. Collecting DNA
The most frequently used method of concentration is ethanol precipitation. In the presence of salt and at a temperature of -20 °C or less, absolute ethanol will efficiently precipitate polymeric nucleic acids. With a concentrated solution of DNA one can use a glass rod to spool the adhering DNA strands. For dilution purposes the precipitated DNA can be collected by centrifugation and r re dissolved in an appropriate volume of water.
4. Measurement of purity and DNA concentration
UV absorbance can also be used to check the purity of a DNA preparation. For a pure sample of DNA the ratio of absorbencies at 260 nm and 280 nm (A260/A280) is 1.8. This is because proteins absorb maximum UV light at A280.A ratio of less than 1.8 is indicative of protein contamination. If the solution is reasonably pure, DNA concentrations can accurately be measured using UV absorbance spectrometry. This is because the base pairs in DNA absorb UV light, therefore the amount of bp is directly proportional to DNA concentration. For DNA, absorbance at A260 (also called optical density, OD) is converted into DNA concentration by following method: A260/OD of 1.0 = a concentration of 50 pg/ml of double-stranded DNA (dsDNA) A260/OD of 1.0 = a concentration of 33 pg/ml of single-stranded DNA (ssDNA or RNA)
Quantification Of Extracted Dna Sample By Spectrophotometry 50 pg/ml of DNA = 1 OD (optical density) Therefore the concentration is calculated by using following equation: OD260 of sample X dilution factor X 50 pg/ml (1 OD) = pg/ml DNA Example; If 5 pl of extracted DNA in 1000pl (1ml) gives an OD260 = 0.14 Dilution factor = 1000 / 5 = 200 0.14 X 200 X 50 = 1400 pg/ml or 1.4 mg/ml
Concentration and purity via OD measurement
Abbildung in dieser Leseprobe nicht enthalten
DNA extraction using QIAGENTM mini columns A costly, however, an effective method of extracting high-quality amplifiable genomic DNA from whole blood, urine, dried blood spot, buffy coat, and tissue biopsy samples. Refrigerated samples and reagents from the kit are brought to room temperature before starting the procedure.
Check the following equipment and reagents are ready:
- Water bath at 56°C.
- Buffer AE or dd.H2O for elution.
- Buffer AWl, Buffer AW2, and QIAGEN Protease.
- If a precipitate has formed in Buffer AL, dissolve by incubating at 70o C
- All centrifugation steps should be carried out at room temperature
1. Pipette 20gl of QIAGEN Protease into the bottom of a 1.5ml microcentrifuge tube.
2. Add a 200 pl sample to the microcentrifuge tube.
3. Add 200gl buffer AL to the sample. Mix by pulse-vortexing for 15sec.
4. Incubate at 56°C for 10 min. DNA yield reaches a maximum after lysis for 10 min at 56°C, but longer incubation times will not have a negative effect on DNA extraction.
5. Briefly centrifuge the 1.5ml microfuge tube to remove drops from the inside of the lid.
6. Add 200gl of ethanol (96-100%) to the sample and mix again by pulse-vortexing. After mixing, briefly centrifuge the 1.5ml microfuge tube to remove drops from the inside of the lid
7. Carefully transfer the mixture from step-6 to the QIAamp spin column (in a 2ml collection tube) without wetting the rim, close the cap, and centrifuge at 6000xg (8000rpm) for 1 min.
Place the QIAamp spin column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate. Do not over tighten caps. If caps are tightened until they snap they may loosen during centrifugation and subsequently damage the centrifuge.
8. Carefully open the QIAamp spin column and add 500gl Buffer AW1 without wetting the rim. Close the cap and centrifuge at 6000xg (8000rpm) for 1min. Place the QIAamp spin column in a clean 2ml collection tube (provided), and discard the collection tube containing the filtrate.
9. Carefully open the QIAamp spin column and add 500g1 Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (20,000xg; 14000rpm) for 3 min. Continue directly with
Step-10, or to eliminate any chance of possible buffer AW2 carryover, perform step 9a, and then continue with step 10. 9a. (optional): Place the QIAamp spin column in a new 2 ml collection tube (not provided) and discard the collection tube containing the filtrate. Centrifuge at full speed for 1min. 10.
Place the QIAamp spin column in a clean 1.5ml microfuge tube (not provided), and discard the collection tube containing the filtrate. Carefully open the QIAamp spin column and add 200li1 Buffer AE or distilled water. Incubate at room temperature for 5min, and then centrifuge at 6000xg (8000rpm) for 1min. *A second elution step with a further 200ll Buffer AE will increase yields by up to 15%. For calculating DNA concentration; Pipette 2pls in a clean 1ml tube and add to it 198pls of water to give 1/100 dilution factor Take the OD at A260 x 100 x 50 = pg/ml of DNA
1-Master mix preparation
Abbildung in dieser Leseprobe nicht enthalten
2-Run PCR Reaction
Abbildung in dieser Leseprobe nicht enthalten
3-Agrose gel preparation
-Prepare 2% Ag arose gel
-Add 4^ Gel red with Loading dye, 10PCR product and 10 |il markers (Ladder)
-Run Electrophoresis for 1 hour at 120V
-Read the result by using UV -light
-It is around 401bp for Spy (S.Typhimurium), 738bp for S. Typhi and 304bp for S.Enteritidis positive result.
Appendix 4: The Susceptibility of each antimicrobial was determined depending on the following the measure of zone inhibition diameter
Abbildung in dieser Leseprobe nicht enthalten
Source: (CLSI, 2019)
Appendix 5 : Plating and biochemical tests record format used for Salmonella isolation.
Sample
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Figure 3: Poor fish processing at the landing site of Lake Fincha
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Figure 4: Salmonella appearance on XLD agar
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Figure 5: Biochemical test results of Salmonella
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Figure 6: Phenotypical characteristics of Salmonella by Biolog.
Abbildung in dieser Leseprobe nicht enthalten
Figure 7: Species Identification of Salmonella by Conventional multiplex PCR
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Figure 8: Salmonella Typhimurim isolated from fish by Conventional multiplex PCR.
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Figure 9: Antimicrobial Susceptibility Test
[...]
- Quote paper
- Ebisa Bane (Author), 2021, Salmonella Isolated from Fishermen and Fish Harvested from Lake Fincha, Ethiopia. Occurrence, Risk Factors and Antimicrobial Susceptibility, Munich, GRIN Verlag, https://www.grin.com/document/1327906
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