Molecular Epidemiology, Risk Factors And Assessment of Antibiotic Resistance of Salmonellae In Commercial Layer Farms In Six Selected States In Nigeria
– Molecular Epidemiology, Risk Factors And Assessment of Antibiotic Resistance of Salmonellae In Commercial Layer Farms In Six Selected States In Nigeria –
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ABSTRACT
Poultry salmonellosis remains a major constraint to poultry production in all parts of Nigeria with farmers still experiencing great losses caused by Salmonella infection despite huge amounts of resources expended on vaccination and medication.
Relatively few African countries report surveillance data on Salmonella and as such, very limited information is available on Salmonella isolation for this continent.
A cross sectional study was carried out to determine the prevalence, risk factors and the circulating Salmonella serovars in commercial layer farms in Nigeria.
The study also identified the different antibiotic resistance patterns seen in the Salmonella species and assessed the mechanisms of their resistance and determined the degree of relatedness within the different serovars isolated.
In all, 358 farms were visited in the six selected States (Imo, Edo, Ogun, Kaduna, Plateau and Bauchi) and 1790 samples comprising of litter, dust, faeces, feed and water were collected and tested for Salmonella according to ISO6579:2002; Annex D protocol.
TABLE OF CONTENTS
Cover page……………………………………………………………………… i
Fly page…………………………………………………………………………. ii
Title Page…………………………………………………………………………… iii
Declaration………………………………………………………………………….iv
Certification………………………………………………………………………… v
Dedication………………………………………………………………………..…vi
Acknowledgements………………………………………………………………… vii
Abstract……………………………………………………………………………..ix
Table of Contents………………………………………………………………….. xii
List of Tables………………………………………………………………………. xvi
List of Figures……………………………………………………………………… xix
List of Plates……………………………………………………………………….. xxii
List of Appendices………………………………………………………………… xxiv
List of Abbreviations………………………………………………………………… xxv
CHAPTER 1: INTRODUCTION……………………………………………….. 1
1.1 Background Information……………………………………………………… 1
1.2 Statement of the Research Problem…………………………………………. 3
1.3 Justification of the Study………….……….………………………………….6
1.4 Aim of the Study……….………………….………………………………….. 7
1.5 Objective of the Study………………………………………………………… 7
1.6 Research Questions….…………………………………….…………….…….8
CHAPTER 2: LITERATURE REVIEW………………………………………. 9
2.1 History of Salmonella………………………….………………………..……..9
2.2 The Aetiology of Salmonella…………………………………………………. 9
2.3 Taxonomy of Salmonella…….………………………………………………… 10
2.4 Nomenclature of Salmonella…….……………….………………………..….. 11
2.5 Antigenic Diversity…….………………………………………………….……15
2.5.1 O (somatic) antigens……….…………………………………………………… 15
2.5.2 H (flagellar) antigens………………………………………………………….. 15
2.5.3 Vi (capsular or surface or virulence) antigens………………………………… 16
2.6 Genome of Salmonella…………..……………………………………………. 17
2.7 Salmonellosis…………………………..……………………………………… 20
2.7.1 Pathogenesis and immunology……………….………………………………. 20
2.7.2 Virulence factors……………………………………………………….……..22
2.7.3 Epidemiology of salmonellosis……………………………………………….25
2.8 Isolation and Identification of Salmonella Isolates……….…..…………….. 30
2.8.1 Culture……………………………………………….……………………….. 30
2.8.2 Biochemical tests………………………………………………………….….. 34
2.8.3 Serology…………………………………………………………………….…35
2.9 Mechanisms of Drug Resistance Salmonella species…………………………36
2.9.1 Chloramphenicol………………………………………………………………36
2.9.2 Co-trimoxazole……………………………………………………………….. 37
2.9.3 Fluoroquinolones……………………………………………………….…….. 38
2.9.4 β-lactam antibiotics……………………………………………………………..40
2.9.5 Ampicillin……………………………………………………………………. 41
2.9.6 Cephalosporins………………………………………………………………. 41
2.10 Trends of Drug Resistance in Salmonella………………………………….. 44
2.10.1 Salmonella enterica serovar Typhi…………………………………………. 45
2.10.2 Non-typhoidal Salmonella enterica serovars………………………………..46
2.11 Typing of Salmonella………………………………………………………… 49
2.11.1 Phenotyping…………………………………………………………………. 49
2.11.2 Genotyping…………………………………………………….…….……… 52
2.11.3 Sequence-based typing……………………………..………………………. 61
2.12 Treatment of Salmonellosis….………………………………………………66
2.12.1 Typhoidal salmonellosis……………………………………………………. 66
2.12.2 Non-typhoidal salmonellosis……………………………………………….. 67
2.13 Prevention and Control of Salmonellosis……………………………………68
CHAPTER 3: MATERIALS AND METHODS………………………….…….. 70
3.1 Study Area……………………………………………………….……………. 70
3.1.1 Ogun State……………………………………………………….…..………. 70
3.1.2 Plateau State….……………………………………………………………… 71
3.1.3 Edo State………………………………………………………………………. 71
3.1.4 Imo State………………………………………………………………………. 71
3.1.5 Bauchi State………………………………………………………………….. 72
3.1.6 Kaduna State………………………………………………………….……… 73
3.2 Sample Size………………………………………………………………….…75
3.3 Sampling Frame…………………………………………………………….…75
3.3.1 Criteria for selection of study areas, farms and samples…………………….. 75
3.4 Sampling Procedure………………………………………………………….. 76
3.4.1 Farm selection…………………………………………………………………75
3.4.2 Sample collection……………………………………………………………..77
3.4.3 Risk factor determination……………………………………………………. 77
3.5 Isolation and Characterization of Salmonella isolates using ISO 6579:2002 Method………….……. 78
3.6 Serotyping………………………………………………………………………79
3.6.1 Microtitre plate method for Salmonella serotyping…………………………..79
3.6.2 Traditional (slide agglutination) serotyping…………………………………. 82
3.6.3 Molecular determination of Salmonella serotypes using a Multiplex,Bead-Based Suspension Array (Bioplex)………… 85
3.7 Phage Typing for S. Enteritidis and S. Typhimurium………………………. 88
3.7.1 Phage for S. Enteritidis………………………………………………………. 88
3.7.2 Phage for S. Typhimurium……………………………………………………88
3.7.3 Phage inoculation……………………………………………………………. 88
3.8 Antimicrobial Susceptibility Profiling…..………………………………….. 91
3.9 Detection of Antimicrobial Resistance Genes………………………………. 92
3.9.1 Procedures for DNA extraction……………………………………………… 92
3.10 Sequencing of gyrA, parC, pmrA and tem Genes……..……………………. 93
3.11 Pulsed Field Gel Electrophoresis (PFGE)……………………..……………93
3.12 Multilocus Variable-Number Tandem-Repeat Analysis (MLVA)………. 96
3.13 Data Analysis……………………….…………………………………….…. 98
3.14 Statistical Analysis for Determining Risk Factors …………………..…….98
CHAPTER 4: RESULTS …………….…………………………………………. 99
4.1 Result of Isolation and Prevalence of Salmonella based on Farm, Sample and Sample Types in Six Selected States of Nigeria………………99
4.2 Result of Circulating Salmonella Serovars in Commercial Layer Farms in the Six Selected States in Nigeria…………………………………99
4.3 Result of the Risk Factors Associated with Salmonella Infection in Commercial Layer Farms in Nigeria……………………………………. 106
4.4 Results of Antimicrobial Susceptibility Patterns of Salmonella Strains isolated in the Commercial layer farms in the Six Selected States in Nigeria…………….. 121
4.5 Result of Antimicrobial Resistance Target Genes of Salmonellae Isolated from Commercial Layer Farms in the Six Selected States in Nigeria….…………125
4.6 Result of Sequence Analysis to Determine Point Mutation(s) inTarget Resistance Genes for Isolates that were Resistant to Quinolones, Polymixins and Beta-lactams……….. 125
4.7 Results showing genetic diversity/relatedness amongst Salmonella Serovars from the Six Selected States in Nigeria using PFGE and MLVA…………… 145
CHAPTER 5: DISCUSSION …..………….……………………………………. 165
CHAPTER 6: CONCLUSION AND RECOMMENDATIONS ……….…….. 181
6.1 Conclusion………………………………………………………………………. 181
6.2 Recommendations……………………………………………………………….182
REFERENCES……………………………………………………………..…….. 184
INTRODUCTION
Background Information
Nigeria is the largest country in sub-Saharan Africa with an estimated population of174.5 million people in 2013 and a population growth rate estimated at three per centannually.
The estimated gross domestic product (GDP) in 2012 was $450.5 billion, ranking the country 31st globally and agriculture accounts for over 38% of non-oil foreign earnings and 70% of the labour force (FAO, 2006a).
The poultry industry in Nigeria is rapidly expanding despite being faced with many problems such as avian influenza, global financial crisis and inadequate credit (FAO, 2008). Nigerian poultry industry increased from 150,700 million birds in 2005 to 192,313 million birds in 2010(FAO, 2015).
Across the different regions of the country, the poultry sector ischaracterized by a low level of production, specialization and generally with biosecuritymeasures at a very weak level (FAO, 2008).
REFERENCES
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Achard, C. and Bensaude, R. (1896). Bulletin et Memoires de la Societe. Medicale desHopitaux de. Paris, 13: 679.
Adesiji, Y.O., Deekshit, V.K., and Karunasagar, I. (2014). Antimicrobial-resistant genesassociated with Salmonella spp. isolated from human, poultry, and seafoodsources. Food Science & Nutrition, 2(4): 436–442.
Adesiji, Y. O. and Fagbami, A. H. (2006). Epidemiology of bacterial zoonosis inNigeria. Nigerian Journal of Health and Biomedical Sciences, 5: 20–25.
Adetosoye, A. I. (1980). Infective drug resistance among Escherichia coli isolated fromclinically healthy domestic livestock.Veterinary Microbiology, 5: 333–342.
Afema, J. A., Mather, A. E. and Sischo, W. M. (2014). Antimicrobial resistance profilesand diversity in Salmonella from humans and cattle, 2004–2011.ZoonosesPublic Health, 62 (7): 506-517.
