Isolation and Identification of Salmonella from the Environment of Traditional Poultry Farms in Khartoum North

Isolation and identification of Salmonella from the environment of traditional poultry farms in Khartoum North

By Hisham Ibrahem MohammedAhmad Saeed B. V. M U of K 2004

Supervisor Dr. Awad Elkarim A. Ibrahem B. V. Sc, M. Sc. & Ph. D.

A thesis submitted for the partial fulfillment of the requirements for Master Degree in Microbiology

Department of Microbiology Faculty of Veterinary Medicine University of Khartoum March 2010

DEDICATION

To my parents,

Wonderful Brothers and Sisters

And all my teachers throughout my life

With respect

ACKNOWLEDGEMENT

To begin with, my gratitude and praise are due to almighty Allah, the Beneficent and the Merciful for the precious gift of health and the capability to accomplish this work.

I am deeply indebted and thankfulness to my supervisor Dr. Awad Elkarim Abdelghaffar Ibrabim for his priceless guidance, close supervision, helpful, patience and contiuous encouragement.

I am deeply thankful to my wonderful friend Reem Merghani Ali Nageela for her continuous support to accomplish this work.

I would like to thank all the technicians of the department of microbiology for their great help.

I am also grateful to my colleagues Ahmed Mohammed saad and Amani Mahmoud.

Finally my thanks extended to everybody he gave me his time, support and help.

ABSTRACT

Avian salmonellosis is a large group of acute or chronic diseases of fowl caused by different species of the genus Salmonella. It is a problem of economic concern to all phases of the poultry industry from production to marketing.

Our study was conducted to investigate the incidence of Salmonella species in the feed and environment of open system poultry farms in Khartoum North area during the period from August to November 2009.

A total of 80 samples were taken from six poultry farms of layers and broilers located in Al-Halfaya, Shambat, Hillat Kuku and Al- Zakiab area. The samples include: poultry feed from feeders (27 samples), litter (27 samples) and drinking water from drinkers (26 samples).

Isolation of Salmonella were carriedout in selective classical medium (DCA) after enrichment in selenite-f-broth. Four salmonella isolates represent (5%) of total samples were recovered; three isolates (75%) from litter samples and one isolate (25%) recovered from water sample, no Salmonellae recovered from feed samples. Three isolates belong to S. enteritidis while the fourth isolate belongs to S. arizonae.

All four Salmonella isolates were recovered from two farms: three isolates recovered from a farm located in Al-Halfaya (layers) and one isolate recovered from a farm located in Shambat, (broilers) no isolates were recovered from Hillat Kuku or Al-Zakyab area farms.

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Other Enterobacteria were also isolated and included 15 (18.75%) Serratia spp., 11 (13.75%) Proteus spp., 8 (10.00%) Citrobacter spp., 1 (1.25%) Kluyvera spp., 1 (1.25%) Enterobacter spp., 1 (1.25%) Yersinia spp., and 1 (1.25%) Hafnia spp.

All isolates were identified to the species level using cultural characteristics and biochemical reactions.

Antimicrobial sensitivity test to the four Salmonella isolates was carried out. Each isolate was tested to 10 different antimicrobial agents using Mueller and Hinton Agar Medium. All isolates found sensitive to chloramphenicol, ceftizoxime, amikacin and resistant to gentamycin, tetracycline, ambicillin\ sulbactam and piperacillin\ tazobactam.

ﻣﻠﺨﺺ اﻷﻃﺮوﺣﺔ

ﺳﺎﻟﻤﻮﻧﻴﻠﻪ اﻟﻄﻴﻮر ﻣﺼﻄﻠﺢ ﻳﻄﻠﻖ ﻋﻠﻰ ﻣﺠﻤﻮﻋﺔ آﺒﻴﺮة ﻣﻦ اﻷﻣﺮاض اﻟﺤﺎدة واﻟﻤﺰﻣﻨﺔ اﻟﺘﻲ ﺗﺴﺒﺒﻬﺎ أﻧﻮاع ﻣﺨﺘﻠﻔﺔ ﺗﻨﺘﻤﻲ إﻟﻰ ﺟﻨﺲ اﻟﺴﺎﻟﻤﻮﻧﻴﻠﻪ . ﻣﻊ اﻟﺘﻮﺳﻊ اﻟﻜﺒﻴﺮ ﻓﻲ ﺻﻨﺎﻋﺔ اﻟﺪواﺟﻦ أﺻﺒﺤﺖ ﺳﺎﻟﻤﻮﻧﻴﻠﻪ اﻟﻄﻴﻮر ﻣﻦ أهﻢ اﻷﻣﺮاض اﻟﺒﻜﺘﻴﺮﻳﺔ اﻟﻤﻨﻘﻮﻟﺔ ﺑﻮاﺳﻄﺔ اﻟﺒﻴﺾ وهﻮ ﻣﺮض ذو أﺑﻌﺎد اﻗﺘﺼﺎدﻳﺔ ﻓﻲ ﺟﻤﻴﻊ ﻣﺮاﺣﻞ ﺻﻨﺎﻋﺔ اﻟﺪواﺟﻦ .

ﺗﺘﻠﺨﺺ اﻟﺪراﺳﺔ ﻓﻲ ﺗﺤﺪﻳﺪ وﺟﻮد أﻧﻮاع ﺟﻨﺲ اﻟﺴﺎﻟﻤﻮﻧﻴﻠﻪ ﻓﻲ ﻋﻠﻒ وﺑﻴﺌﺔ اﻟﺪواﺟﻦ ﻓﻲ ﻣﺰارع ﺗﺮﺑﻴﺔ اﻟﺪواﺟﻦ ﻷﻏﺮاض إﻧﺘﺎج اﻟﺒﻴﺾ واﻟﻠﺤﻢ ﺑﻤﻨﻄﻘﺔ اﻟﺨﺮﻃﻮم ﺑﺤﺮي ﻓﻲ اﻟﻔﺘﺮة ﻣﻦ أﻏﺴﻄﺲ إﻟﻰ ﻧﻮﻓﻤﺒﺮ 2009 .

ﺗﻢ ﺟﻤﻊ 80ﻋﻴﻨﺔ ( 27 ﻋﻠﻒ ، 27 ﻓﺮﺷﺔ ، 26 ﻣﺎء ﺷﺮب ) ﻣﻦ 6 ﻣﺰارع ﺑﻨﻈﺎم اﻟﺘﺮﺑﻴﺔ اﻟﻤﻔﺘﻮح ﻓﻲ ﻣﻨﺎﻃﻖ اﻟﺤﻠﻔﺎﻳﺔ ، ﺣﻠﺔ آﻮآﻮ ، ﺷﻤﺒﺎت ، وﻣﻨﻄﻘﺔ اﻟﺰاآﻴﺎب .

ﺗﻢ ﻋﺰل 4 ﻋﺰﻻت ﻣﻦ ﺑﻜﺘﺮﻳﺎ اﻟﺴﺎﻟﻤﻮﻧﻴﻠﻪ ( 5% ) وﺻُﻨﻔﺖ آﺎﻵﺗﻲ :

3 ﻋﺰﻻت ﻣﻦ اﻟﺴﺎﻟﻤﻮﻧﻴﻠﻪ اﻟﻤﻠﻬﺒﺔ ﻟﻸﻣﻌﺎء وﻋﺰﻟﺔ واﺣﺪة ﻣﻦ اﻟﺴﺎﻟﻤﻮﻧﻴﻠﻪ اﻷرزوﻧﻴﺔ . ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻰ 3 ﻋﺰﻻت ﻣﻦ ﻋﻴﻨﺎت اﻟﻔﺮﺷﺔ وﻋﺰﻟﺔ واﺣﺪة ﻣﻦ ﻋﻴﻨﺎت ﻣﺎء اﻟﺸﺮب وﻟﻢ ﻳﺘﻢ اﻟﺘﺤﺼﻞ ﻋﻠﻰ ﻋﺰﻻت اﻟﺴﺎﻟﻤﻮﻧﻴﻠﻪ ﻣﻦ اﻟﻌﻠﻒ . ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻰ اﻷرﺑﻊ ﻋﺰﻻت ﻟﻠﺴﺎﻟﻤﻮﻧﻴﻠﻪ ﻣﻦ ﻣﺰرﻋﺘﻴﻦ ﻓﻘﻂ : ﻣﺰرﻋﺔ ﻹﻧﺘﺎج اﻟﺒﻴﺾ ﻓﻲ اﻟﺤﻠﻔﺎﻳﺔ وأﺧﺮى ﻹﻧﺘﺎج اﻟﻠﺤﻢ ﻓﻲ ﺷﻤﺒﺎت .

ﺗﻢ ﻋﺰل اﻟﺒﻜﺘﺮﻳﺎ وﺻُﻨﻔﺖ آﺎﻵﺗﻲ :

ﺳﺮﻳﺸﻴﺎ 15 (18.75%) ، ﺑﺮوﺗﻴﺲ 11 (13.75%) ، ﺳﺘﺮوﺑﺎآﺘﺮ 8 (%10) ، آﻠﻮﻓﻴﺮا 1 (1.25%) ، اﻧﺘﻴﺮوﺑﺎآﺘﺮ 1 (1.25%) و هﺎﻓﻨﻴﺎ 1 (1.25%) . ﺟﻤﻴﻊ اﻟﻌﺰﻻت ﺗﻢ ﺗﺼﻨﻴﻔﻬﺎ ﺣﺘﻰ ﻣﺮﺣﻠﺔ اﻟﻨﻮع ا ﻋ ﺘ ﻤ ﺎ د اً ﻋﻠﻰ اﻟﺨﺼﺎﺋﺺ اﻟﻤﺰرﻋﻴﺔ واﻻﺧﺘﺒﺎرات اﻟﺒﻴﻮآﻴﻤﻴﺎﺋﻴﺔ .

ﺗﻢ إﺟﺮاء اﺧﺘﺒﺎر اﻟﺤﺴﺎﺳﻴﺔ ﻟﻠﺴﺎﻟﻤﻮﻧﻴﻠﻪ ﺑﺎﺳﺘﺨﺪام 10 ﻣﻀﺎدات ﺣﻴﻮﻳﺔ . ﺟﻤﻴﻊ ﻋﺰﻻت اﻟﺴﺎﻟﻤﻮﻧﻴﻠﻪ أﻇﻬﺮت ﺣﺴﺎﺳﻴﺔ ﻟﻠﻜﻠﻮراﻣﻔﻴﻨﻴﻜﻮل واﻟﺴﻔﺘﻴﺰوآﺰﻳﻢ و اﻷﻣﻴﻜﺎﺳﻴﻦ وأﻇﻬﺮت ﻣﻘﺎوﻣﺔ آﺎﻣﻠﺔ ﻟﻠﺠﻨﺘﺎﻣﻴﺴﻴﻦ و اﻟﺘﻴﺘﺮﺳﻴﻜﻠﻴﻦ و اﻷﻣﺒﻴﺴﻠﻴﻦ ﻣﻊ اﻟﺴﺎﻟﺒﺎآﺘﺎم واﻟﺒﻴﺒﺮاﺳﻴﻠﻴﻦ ﻣﻊ اﻟﺘﺎزوﺑﺎآﺘﺎم .

List of contents Subject Page Dedication …………………………………………………... . i Acknowledgment ………………………………………….... .ii Abstract……………………………………………………… .iii Arabic Abstract …………………………………………….. .v List of Contents …………………………………………….. . iv List of Tables ……………………………………………….. .x List of Figure ……………………………………………….. . xi Introduction ………………………………………………... 1 Chapter One: Literature Review ………………………….. 3 1.1History of Salmonella ………………………………….. 3 1.2 Classification of Salmonella …………………………... 3 1.3 Morphology of Salmonella …………………………… 5 1.4 Antigenic Structure …………………………………….. 5 1.5 Prevalence of Salmonella …………………………….. 7 1.5.1 Prevalence in Poultry …………………………..…… 7 1.5.1.1 Isolation of Salmonella from different poultry sources ……………………………………………….……... 7 1.5.1.1.1 Chicks …………………………………………... 7 1.5.1.1.2 Poultry flocks …………………………………… 8 1.5.1.1.3 Poultry feed …………………………………….. 9 1.5.1.1.4 Poultry carcasses and other poultry sources .…… 9 1.5.2 Prevalence in animals……………………..………..… 11 1.5.3 Prevalence in man……………………….…………… 12 1.6 Pathogenicity of Salmonella ………………....………… 14 1.7 Laboratory diagnosis………………………....………… 17

1.7.1 Isolation of Salmonella ……………………………… 17 1.7.1.1 Cultural characteristics …………………………… 17 1.7.1.1.1 Enrichment media ……………………………… 18 1.7.1.1.2 Differential and selective solid media …..……… 19 1.7.1.2 Biochemical reactions ……………………..……… 22 1.7.2 Serological tests ……………………………..……… 23 1.8 Drug susceptibility ………………………………..…… 24 1.9 Control and treatment ………………………..………… 26 1.10 Salmonella vaccine ………………………...………… 28 Chapter Two: Materials and Methods: ………...…………… 30 2.1 Sampling ………………………………….…………… 30 2.1.1 Source of specimens ………………………………… 30 2.1.2 Collection of specimens ………………..…………… 30 2.1.2.1 Feed samples ……………………………………… 31 2.1.2.2 Litter samples ……………………………………… 31 2.1.2.3 Drinking water sample …………………………… 33 2.1.3 Transport and storage of samples …………..……… 33 2.2 Bacteriological investigation ………………………… 33 2.2.1 Culture media ……………………………………… 33 2.2.1.1 Liquid media …………………………………….. 33 2.2.1.2 Semi-solid media ………………………………… 35 2.2.1.3 Solid media ……………………………………… 35 2.2.2 Solutions and Reagents …………………………… 38 2.2.2.1 Normal saline solution …………………………… 38 2.2.2.2 Methyl Red ……………………………………… 38 2.2.2.3 Kovac’s reagent ………………………………… 38 2.2.2.4 Oxidase test reagent ……………………………… 39

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2.2.2.5 Potassium Hydroxide solution ……………………. 39 2.2.2.6 Andrade’s indicator ……………………………… 39 2.2.2.7 Voges- Proskauer (V.P) test reagent …………….. 39 2.2.2.8 Lead acetate paper ……………………………… 39 2.2.2.9 Bromo-thymol blue ………………………………. 40 2.2.2.10 Phenol red ……………………………………….. 40 2.2.3 Sterilization procedures …………………………… 40 2.2.3.1 Hot air oven ……………………………………… 40 2.2.3.2 Autoclaving ………………………………………. 40 2.2.3.3 Disinfectants and antiseptics …………………… 40 2.2.3.4 U. V Light ……………………………………….. 40 2.2.4 Cultivation of samples ……………………………… 41 2.2.4.1 Inoculation of enrichment medium ……………… 41 2.2.4.2 Inoculation of plates ……………………………… 41 2.2.4.3 Purification and storage of isolates ……………… 42 2.2.5 Identification of the isolated bacteria ……………… 42 2.2.5.1 Microscopic examination ………………………… 42 2.2.5.2 Biochemical tests for identification of bacteria … 43 2.2.5.2.1 Primary biochemical tests ……………………. 43 2.2.5.2.2 Secondary biochemical tests …………………… 44 2.2.6 Antimicrobial sensitivity test ……………………… 46 Chapter Three: Results …………………………………… 49 3.1 Isolation of bacteria ………………………………….. 49 3.2 Site of isolation ………………………………………. 49 3.3 Properties of Salmonella …………………………….. 50 3.3.1 Cultural properties …………………………………. 50 3.3.1.1 Growth in liquid media …………………………. 50

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3.3.1.2 Growth on solid media …………………………… 50 3.3.2 Microscopic properties …………………………….. 50 3.3.3 Biochemical reactions ………………………………. 51 3.4 Sensitivity to antimicrobial agents …………………… 51 Chapter four: Discussions ………………………………… 59 Conclusion ……………………………………………….. 63 Recommendations ………………………………………… 63 References ………………………………………………. 64

List of Tables Table title Page Origin, type and number of samples collected in the study…... 32 Antibiotics used in sensitivity testing ………………………... 47 Standard zone of inhibition to different antimicrobial agents... 48 Isolated Enterobacteria from different samples in the study … 52 Isolated Salmonellae from different samples and areas in Khartoum North………………………………………………. 53 Sensitivity of Salmonella isolates to different Antimicrobial Agents…………………………………………………………. 54

List of Figure

Figure Title Page Growth of Salmonella on Nutrient Agar……………………. 55 Growth of Salmonella on Desoxychocolate Citrate Agar 55 (DCA)……………………………………………………….. Growth of Salmonella on Triple Sugar Iron Agar (TSI)……. 56 The sensitivity of Salmonella to different antibiotics …….. 57 The sensitivity of Salmonella to different antibiotics………. 58

INTRODUCTION

Members of the family Enterobacteriaceae are gram- negative, non-spore forming rods. Some of them are human and animal pathogens producing intestinal infection and food poisoning. The genera of pathogenic importance in poultry include Salmonella and Escherichia (Holt et al., 1994).

Avian salmonellosis is an inclusive term designating a large group of acute or chronic diseases of fowl caused by different species of the genus Salmonella including S. pullorum (Pullorum disease), S.gallinarum (Fowl typhoid), S. arizonae (Arizonae infection), S. enteritidis and others (Paratyphoid infection) (Carter and Wise, 2004).

Paratyphoid infections are economically among the most important bacterial disease of the hatching industry and result in high death losses among all types of young poultry (Hofstad et al., 1978). In addition, the occurrence of this disease in valuable breeding stocks is extremely costly. Also fertility, hatchability and egg production may be seriously impaired (Graham and Michael 1936; Pomeroy and Fenstermacher 1941).

Adult birds infected with paratyphoid organisms generally show no outward symptoms; however, they may serve as intestinal carriers of the infection over long periods of time and serve as the chief source of paratyphoid infections of most species of poultry (Olesuik et al., 1969; Hofstad et al., 1978).

Fecal contamination of egg shells with paratyphoid organisms during the process of laying or from contaminated nests, floors, or

1 incubators after laying is of foremost importance in the spread of the disease. Also the disease may be transmitted directly to young birds from older fowl that are chronic intestinal carriers of the infection but exhibit no visible symptoms (Hofstad et al., 1978).

Evidence has been presented that poultry feeds may be a common and very important source of paratyphoid organisms. The level of Salmonella contamination in poultry feeds is normally low; however, it has been shown that even one organism per 15 grams of feed can produce infection (Harry and Brown, 1974).

Salmonellosis in poultry resulted in continuous increase of public health problems as stated by Corrier et al., (1990). Contamination of poultry meat with Salmonella was investigated by many scientists in Sudan as well as in many countries. In Sudan Mamon et al., (1992) succeeded to isolate 21 Salmonella enteritidis from embryonated eggs. Yagoub and Mohammed (1987) studied the occurrence of Salmonella in poultry carcasses in Khartoum state; 23 serotypes were identified and most of them were S. monas and S. amek.

The objectives of this study were:

1- To investigate the contamination of poultry environment: feed, drinking water and poultry litter with Salmonella species in Khartoum North area poultry farms. 2- To determine the antimicrobial sensitivity of Salmonella isolates to most common used antimicrobial agents in the Sudan.

CHAPTER ONE

LITERATURE REVIEW

1.1 History of Salmonella:

Salmon and Smith were the first to isolate Salmonella from pigs in (1885) (cited by Ryan and Ray, 2004). The typhoid bacillus was first isolated in 1884, when the German microbiologist Gaffkey obtained Salmonella typhi from human spleen (Scherer and Miller, 2004). Salmonella is an important genus of the family Enterobacteriaceae. Members of the genus are Gram-negative, facultative anaerobes and inhabit the intestinal tract of man and animals. They may be recovered from a wide range of hosts such as poultry, swine, human, foods and environment. Members of the genus Salmonella may be pathogenic to wild or domestic animals and human (Holt et al., 1994). It's important pathogen to the food industry and has been frequently identified as the etiological agent of food-borne out breaks (Siqueira et al., 2003; Zhao et al., 2001). In human they cause enteritis, enteric fever and systemic infection (Brook et al., 2004).

1.2 Classification of Salmonella:

The scientific classification of salmonella was described by Hafez (2005) as follow: Domain: Bacteria, Kingdom: Monera, Phylum: Proteobacteria, Class: Gamma proteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae and Genus: Salmonella.

The members of the genus Salmonella were originally classified on the basis epidemiology, host range, biochemical reactions and structures of the O, H and Vi antigens (Brook et al., 2004).

Recent advances in Salmonella taxonomy divide the genus in to two species: Salmonella bongori and Salmonella enterica (Le Minor and Popoff, 1987). S. bongori contains less than 10 serovars while S. enterica contains more than 2500 serovars and are divided into six subspecies namely enterica, salamae, arizonae, diarizonae, houtenae and indica.

All centers of disease control and prevention recommended that Salmonella species should be named by their genus and serovar e.g. Salmonella typhi instead of Salmonella enterica subspecies enterica serovar typhi.

Most commonly, the Salmonella was classified according to serology. The division is first by the somatic (O) antigen and by the flagellar (H) antigen. (O) antigen is of lipopolysaccharide nature and (H) antigen of protein nature (Kauffmann White Scheme 1960).

The genus Salmonella can roughly be classified into three groups (Hafez and Jodas 2000). First group includes highly host adapted and invasive serovars such as S. gallinarum, S. pullorum in poultry and S. typhi in human. Second group includes non-host adapted and invasive serovars such as S. typhimurium, S. arizonae and S. enteritidis. Third group contains non-host adapted and non invasive serovars, most of these serovars are harmless for animals and human.

1.3Morphology of Salmonella: Salmonellae are Gram-negative straight rods, (0.7- 1.5 x 2.0 – 5.0), conferring the general definition of the family Entrobacteriaceae, (Collee et al., 1996), non-acid- fast, non-capsulated and non- spore forming. Most serotypes are motile with peritrichous flagella, but Gallinarum- Pullorum is non- motile; non- motile variants (OH→ O variation) are occasionally found in other serotypes. Most strains of most serotypes form type 1 fimbrae (mannose- sensitive, haemagglutinating); Gallinarum- Pullorum and a few strains in other serotypes either form type 2 fimbriae (non- haemagglutinating) or are non- immediate; most strains of S. paratyphy A are non- fimbriated (Duguid et al., 1966).

1.4 Antigenic structure:

Although the principal varieties of enteric bacilli can be identified by their reactions in differential media, final identification of many species, as well as many strains, is based on antigenic structure. However, strains with the same antigenic pattern may exhibit different metabolic reactions (fermentative variants or biotypes), three kinds of surface antigens (H, O and K or Vi), determine the organism's reaction with the specific antiserum (Davis et al., 1990).

Salmonella, like other Gram-negative bacteria, has somatic (O) antigens, which are lipopolysaccharide components of the cell wall, and flagellar (H) antigens, which are proteins (Mandel et al., 1985; Collee et al., 1996 and Brook et al., 2004).

Capsular antigens are called K Ag (German. Kapsel), and a specific capsular antigen of S. typhi is called Vi due to it's role in virulence. Fimbrial antigens previously designated as K antigens before their structure was recognized, and they now bear descriptive names (CFA i and ii: colonization factors and antigens 1 and 2) (Davis et al., 1990).

There are about 60 (O) antigens, and many H antigens, each of which is designated by number and letter (Mandel et al., 1985; Paniker and Vilma, 1997; Brooks et al., 2004).

The polysacharide Vi Ag in certain species is usually too thin to be seen as capsule. However, it does inhibit O agglutination unless it is destroyed (along with H-Ag ) by boiling for two hours .

The (O) Ags in smooth (S) strains cover the underline R Ag which becomes accessible to antibodies in rough (R) mutants. The change from S to R may take place without the loss of flagellar or Vi Ags. Rough strains tend to agglutinate spontaneously unless suspended in media of proper ionic strength (Davis et al., 1990). The smooth- to – rough variation is associated with change in colony morphology and loss of the O-Ag of virulence. The colony becomes large, rough and irregular. R forms may be common in laboratory strains maintained by serial sub-cultivation (David et al., 1989).

Mucoid colonies, associated with development of a new mucoid or M-Ag, have been described with Salmonella paratyphi B and some other species (Ananthanarayan and Paniker, 1997).

1.5 Prevalence of Salmonella:

1.5.1 Prevalence in poultry:

1.5.1.1 Isolation of Salmonella from different poultry sources:

1.5.1.1.1 Chicks:

Nicklas (1987) reported that 50 deliveries of one day- old chicks were examined for Salmonella as a part of routine microbiological monitoring. Paper floor inserts and faeces from the transport boxes were immersed in peptone water and then cultured in two different enrichment media. Salmonella was isolated from 6of the 50 samples, one isolate was identified as S. muenchen and the other 5 as S. serovar siegburg.

Al Obaidi et al., (1987) stated that in a survey over 18 months, S. neuikerk (8 isolates) and S. montevideo (6 isolates) were recovered from 30 day- old chicks. S. neuikerk was also recovered from 3 of 30, 7 day- old chicks and from 2 of 20, 30 day- old chicks. S. Virchow was recovered from one chick in each of the two later groups. Salmonella was not isolated from chicks more than 45 day- old.

Only S. java was recovered from litters in pens containing chicks 7 and 30 days old. It was concluded that antibiotics in feed increase the rate of isolation of Salmonella from these birds.

Choudhery et al., (1993) isolated Salmonella species from 9 (10.57%) infected yolk sacs of 20 chicks and 80 dead chicks on 85 farms.

1.5.1.1.2 Poultry flocks:

Desoutter (1986) reported that 20 outbreaks of salmonellosis in poultry have been observed since 1981. S. typhimurium was isolated in 50% of cases, S. London of 20%, S. muenchen in 20% and S. Arizona in 10%, while S. pullorum- gallinarum has never been isolated.

Majid et al., (1991) found that 18 (13.5%) of 133 commercial poultry flocks were affected by salmonellosis. Prevalence was higher in flocks with poor management conditions. Of 18 typed isolates, 10 were S. gallinarum or S. pullorum.

Pacini et al., (1993) reported that the incidence of different serotypes of Salmlonela isolated from faecal samples of poultry during 1984-1992 showed a decrease in frequency of S. infantis and S. typhimurium and an increase in S. enteritidis.

Menzies et al., (1994) found that the predominant serotypes in poultry during 1979- 1994 were S. typhimurium (22%) and S. entritidis (11.7%). There was no significant annual trend in avian salmonellosis, although a peak incidence occurred between 1986- 1987 caused by an increased outbreaks involving S. enteritidis and S. typhimurium.

In Sudan, Ezdihar (1996) examined 610 samples from infected chickens and reported the isolation of 14 bacterial genera which included Klebsiella, Citrobacter, Serratia, Morganella, Enterobacter, E. coli, Salmonella, Proteus, yersinia, Edwardsiella, Hafnia, Acinetobacter and Shigella.

1.5.1.1.3 Poultry feed:

Salmonella have been isolated from poultry feed stored at 25C after 16 month of storage (Williams and Benson, 1978). Survival and heat resistance of Salmonella in meat, bone meat, dry milk and poultry feed is related to moisture content and relative humidity (Carlson and Snoeyenbos, 1970). The only feed ingredient that is resistant to contamination by Salmonella is liquid animal and vegetable fat (Harris et al., 1997). Fatty acids have been shown to inhibit the growth of gram- negative bacteria (Khan, 1969).

Jardy and Michard (1992) tested samples of row poultry feed components for Salmonella. The most commonly isolated serovars were S. senftenberg, S. rissen, S. tennesee, S. landott, S. mbandaka, S. agona and S. havana.

1.5.1.1.4 Poultry carcasses and other poultry sources:

Yagoub and Mohammed (1986) determine that 58 Salmonella strains were isolated from 1488 samples from slaughtered chicks over 18 months in Khartoum North and Omdurman.

Twenty three serotypes were identified, the most common serotypes were S. monas (25.6%) and S. amek (16.3%) and of serotypes except S. uganda had previously been isolated in Sudan (Khan, 1969).

Baumgartner et al., (1992) isolated 132 Salmonella isolates from 945 broiler carcasses, 47 (36.2%) were S. infantis, 39 (30%) S. typhimurium and 25 (19.2%) S. enteritidis.

Mrdjen et al., (1992) examined 658 samples (eggs, chicks, and broilers), and isolated 93 Salmonella strains:

26 (28%) were S. enteritidis, 24 (26%) S. typhimurium, 23(25%) S. verchow, 12 (13%) S. infantis, 4 (5%) S. lindenburg, 2(3%) S. gallinarum- pullorum, 1 each of S. berdeney and S. manbattan.

Orhan and Gruler (1993) isolated Salmonella from internal organs, cloacal swabs, feed samples and eggs. These strains were identified as S. gallinarum (25) and S. enteritidis (13) while 63 strains of S. pullorum were isolated by Pan et al., (1993).

Choudhury et al., (1993) isolated Salmonella from 9 (10.6%) infected yolk sacs of 20 sick and 80 dead chicks. Also S. enteritidis phage type 4 was isolated from the reproductive tract of 10 of 37 laying hens (Hoop and Pospischil).

Pope (1994) reported that S. enteritidis was isolated from environmental samples of (2.7%) of layer flocks and (3%) of broiler flocks.

Salmonella may be isolated from most body tissues including liver, heart, gizzard and cooked chicken products (Jerng Klinchan et al., 1994), small intestine, caeca (Wieliczko, 1994), eggs shell fragments, external rinses, intestinal tracts from one day- old chicks (Bailey et al., 1994).

Enrichment broth was considered by most workers as an essential part of Salmonella isolation. Delayed secondary enrichment

10 was required to isolate Salmonella from 36(15%) of the 226 Salmonella positive chickens and to detect 20% of the total isolation.

The optimum incubation time for Salmonella enrichment cultures were obtained by inoculation of enrichment broth on to plating media after 23 hrs at 37C, after 48 hrs at 37C, after 3 days delayed secondary enrichment (DSE) and after a 5- day DSE procedure. Inoculation of the enrichment broth onto plating media after 24 hrs incubation followed by 5-day DSE, made the detection of 96-98% of Salmonella positive samples possible and was the best combination of condition (Waltman et al., 1993).

1.5.2 Prevalence in animals:

Buxton and Fraser (1977) reported that animals were affected by different serotypes of Salmonella species. The serotypes usually affecting horses are S. abortus equi and S. typhimurium. While S. Dublin and S. typhimurium are the most common causes of bovine salmonellosis which affect cattle of all ages, and the disease may be acute or chronic. Sheep and goats are affected by S. abortus ovis and S. typhimurium and other serotypes including S. dublin which have been recorded less frequent. Pigs constitute one of most important reservoirs of Salmonellae and are susceptible to the disease caused by a wide variety of serotypes. Most important of these are S. choleraesuis and S. typhimurium.

The transmission of Salmonella spp. to animal's feed was noted by Jones et al., (1982) who detected the same serotypes (S.ser. mbandaka) in both cattle and unopened bags of vegetable fat on the same farm site. However, Grimont et al., (2000) noted that the habitat

11 of Salmonella spp. is limited to the digestive tract of animals and humans, and that it's presence in other environments may be limited to faecal contamination.

Marx (1969) noted that S.enteritidis was isolated from field mice (Apodemus sylvaticus) as early as 1900. Later,Singer et al., (1992) isolated S. enteritidis again from mice (Family: muridae). Other Salmonella serovars such as S. derby and S. typhimurium were isolated from rats (Schnurrenberger et al., 1968).

In birds, Salmonella was isolated in a study from racing pigeons (Adesiyun et al., 1998) but not in wild pigeons (Nielsen and Clausen, 1975). Salmonella isolated from wild birds such as crows and gulls (Kapperud and Rosef 1983; Devis and Murray, 1991). Evidence also exists that Salmonella may survive in the intestinal tracts of insects (Everard et al., 1979). Jones et al., (1991) and Kopanic et al., (1994) suggested that insects may be vectors for the transmission of Salmonella spp.

1.5.3 Prevalence in man:

The incidence of human salmonellosis has increased greatly over the past 20 years and this can mostly be attributed to epidemics of S. enteritidis in poultry in numerous countries (Barrow et al., 2003; Guard- Petter, 2001). The association between egg consumption and S. enteritidis outbreaks is a serious international economic and public health problem (Center of Disease Control, 2000 and 2003; Guard Petter, 2001; Patrick et al., 2004).

Transmission to hen may originate from contaminated food or water or by contact with wild animals. But the main concern with this bacterium is the existence of silence carriers. These animals can, in turn, transmit the bacterium to their flock- mates through horizontal transmission or to their off- spring by vertical transmission. However, they are difficult to be distinguished from healthy animals, thus are responsible for transmission to human.

Zhao et al., (2001) and Siqueira et al., (2003) reported the occurrence of 1.4 million cases of human salmonellosis in United States of America.

The transmission of Salmonella is usually associated with consumption of contaminated food (Soumt et al., 1999). However, a great number of outbreaks might be associated with contaminated water, which is known to be an important transmission route (Fertado et al., 1998).

Mead et al., (1999) and Murray (2000) stated that 95% of salmonellosis cases originate from food materials.

In a comparative study in England, Humphrey (2000) reported that 30% to 80% Salmonella spp. were isolated from alfalfa seeds, chocolate, cheddar cheese, red meat, salad, milk and vegetables (Inami and Moler, 1999; Humphrey 2000).

Kotova et al., (1988) conducted study on human developing the Salmonella carrier state (S. enteritidis and S. dublin) after acute salmonellosis and as a result of occupational exposure to poultry. The Salmonella species most frequently isolated from poultry employees

13 was S. typhimurium, while S. newport, S. enteritidis and S. Dublin were also isolated.

In Sudan, Seed Ahmed, (2007) conducted study on Salmonella carrier state among human, he has tested 5493 stool samples taken from food handlers working in food service in Khartoum, only S. paratyphi B was isolated from 17 samples.

1.6 Pathogenicity of Salmonella:

The pathogenesis of Salmonella has been extensively studied in the mouse (Haraga et al., 2008). In susceptible mice, Salmonella causes an acute systemic disease with limited intestinal manifestations (Santos et al, 2001). Recently, a model of Salmonella enterocolitis has been developed in streptomycin-treated mice (Barthel et al., 2003).

Studies using these mice and other animal models of Salmonella diseases have yielded substantial data about the molecular players involved at different levels. The Salmonella pathogenicity islands (SPIs) I and ІI are two major virulence determinants of S. enterica. They encode type III secretion systems (T3SS) that form syringe-like organelles on the surface of gram-negative bacteria and enable the injection of effector proteins directly into the cytosol of eukaryotic cells (Galan, 2001; Waterman and Holden, 2003). These effectors ultimately manipulate the cellular functions of the infected host and facilitate the progression of the infection. SPI (I) and SPI (II) play several roles in different organs within the host. SPI (I) primarily promotes the invasion of non-phagocytic intestinal epithelial cells and the initiation of the inflammatory responses in the intestines (Coombes et al., 2005; Hapfelmeier et al., 2004). It is also involved in

14 the survival and persistence of Salmonella in the systemic compartment of the host (Brawn et al., 2007; Steele-Mortimer et al., 2002).

The first characterized role of SPI (II) was its ability to promote Salmonella survival and multiplication in phagocytic cells that constitute the main reservoirs for dissemination of the bacteria into systemic organs (Waterman and Holden, 2003). SPI (II) also plays an important role in the intestinal phase of Salmonella infection in mice (Coombes et al., 2005; Cobum et al., 2005; Hapfelmeier et al., 2005).

The regulation of SPI (I) and SPI (II) gene expression involves numerous transcriptional regulators located both inside and outside these pathogenicity islands. The regulation of SPI (I) is particularly complex. SPI (I) encodes for the five regulators HilA, HilC, HilD, InvF, and SprB. The first four of which are involved in regulatory pathways that lead to the activation of SPI (I) genes and of genes encoding T3SS effectors located outside SPI (I). In contrast to SPI (I) the regulation of SPI (II) genes is simpler with the SsrAB two- component system being the only transcriptional regulator encoded within SPI (II) that activates the expression of SPI (II) genes and of genes encoding T3SS effectors located outside SPI (II). Interestingly, SPI (I) regulators can regulate SPI (II) genes. These include HilA that binds and represses the promoter of ssaH (Thijs et al., 2007), and HilD that binds and activates the promoter of the ssrAB operon (Bustamante et al, 2008). In contrast, SsrAB has never been shown to act on the expression of SPI (I) genes.

Few studies have investigated the role of SPI (1) and SPI (II) during the infection of chickens. In studies using Typhimurium, two approaches have provided data about the roles of SPI (I) and SPI (II). The first approach compared colonization in chickens by infecting with single strains and enumerating colonies from internal organs. Porter and Curtiss (Porter et al., 1997) found that mutations in structural genes of the SPI (I) T3SS resulted in a reduction of the colonization of the intestines in day-old chickens. Jones et al., (2007) generated strains with deletions of spaS and ssaU, genes that encode structural proteins of the SPI (I) and SPI (II) T3SS respectively, and compared their ability to colonize the cecum and liver in one-day and one-week old chickens to that of wild type. They concluded that both SPI (I) and SPI (II) play major roles in both the intestinal and the systemic compartments, with SPI (II) contributing more than SPI (I) in both compartments. The second approach screened random transposon libraries for reduced recovery from the chicken gastrointestinal tract through cloacal swabbing. Turner et al., (1998) analyzed a library of 2,800 mutants for intestinal colonization in chickens. Among the mutants that showed reduced intestinal colonization they found one in which the SPI (I) gene sipC was inactivated. No mutations in SPI (II) genes were identified in this screen.

Morgan et al., (2004) screened a library of 1,045 mutants in chickens and found two mutations in SPI (I) genes and one in a SPI (II) gene that led to a reduction in colonization ability. The SPI (I) mutants were unable to be recovered from 50% or 100% of the day old birds tested, while the single SPI (II) gene was unable to be

16 recovered in only 33%. In this study fourteen strains with mutations in SPI (I) and fifteen strains with mutations in SPI (II) did not show any defect in colonization. The authors of these two studies concluded that SPI (I) and SPI (II) play a marginal role in the colonization of chicken intestines by S.typhimurium.

Dieye et al., (2009) reported that SPI (I) contributes to the colonization of both the cecum and spleen of the chicken. In contrast, SPI (II) contributes to colonization of the spleen but not the cecum and, in the absence of SPI (1) inhibits cecal colonization. Additionally, they show that the contribution of SPI (I) in the spleen is greater than that of SPI (II). These results differ from those observed during the infection of mice by Typhimurium, where SPI (II) plays a major role during systemic colonization.

1.7 Laboratory diagnosis:

1.7.1 Isolation of Salmonella:

1.7.1.1 Cultural characteristics:

Salmonella are facultative anaerobic. The optimum growth temperature is 37ºC, but some growth is observed in a range from about 5- 45ºC. Salmonella can grow within a pH range of approximately 4.0 to 9.0, with an optimum pH around 7.0 (Cruickshank 1972).

1.7.1.1.1 Enrichment media:

These are liquid media used to assist isolation of Salmonellae from feces, sewage, food stuffs and other materials containing a mixed bacterial flora. A larger amount of the material can be inoculated into an enrichment medium than on to an agar plate, so facilitating the isolation of Salmonellae when these are present only in small numbers. During incubation any Salmonellae multiply rapidly, while E. coli and most other bacteria are inhibited. After 18- 24 hours the enriched culture is plated onto a differential agar medium e.g. Desoxycholate- Citrate Agar (DCA) or Xylose Lysine Desoxycholate (XLD), on which the production of Salmonella- like colonies may be observed (Barrow and Feltham, 1993). a- Selenite broth:

Selenite broth is an enrichment broth medium used for the isolation of Salmonella and Shigella species. Casein and meat peptones provide nutrients. Selenite inhibits enterococci and coliform those are part of the normal flora if they are subcultuered within 12 to 18 hrs. However, reduction of selenite produces an alkaline condition that may inhibit the recovery of Salmonella. Lactose and phosphate buffers are added to allow stability of the pH. When fermenting organisms produce acid, the acid neutralizes the effect of the selenite reduction and subsequent alkalinization. Cystine added to selenite broth enhances the recovery of Salmonella (Murray et al., 2005).

1.7.1.1.2 Differential and selective solid media:

These media are valuable for the isolation of Salmonellae from feces and other materials contaminated with many bacteria of other kinds. They include: a- Mc Conkey Bile- Salt lactose Agar:

After 18 to 24 hrs the colonies are pale yellow or nearly colourless, 1-3 mm in diameter, and easily distinguished from the pink- red colonies of lactose fermenting commensal coliform bacilli, e.g. Escherichia coli which also grow well on this unselective differential (indicator) medium (Koneman et al.,1997). b- Brilliant Green Mc Conkey Agar:

The addition to Mac Conkey agar of brilliant green 0.004 g\liter, which is inhibitory to E. coli, Proteus species and the other commensal enterobacteria likely to out number the Salmonellae in faeces, makes this an excellent selective aswell as differential medium for Salmonellae except S. typhi which is not grow well on it.

Salmonellae appear as low convex, pale- green translucent colonies 1-3 mm in diameter. Lactose fermenting bacteria, including rare strains of Salmonella serotypes, produce blue- purple colonies (Murry et al., 2005).

19 c- Leifson,s Deoxycholate- Citrate Agar (DCA):

The colonies of Salmonellae on DCA are similar to or slightly smaller in size than those on Mac Conkey agar. They are pale, nearly colorless, smooth, shiny and translucent. Sometimes they have a black center and sometimes a zone of cleared medium surrounds them, but these characters may required 48 hrs of incubation for their development. Salmonellae are easily distinguished from the opaque pink colonies of lactose- fermenting coliform bacilli, which are largely inhibited on this selective differential medium (Cheesbrough, 2000). d- Wilson and Blair's Brilliant- green Bismuth Sulphite Agar (BBSA):

This medium is particularly valuable for the isolation of S. typhi. Cultures should be examined after 24 hrs, then after 48 hrs. Crowded colonies about 1 mm diameter may appear green or pale- brown. Larger discrete colonies have a black center and clear edge (Barrow and Feltham, 1993). e- Taylor's Xylose Lysine Deoxycholate (XLD):

It is a popular medium for the primary plating of faeces from suspected Salmonella and Shigella infection. It gives colony appearances that distinguish Salmonellae from Shigellae, and these pathogens from from the many non- lactose fermenting strains of non- pathogenic enterobacteria which form pale colonies similar to theirs on Mac Conkey and DCA. Colonies of Salmonellae and Shigellae are red (alkaline to phenol red) because Shigellae do not form acid from xylose, lactose and sucrose in the medium within 24 hrs and because

Salmonellae neutralize the acid they form from the limited amount of xylose by decarboxylating the lysine. Most Salmonellae (and Edwardsiellae) are distinguished from the Shigellae because they produce hydrogen sulphide, which reacts with ferric ammonium citrate in the medium to produce black centers in their red colonies (Cheesbrough, 2000). f- Rambach,s Agar:

This recently described medium (1990), which contains propylene glycol (PG) and a novel chromogenic substrate (Merk) to detect β- glactosidase activity, is claimed to allow detection of 98% of ,customary, Salmonellae. Non- typhoidal Salmonellae (β- glactosidase- negative) form acid from the metabolism of PG and, with suitable pH indicator, grow as red colonies. (Dusch and Altwegg, 1995). g- Salmonella- Shigella Agar:

It is a selective and differential medium used for the isolation and differentiation of Salmonella and Shigella from clinical specimens and other sources. The nutritive base contains animal and casein peptones and beef extract. The selective agents are bile salts, citrates, and brilliant green dye, which inhibit gram- positive organisms.

The high degree of selectivity of the medium results in the inhibition of some strains of Shigella, and the medium is not recommended as a primary medium for isolation of this species. The medium contains only lactose and thus differentiates organisms on the basis of lactose fermentation. The formation of acid on fermentation

21 of lactose causes the neutral red indicator to make red colonies. Non- lactose fermenting organisms are clear on the medium. As with Hektoen enteric agar, sodium thiosulfate and ammonium citrate allow the differentiation of organisms that produce hydrogen sulphide. Lactose fermenters, such as E. coli, have colonies, which are pink with a precipitate; Shigella appears transparent or amber with black centers. Some strains of Shigella dysenteriae are inhibited (Murray et al., 2005).

1.7.1.2 Biochemical reactions:

All serotypes of the genus Salmonella and those of the former genus 'Arizona' are now considered to belong to one species for which the name Salmonella enterica has been proposed, it comprises species which, historically, have been numbered but now are named (Le Minor, 1985).

Carbohydrates are generally fermented with the production of acid and gas. S. typhi, S. gallinarum and rare anaerogenic variants in other serotypes, e.g. S. typhimurium, form only acid. Typically, glucose, mannitol, arabinose, maltose, dulcitol and sorbitol are fermented, but not lactose, sucrose, salicin or adonitol; the ONPG test for β- glactosidase is negative. Among exceptional strains, choleraesuis and some strains of S. typhi do not ferment arabinose (Duguid et al., 1975.).

Many strains of Salmonella arizonae ferment lactose rapidly or slowly as well as having activity to β- glactosidase enzyme (Holt et al., 1994).

Salmonella decarboxylate the amino acids lysine, ornithine and arginine, but not glutamic acid. S. typhi is exceptional in lacking ornithine decarboxylase and S. paratyphi in lacking lysine decarboxylase (Collee et al., 1996).

Most Salmonellae have the following reactions: indole not produced. Methyl- red positive. Acetyl- methyl carbinol not produced (e.g. Voges- proskauer negative). Citrate utilized except by S. typhy and S. paratyphy A. Malonate not utilized. Gluconate not utilized. Urease not produced. Phenylalanine deaminase not produced. Hydrogen sulphide produced in ferrous chloride gelatin medium, except by S. paratyphi A, S. choleraesuis, S. typhisuis and S. sendai. No growth in KCN medium. Gelatin not liquefied.

1.7.2 Serological tests:

These are satisfactory for establishing the presence and estimating the prevalence of the infection within a flock. The tests that are readily applied include tube agglutination (TA) test, rapid serum agglutination (RSA) test, stained antigen whole blood (WB) test and micro-agglutination (MA) test (Gast, 1997). Other serological tests include micro-antiglobulin (Coombs), immunodifusion, haemagglutination and enzyme-linked immunosorbent assay (ELISA).

The rapid serum agglutination test can be used under field condition and the reaction can be identified immediately. Chickens can be tested at any age, although some authorities specify a minimum age of 4 months (Wray, 2000).

1.8 Drug susceptibility:

An increase of Salmonella strains showing resistance and multiple resistances against different antibiotics have been found from isolates from poultry in recent years. Kheir El-Din et al, (1987) examined in vitro the sensitivity of 89 isolates of S. gallinarum, S. pullorum, S. virchow and S. newport against 11 antibiotics. The result reveal that 70- 80% of the isolates were sensitive to flumaquine and chloramphenicol, and that 38- 57% were moderately sensitive to nitrofurantoin, ampicillin and neomycin and only 15- 18% were weakly sensitive to lincomycin and streptomycin, but completely resistant to erythromycin, penicillin, tetracycline and trimethoprim.

Bolinski et al., (1994) reported that flumaquin inhibited 86% of Salmonella strains, followed by apramycin, ampicillin, oxitetracycline and gentamycin. The minimum inhibitory concentration of flumaquine varied between 1.0 and 5.0 µg /ml. On the other hand, Ghosh, (1988) found that 36 strains of S. virchow were highly sensitive to gentamycin, streptomycin and kanamycin but resistant to bacitracin, penicillin, sulphaphenazole and tetracycline.

Lee et al., (1993) determined that 57% of 105 Salmonella isolates were resistant to one or more antimicrobial agents and 45% were resistant to two or more agents. Highest resistance was to tetracycline 45%, streptomycin 41%, sulfisoxazole 19% and gentamycin 10%.

Jacobs et al, (1994) reported that 7.5% of 94 Salmonella isolates were resistant to nalidixic acid and fumaquine but did not to ciprofloxacin.

Roliniski et al, (1994) determined that 52.98% of S. enteritidis and S. typhimurium were resistant to nitrofurans, oxytetracycline, sulphonamides alone and with trimthoprim. The similar levels of resistance (49.84%) were shown by S. gallinarum isolates to oxytetracycline and sulphonamides alone and with trimethoprim and only 8% were resistant to nitrofurans.

Esaki et al., (2004) isolated 94 Salmonella strains of 10 serotypes from different poultry farms (broiler and lying hens). 39 of them were resistant to flumaquine, nalidixic acid and oxolinic acid. All strains were sensitive to ciprofloxacin. The most frequent serotypes were S. enteritidis and S. heidelberg.

Currently, S. typhi isolates resistant six different antimicrobial agents prevail in highly endemic typhoid areas, particularly China, Pakistan and India. These strains of S. typhi carry a 120- kb plasmid that encodes resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, tetracycline and trimethoprim. In addition, S. typhi strains isolated from recent outbreaks in Tadjikistan and Pakistan have also acquired resistance to ciprofloxacin (a fluoroquinolone), one of the preferred antibiotics for treatment of typhoid fever (Hampton et al., 1998).

Resistance of non- typhoidal Salmonellae is also a growing health problem. Particularly troubling is the penta- resistant strains of S. typhimurium known as DT104 (Definitive phage type 104), which emerged in Great Britain in 1984 and was reported in 1997 to have been isolated in the United States of America (CDC, 1997). This strain has been isolated from numerous species of animals and is

25 resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline (R type ACSSuT). In addition, there have been reports of resistance to two other antibiotics, trimethoprim and fluoroquinolones, in Great Britain (Threlfall et al., 1996).

Fluoroquinolones are the drugs of choice for an invasive extraintestinal infection in adults, whereas extended-spectrum cephalosporins (ESC) are preferentially used to treat salmonellosis in children (Fey et al., 2000). However, the use of these drugs is becoming compromised by the increasing development of the ESC and quinolone resistance all over the world.Treatment failures due to in vivo acquisition of an extended spectrum β-lactamase (ESBL) gene (Su et al., 2003) or a reduced susceptibility to ciprofloxacin (Aarestrup et al., 2003) in Salmonella are now well established.

Salmonella strains resistant to ESC have been reported since the late 1980s, and their numbers have increased ever since. Two major resistance mechanisms have been identified in Salmonella. In the first, the isolates express plasmid-mediated AmpC-like β-lactamases that hydrolyze the cephamycins, extended-spectrum cephalosporins, and monobactams. In the second mechanism, the Salmonella strains express an ESBL that is able to hydrolyze monobactams and oxyimino cephalosporins (such as cefotaxime and ceftazidime) but not the cephamycins (Miragou et al., 2004).

1.9 Control and treatment:

Reddy et al, (1987) determined synergy between sulphadiazine and trimethoprim against S. gallinarum. The minimum inhibitory concentrations (MIC) of the two drugs used separately were 15.2 and

0.8 µg\ml respectively, and when combined they were 1.9 and 0.1 µg\ml. Trimethoprim plus sulphadiazine added to the drinking water at 60+300 mg\L for 7 days reduced liver and spleen lesions and the number of bacterial isolates from chicks experimentally infected with 2×109 S. gallinarum.

Humphrey and Laning (1988) evidenced that treatment of feed given to lying hens with 0.5% formic acid significantly reduced the isolation rate of Salmonella and was associated with reduction in the incidence of infection in newly- hatched chicks. These improvements were not sustained until slaughtered, as growing birds acquired Salmonella, probably from feed which was not acid treated. These finding indicate that formic acid treatment of chicken food could have important benefits for the public health.

The administration of injectable antibiotics such as gentamycin in the hatchery played a pivotal role in controlling the spread of S. arizonae in turkey pouts (Shivaprasad et al., 1998). Antibiotics have been used to control S. enteritidis infection in several experimental and commercial contexts. Treatment of chicks with polymyxin B sulphate and trimethoprim both prevent and cleared experimental infection (Goodnough and Johnson, 1991). Administration of flavophospholipol or salinomycin sodium as feed additives reduced faecal shedding (Bolder et al, 1999). Provision of a competitive exclusion culture to restore a protective normal microflora after treatment with enrofloxacin reduced the isolation of S. enteritidis from broiler breeders and their environment (Reynolds et al., 1997).

Mcllory et al., (1989) reported that antibiotics were used effectively both as therapeutic and prophylactic agents as a part of control efforts for S. enteritidis in broiler and broiler breeder's flocks in Northern Ireland.

To control this zoonosis, a number of prophylactic means have been developed. Vaccination has a general effect and may reduce animal contamination and rate of excretion of the bacterium through faeces (Zhang-Barber et al., 1999). Other methods aimed to reduce the introduction of the bacterium into the gut, which is based on the on the early implementation of an adult- type intestinal flora which compete with S. entritidis (Rabsch et al., 2000) or acidification of feed which deters bacterial growth. Genetic methods may also be successful in increasing resistance to systemic disease or carrier state (Beaumonal et al., 1999), thus reducing the need for antibiotic treatments and risk of antibiotic resistance.

1.10 Salmonella vaccine:

Worldwide, salmonellosis is a serious medical and veterinary problem and raises great concern in the food industry. Vaccination is potentially an effective tool for the prevention of salmonellosis. Whole-cell killed vaccines and subunit vaccines were used with variable results for the prevention of Salmonella infection in humans and animals (Mastroeni et al., 2001). Recent advances in the genetics of Salmonella led to the development of attenuated Salmonella vaccine strains with single or multiple defined mutations in the bacterial genome (Mastroeni et al., 2001). Live attenuated Salmonella vaccines were also used successfully as carriers for the delivery of

28 heterologous antigens to the immune system. Flagellin, the major structural protein of flagella, is used for the serotyping Salmonella. Purified flagellin induces a high systemic, humoral and mucosal immune response in C3H/HeJ mice (Strindelius et al., 2004).

While mice immunized intraperitoneally with flagellin show a strong systemic (immunoglobulin G IgG) response against flagellin, mice immunized mucosally with the strain did not (Strindelius et al.,

2004).

Nevertheless, flagella elicit a strong immune response in chickens (Mazumoto et al., 2004; Van Zijdirveld et al., 1992) and are useful serological markers that carry the serotype-specific H-antigenic determinants (Kauffmann, 1964) in the central variable domain of the protein (Van Asten et al., 1995). Therefore, deletion of fliC, the gene that codes for flagellin (FliC) should allow serological differentiation between animals immunized with the _fliC vaccine strain and animals infected by wild-type S. enterica serovar Enteritidis. Specific antibodies against S. enterica serovar Enteritidis FliC were detected in sera of spray-inoculated young chickens but not in sera of young chickens inoculated orally with S. enteric serovar Enteritidis (Mazumoto et al., 2004).

Chapter two

Materials and Methods

2.1 Sampling:

2.1.1. Source of specimens:

The specimens were taken from poultry farms (layers and broilers) in Khartoum North area. Samples (feed, litter and drinking water) were selected from six poultry farms (three broiler farms and three layer farms) during the period between August to November 2009.

Sites in the area from which samples were collected include (Table 1):

- Two farms in Shambat area.

- Animal production research center, Kuku (broilers and layers).

- A farm in Al-Halfaya area (layers).

- A farm in Al-Zakyab (broilers).

2.1.2. Collection of specimens:

A total of eighty samples were collected. These samples comprise 27 feed samples, 27 litter samples and 26 samples of drinking water (table 1):

2.1.2.1. Feed samples:

A mount of 10 grams feed was taken for every sample from poultry feeders. Every sample taken from 5 different feeders in the house. Then samples placed in sterile containers. Sterile spoons were used for sample collection.

2.1.2.2. Litter samples:

Litter samples were collected in a mount of 10 grams for every sample and then placed in sterile containers. Sterile spoons were used for sample collection.

Table (1): Origin, type and number of samples collected in the study

No. and type of samples examined source feed litter water

Amr farm, Shambat 4 4 4 (broilers) Dr. Wafa farm, Shambat (layer) 5 5 5

Animal production research center, 10 10 10 Kuku (tow farms, broilers and layers) Dr. Haytham farm, AlHalfaya 3 4 3 (layer) Dr. Jalal farm, AlZakyab 5 4 5 (broilers)

2.1.2.3. Drinking water samples:

30 ml of water samples were collected from drinkers. Every sample taken from 5 different drinkers in the house. Then samples placed in sterile containers.

2.1.3. Transport and storage of samples:

All samples were placed on ice in a thermos flask immediately after collection and transported to the laboratory of bacteriology in Department of Microbiology (Faculty of Vet. Medicine) and kept at 4º C.

2.2. Bacteriological investigation:

2.2.1. Culture media:

2.2.1.1. Liquid media: a. Peptone water (Oxoid Ltd, England):

This medium was used as base of carbohydrates utilization tests and for other purposes. It was composed of 10 grams peptone, 5 grams sodium chloride. It was prepared by dissolving 15 grams of powder in 1 liter distilled water, the pH was adjusted to 7.4, then mixed well and distributed into test tubes 5 ml each and sterilized by autoclaving at 121º C for 15 minutes, then stored in the refrigerator at 4º C until used. b. Nutrient broth (Oxoid Ltd, England):

It was composed of 1.0 gram of lab-lemco powder, 2 grams yeast extract, 5.0 grams peptone and 5.0 grams sodium chloride. It

33 was prepared by adding 13 grams to 1 liter DW, the pH was adjusted to 7.4, then mixed well and distributed in 3 ml amounts into bijou bottles and sterilized by autoclaving at 121º C for 15 minutes, then stored in the refrigerator at 4º C until used. c. Selenite-F-broth (Oxoid Ltd, England):

According to manufacturer, the medium was prepared by dissolving 5.0 grams peptone, 4.0 grams mannitol, 10 grams disodium hydrogen phosphate and 4.0 grams sodium hydrogen Selenite in one liter of distilled water, the pH was adjusted to 7.0 and sterilized by steaming for 20 minutes, mixed well and dispensed into sterile containers. d. Methyl Red-Voges Proskauer medium (MR-VP) (Oxoid Ltd, England):

This medium contains (grams per liter) peptone P (Oxoid L49) 5 grams, dextrose 5 grams and phosphate buffer 5 grams.

It was prepared by adding 15 gram of powder to 1 liter of DW, mixed well, the pH adjusted into 7.5, distributed into test tubes in 5ml amount and sterilized by autoclaving at 121º C for 15 minutes. e. Peptone water sugars:

This medium composed of peptone water and different sugars. The pH of the peptone water (900 ml) was adjusted to 7.1-7.3 before 10 ml of Andrade’s indicator added, then 100 ml of 10% sugar solution (glucose or sucrose or mannitol) were added to the mixture, mixed well and distributed in 2 ml amounts into sterile test tubes

34 containing inverted Durham’s tube, then sterilized by steaming for 30 minutes and stored in the refrigerator at 4º C until used.

2.2.1.2 Semi- solid media: a. Hugh and Liefson’s (O\F) medium:

This media contain peptone, NaCl, K2Hpo4, agar and bromothyonol blue as an indicator. It was prepared according to Cowan and Steel (1995) by adding the solids in 1 liter DW and boiled to dissolve completely. The pH adjusted to 7.1 and the medium was filtered then the indicator was added followed by sterilization at 115º C for 20 minutes. Sterile glucose solution was then added to give final concentration of 1%, mixed and distributed aseptically in 10 ml volumes into sterile test tubes of not more than 16mm diameter. b. Motility media (Oxoid Ltd, England):

This media prepared by adding 13 grams of nutrient broth 7.5 grams of Oxoid agar No. 1 and dissolved in one liter of distilled water by heating to 100º C. The pH was adjusted to 7.2 and poured into test tubes (U shape). The tubes were sterilized by autoclaving at 121º C for 15 minutes, then the media were cooled to use for motility test.

2.2.1.3. Solid media: a. Nutrient agar (Oxoid Ltd, England):

It consists of (grams per liter) lab-lemco powder 1.0 gram, yeast extract 2 grams, peptone 5 grams, sodium chloride 5 grams and agar 15 grams.

28 grams of medium were added to 1 liter of distilled water and boiled to dissolve completely, the pH was adjusted to 7.4, and then the medium was sterilized by autoclaving at 121º C for 15 minutes and distributed aseptically in 15 ml amounts into sterile Petri dishes. Nutrient agar slops were also prepared and stored in refrigerator at 4º C until used. b. Triple sugar Iron Agar medium (TSI) (Oxoid):

It contains (grams per liter) Lab-Lemco powder (Oxiod L29) 3 grams, yeast extract (Oxoid L20) 3 grams, peptone (Oxoid L37) 20 grams, sodium chloride 5 grams, lactose 10 grams, sucrose 10 grams, dextrose 1 gram, ferric citrate 0.3, sodium thiosulfate 0.3, phenol red 0.025 gram and agar No. 3 (Oxoid L13) 12 grams.

Triple sugar iron agar was prepared by adding 65 gram of powder to 1 liter of DW, the pH adjusted into 7.4, then boiled to dissolve completely, mixed well, distributed in 5 ml amount into McCarteny bottles and sterilized by autoclaving at 121º C for 15 minutes. The medium was allowed to set in a slope position about one inch butt and stored at 4º C. c. Desoxycholate Citrate Agar (DCA) (Oxoid Ltd, England ):

This medium contains (grams per liter) Lab-lemco powder (Oxoid L29) 5 grams, peptone (Oxoid L37) 5 grams, lactose 10 grams, sodium citrate 8.5 grams, sodium thiosulfate 5.4 grams, ferric citrate 1 gram, sodium desoxycholate 5 grams, neutral red 0.02 gram and agar No. 3 (Oxoid L13) 12 grams.

It was prepared by suspending 52 gram of powder in 1 liter of DW, the pH adjusted into 7.3, then boiled over flame to dissolve completely, agitated to prevent charring, and dispensed into sterile petri-dishes in portions of 15ml and stored at 4º C. d. Christensen’s Urea Agar (Oxoid Ltd, England):

The medium was composed of (grams per liter) peptone 1.0 gram, dextrose 1.0 gram, sodium chloride 5.0 grams, disodium phosphate 1.2 grams, potassium dihydrogen phosphate 0.8 gram, phenol red 0.012 gram and agar 15 grams. According to the manufacturer instructions, 2.4 grams of dehydrated medium were dissolved in 95 ml of distilled water by boiling, pH was adjusted to 6.8, sterilized by autoclaving at 115º C for 20 minutes, then cooled to 50º C and aseptically 5 ml of sterile 40% urea solution were added. The medium was poured into sterile screw-capped bottles 10 ml each, and then allowed to set in the slope position. e. Simmon’s Citrate Agar (Oxoid Ltd, England):

It consist of (grams per liter) 0.2 gram of magnesium sulphate, ammonium dihydrogen phosphate 0.2 gram, sodium ammonium phosphate 1.0 gram, sodium citrate 2.0 grams, sodium chloride 5 grams, bromo-thymol blue 0.08 gram and agar 15 grams. 23 grams of dehydrated Simmon’s citrate agar were suspended in one liter of distilled water, boiled to dissolved completely, the pH was adjusted to 7.0 and sterilized by autoclaving at 121ºC for 15 minutes. It was then poured into sterile screw-capped bottles and allowed to set in the slope position.

37 f. Mueller and Hinton Agar (Oxoid Ltd, England):

This medium used for cultivation of Niesseria and antimicrobial susceptibility testing. It contains of (grams per liter) beef infusion from 300 grams, casein hydrolysate 17.5 grams and agar No 1 10.0 grams, and pH adjusted into 7.4.

35 grams of powder were suspended in 1 liter of distilled water, boiled to dissolved completely, then sterilized by autoclaving at 121ºC for 15 minutes.

2.2.2. Solutions and Reagents:

2.2.2.1. Normal saline solution:

This was prepared by dissolving 8.5 gram of sodium chloride in 1 liter of DW (Cowan and Steel, 1985).

2.2.2.2. Methyle Red solution:

This solution was prepared by dissolving 0.04 gram of methyl red powder in 10 ml ethanol and diluted with distilled water to 100 ml (Barrow and Feltham, 1993).

2.2.2.3. Kovac’s reagent:

This reagent was prepared for indol test. 5 gram of p-dimethyl aminobenzaldehyde was dissolved in 75 ml of amylalcohol by warming in water bath (50-55c), and then cooled and 25 ml of HCl was added. It was protected from light and stored at 4º C (Barrow and Feltham, 1993).

2.2.2.4. Oxidase test reagent:

This reagent was prepared by dissolving 0.1 gram tetramethyl- p-phenylene diamine dihydrochloride in 10 ml distilled water (Barrow and Feltham, 1993). It’s prepared immediately before use because it easily oxidized.

2.2.2.5. Potassium Hydroxide solution:

This was prepared by dissolving 40 gram of pure potassium hydroxide in 100 ml DW.

2.2.2.6. Andrade’s Indicator:

This was prepared according to Baker and Silverton (1980) by dissolving 5 grams of acid fuchsin powder in 1 liter of DW, and then 150 ml of NaOH was added to the solution mixed and allowed to stand at room temperature for 24 hours.

2.2.2.7 Voges- Proskauer (V.P) test reagent:

This reagent was prepared by mixing 40% potassium hydroxide (KOH) with 5% alph-naphthol in absolute ethanol.

2.2.2.8 Lead acetate paper:

It was prepared from a filter paper cut into strips of 5-10 mm wide and 50-60 mm long and impregnated with the hot saturated lead acetate solution, dried at 50-60ºC and stored at screw-capped containers. It was used for detection of H2S production.

2.2.2.9 Bromothymol blue:

It was used for citrate medium and (OF) medium. A total of 0.2 gram of the powder was dissolved in 100 ml of distilled water.

2.2.2.10 Phenol red:

It was used for urea agar base medium as 0.2%.

2.2.3. Sterilization procedures:

2.2.3.1. Hot air oven:

Glassware (flasks, test tube, pipettes and petri dishes) and metal instruments (scissors and forceps) were sterilized in hot air oven at 160º C for 2 hours.

2.2.3.2. Autoclaving:

Culture media and discarded cultures were sterilized by autoclaving at 121ºC for 20 minutes while glassware with plastic covers was autoclaved at 121ºC for 15 minutes.

2.2.3.3. Disinfectants and antiseptics:

70% alcohol was used to disinfect the surfaces of benches before and after use.

2.2.3.4. U. V. light:

It was used to sterilize the vacuum of media pouring room and laminar-flow cabinets.

2.2.4. Cultivation of samples:

2.2.4.1. Inoculation of enrichment medium: a. Feed samples:

10 grams of feed sample was inoculated into medical bottle containing 100 ml of selenite-f-broth and then incubated aerobically at 37ºC for 24 hours. b. Litter samples:

10 grams of litter was inoculated into medical bottle containing 100 ml of selenite-f-broth and then incubated aerobically at 37ºC for 24 hours. c. Water samples:

30 ml of water samples were centrifuged (5000 rounds per minute for 5 minutes), 1 ml of sediment was inoculated into test tube containing selenite-f- broth and then incubated aerobically at 4º C for 24 hours.

2.2.4.2. Inoculation of plates:

A loop of the inoculated selenite-f-broth was streaked on a plate of deoxycholate citrate agar and incubated aerobically at 37º C for 24 hours.

2.2.4.3. Purification and storage of isolates:

Non- lactose fermenter colonies were purified by repeated subculture on nutrient agar. Pure isolates were stored on nutrient agar slopes in the refrigerator at 4º C.

2.2.5. Identification of the isolated bacteria:

Identification of purified isolates was performed according to Cowan and Steel (1985).

2.2.5.1. Microscopic Examination: a. Gram’s stain:

Smears were prepared from the culture by emulsifying a part of a colony in a drop of normal saline on a glass slide, dried and fixed by heating. Then the slides were flooded by crystal violet for 1 minute and then washed with tap water. Iodine solution was applied for 1 minute, and then the slide was washed with tap water. The smear was then decolorized with few drops of acetone for seconds and washed immediately with water. Then the smear was flooded with diluted carbol fuchsin for 30 seconds and washed with tap water. Slides were then blotted with filter paper and examined under oil immersion lens. Gram-positive bacterial cells appeared violet in color while that of gram-negative bacteria appeared red.

2.2.5.2. Biochemical tests for identification of bacteria:

2.2.5.2.1 Primary biochemical tests: a. Oxidase Test:

The test was carried out according to Cruickshank (1972). Strips of filter paper were soaked in 10% solution of tetramethyle -p- phenylene diamine dihydrochloride in a petri dish and then left to dry. Then a fresh young test culture, on nutrient agar, was picked up with a sterile glass rod and streaked on that filter paper. A dark purple color that developed within five to ten seconds was considered positive reaction. b. Catalase Test:

Catalase test was carried out according to Cowan and Steel (1985). A drop of 3% aqueous solution of hydrogen peroxide was placed on a clean microscope slide. A colony of test culture, on nutrient agar was then placed on the hydrogen peroxide drop. The test was considered positive when gas bubbles appeared on the surface of the culture material. c. Glucose utilization Test:

The sugar media were inoculated with the test organism and incubated at 37ºC over night. They were examined daily for 7 days. Acid production was indicated by the development of pink color in the medium, Gas production was indicated by air trapped in the Durham’s tube.

43 d. Oxidation-Fermentation (O/F) Test:

The test was made by growing the test culture in tow tubes of Hugh and Lifeson’s medium. A layer of soft paraffin was added to one tube to a depth of about 1 cm. Both tubes were incubated at 37º C and examined daily. Oxidizer organisms showed acid production in the upper part of medium in the paraffin-covered tube and at the bottom in the open tube. e. Motility (Oxoid Ltd, England):

Motility medium (Semi-solid medium in U- shape tube) was inoculated at the top of one end of the tube with tested organism and incubated at 37ºC for about 4 days. Positive test was indicated by presence of growth in the other sides of the tube.

2.2.5.2.2 Secondary biochemical tests: a. Urease Test:

Suspected Salmonella colonies were streaked on urea agar slope, incubated 37º C for 2 days. A positive reaction was indicated by a change of color to pink. b. Indole Test:

The test culture was inoculated into peptone water medium and incubated at 37º C for 48 hours. 1 ml of Kovacs’s reagent was run down to the side of the tube. A pink ring which appeared on the surface within 1 minute indicated positive reaction.

44 c. Methyl Red (MR) Test:

The test organism was inoculated in glucose phosphate peptone water, incubated 37º C for 2 days. Five drops of methyl red reagent were added. A positive reaction was indicated by appearance of a red color. d. Voges Proskauer (V.P) Test:

The test organism was inoculated in glucose phosphate peptone water, and then 3 ml of 5% alcoholic solution of α-naphthol and 1ml of 40% KOH aqueous solution was added. A positive reaction was indicated by development of bright pink color within 30 minutes. e. Citrate utilization:

An isolate colony from nutrient agar was picked up with a straight wire, then inoculated in Simmon’s citrate agar and incubated at 37º C and examined daily. A positive test was indicated by change of color from green to blue. f. Hydrogen sulphide (H2S) Production:

The test culture was inoculated by stabbing the butt and streaking the slope of triple sugar iron agar in McCarteny bottles and incubated at 37ºC for 2 days. A positive reaction was indicated by development of a black color. g. Sugar fermentation test:

The sugar media were inoculated with the test organism and incubated at 37ºC overnight. They were examined daily for seven

45 days. Acid production was indicated by development of pink color in the medium, Gas production was indicated by air trapped in the Durham’s tube. The sugars used in these tests were lactose, salicin, sucrose, maltose, manitol, rafinose, inositol, xylose and sorbitol.

2.2.6 Antimicrobial sensitivity test:

Sensitivity of Salmonella isolates to a number of antimicrobial agents (Table 2) was determined by the standard disk diffusion method (Buxton and Fraser, 1977). Each isolate was tested to 10 different antimicrobial agents used for Gram-negative bacteria. Colonies from each isolate were emulsified in 2 ml nutrient broth and shaken thoroughly to obtain a homogenous suspension of the test culture. The plates were then flooded with the bacterial suspension, tipped in different directions to cover the whole surface with the suspension. Excess fluid was aspirated and the plates were left for 15 minutes to dry.

The antimicrobial disks were placed on the agar medium by using sterile forceps. The plates were then incubated at 37ºC and examined after 24 hours for zones of inhibition which were measured in mm. The isolates were described as resistant, intermediate and sensitive to different antimicrobial agents according to Bauer et al., (1966) (Table 3).

Table (2): Antibacterial used in antimicrobial sensitivity test

Antimicrobial Code Conc\ disc

Ambicillin\Sulbactam Axiom 20 mcg

Co- Trimoxazole Axiom 25 mcg

Cefotaxime Axiom 30 mcg

Piperacillin\Tazobactam Axiom 100\10 mcg

Chloramphenicol Axiom 30 mcg

Ciprofloxacine Axiom 5 mcg

Ceftizoxime Axiom 30 mcg

Tetracycline Axiom 30 mcg

Gentamycin Axiom 10 mcg

Amikacin Axiom 30 mcg

Table (3): Standard zone of inhibition to different antimicrobial agents

Zone of inhibition (Diameter in mm) Antimicrobial agent Code Disk potency Resistant Intermediate Sensitive Ampicillin\Sulbactam AS 20 mcg. 11 or less 12- 14 15 or more

Co- Trimoxazole BA 25 mcg. 10 or less 11- 15 16 or more

Cefotaxime CF 30 mcg 14 or less 15- 22 23 or more

Piperacillin\Tazobacta TZP 100\10 17 or less 18- 20 21 or more m mcg. Chloramphenicol CH 30 mcg. 12 or less 13- 17 18 or more

Ciprofloxacine CP 5 mcg. 15 or less 16- 20 21 or more

Ceftizoxime CI 30 mcg. 14 or less 15- 19 20 or more

Tetracycline TE 30 mcg. 14 or less 15- 18 19 or more

Gentamycin GM 10 mcg. 13 or less 14- 15 16 or more

Amikacin AK 30 mcg. 14 or less 15- 16 17 or more

CHAPTER THREE RESULTS

3.1 Isolation of bacteria:

A total of 80 samples were subjected to bacteriological examinations. Forty-three Gram-negative Enterobacteria were isolated from 80 samples; 12 samples showed no bacterial growth, 20 samples did not give typical reactions of Enterobacteria with oxidase, catalase, OF and glucose fermentation so they were not further identified, 5 samples gave only lactose fermenter colonies (pink colonies) in DCA. All samples which gave positive results for catalase and OF test and negative result for oxidase test and ferment glucose with gas were further identified.

The isolated Enterobacteria belong to eight genera which included Serratia species (15), Proteus species (11), Citrobacter species (8), Salmonella species (5), Yersinia species (1), Kluyvera species (1) Enterobacter species (1) and Hafnia species (1) (Table 4).

3.2 Site of isolation:

Three isolates of Salmonella were recovered from litter from Al-Halfaya area farm (layers). These three isolates included tow isolates of S. enteritidis, and one isolate of S. arizonae. One isolate of S. enteritidis obtained from poultry drinking water taken from drinkers in Shambat farm (broilers). No Salmonella isolates obtained from Animal production research center farms or from Al-Zakyab farm (Table 5).

3.3 Properties of Salmonella:

3.3.1 Cultural properties:

3.3.1.1 Growth in liquid media:

Growth in selenite-f-broth was detected by brown precipitate in the medium after 24 hours of incubation at 37ºC.

Growth in nutrient broth and peptone water was indicated by the formation of turbidity and slight white sediment after 24 hours of incubation at 37ºC.

3.3.1.2 Growth on solid media:

On nutrient agar Salmonella colonies were moderately large (2- 4 mm), circular with smooth surface and grayish- white in color after 24 hours at 37ºC (Figure 1).

Growth on deoxycholate citrate agar showed slight opaque dome- shaped colonies measured (2-4 mm) with central black spots (indicated production of hydrogen sulfide) surrounded by a zone of clearance after 48 hours at 37ºC (Figure 2).

On triple sugar iron agar Salmonella colonies produced hydrogen sulfide which was indicated by black discoloration, gas production causes bubbles in the agar, and pH change was indicated by production of red color in the slant (Figure 3).

3.3.2 Microscopic properties:

All Salmonella isolates were found Gram-negative, short rods occurred singly or in groups.

3.3.3 Biochemical reactions:

Salmonella isolates were oxidase and urease negative. They produced gas from glucose and manitol, while sucrose, salicin and lactose were not fermented. Hydrogen sulfide was produced by the isolates (Table 5).

3.4 Sensitivity to antimicrobial agents:

Sensitivity test to the four Salmonella isolates against 10 antibacterial agents was carried out. All isolates found sensitive to chloramphenicol, ceftizoxime, amikacin and resistant to gentamycin, tetracycline, ambicillin\sulbactam and piperacillin\tazobactam. All isolates were found sensitive to co- trimoxazole except one isolate of S. enteritidis found sensitive; two isolates of S. enteritidis were resistant to cefotaxime while other two isolates were moderately sensitive to this agent; all isolates found resistant to ciprofloxacin except one isolate of S. enteritidis (Figure 4).

Table (4): Isolated enterobacteria from different samples in the study.

Bacteria spp. No. of isolates Rate of isolation

18.75 % Serratia spp 15 13.75 % Proteus spp 11 10.00 % Citrobacter spp. 8 5.00 % Salmonella spp. 4 1.25 % Kluyvera spp. 1 1.25 % Yersinia spp. 1 1.25 % Enterobacter spp. 1 1.25 % Hfnia spp. 1 15.00% No growth 12

Other bacteria 21 26.25%

Table (5): Isolated Salmonellae from different samples and areas in Khartoum North.

No. and types of samples from which salmonellae were isolated No. of Species of Source of samples Salmonella Salmonella isolates isolates

Feed Litter Water

Al-Halfaya farm (layer) - 3 - 4 S. enteritidis S. arizonae

Shambat farm (broiler) - - 1 1 S. enteritidis

Table (8): Sensitivity of Salmonella isolates to different antimicrobial agents

Name of AS BA CF TZP CH CP CI TE GM AK isolate

HAR R S IN R S R S R R S

HEN1 R S IN R S R S R R S

HEN2 R R R R S R S R R S

SHEN R S R R S IN S R R S

Key:

(S) Sensitive. (R) resistant. (IN) intermedia

(BA) Co-Trimoxazole. (CF) Cefotaxime (AK) Amikacin

(CP) Ciprofloxacin. (CI)Ceftizoxime (TE) Tetracycline. (GM) Gentamycin. (AS) Ampicillin\Sulbactam.

(CH) Chloramphenicol (TZP) Piperacillin\Tazobactam .

HAR (S. arizonae from Halfaya) HEN1 (S. enteritidis from Halfaya)

HEN2 (S. enteritidis from Halfaya) SHEN (S. enteritidis from Shambat)

Figure (1) Growth of Salmonella on Nutrient Agar

Figure (2) Growth of Salmonella on Desoxychocolate Citrate Agar

Figure (3) Growth of Salmonella on Triple Sugar Iron Agar (TSI)

Figure (4) the sensitivity of Salmonella to different antibiotics

Figure (5) Sensitivity of Salmonella isolates to different antibiotics 30

20 inhibition 15 of

10 (mm)

Zone 5

0 AS BA CF TZP CHAntibiotics CP CI TE GM AK HAR (S.arizonae from Al‐Halfaya) HEN1 (S. enteritidis from Al‐Halfaya) HEN2 (S.entretidis from Al‐Halfaya) SHEN ( S.entretidis from Shambat)

(AS) Ampicillin\Sulbactam 20mcg (BA) Co‐ Trimoxazole 25mcg (CF) Cefotaxime 30mcg (TZP) Piperacillin \Tazobactam 100\10mcg (CH) Chloramphenicol 30mcg (CP) Ciprofloxacine 5mcg (CI) Ceftizoxime 30mcg (TE) Tetracycline 30mcg (GM) Gentamycin 10mcg (AK) Amikacin 30mcg

CHAPTER FOUR DISCUSSION

Salmonellosis is a major public health concern and continues to have a serious economic importance in the poultry industry in all countries (Morales and McDowell, 1999). With the great expansion of the poultry industry, the wide spread occurrence of the avian salmonellosis has ranked it as one of the most important egg- borne bacterial diseases of poultry.

The present study was conducted to investigate the contamination of poultry feed and poultry environment with Salmonellae in poultry traditional farms in Khartoum North. Salmonellae were isolated together with other bacterial genera as Serratia, Proteus, Citrobacter, Enterobacter, Yersinia, Kluyvera and Hafnia. Although all collected samples in the study were cultured first in the selenite-f- broth, gram- negative bacteria other than Salmonella were isolated. This can be explained by the fact that selenite-f-broth enriches the growth of Salmonella and Shigella but do not kill other enteric bacteria which under other conditions (subculture in DCA) can grow.

In this study Serratia represented the most dominant isolate and counted for (18.75%), followed by Proteus (13.75%), Citrobacter (10.00%), Salmonella (5.00%), Yersinia (1.25%), Enterobacter (1.25%), Kluyvera (1.25%) and Hafnia (1.25%).

The Salmonella isolation rate (5%) was comparable to that reported in other studies.

Yagoab and Mohammed (1987) examined 1488 samples and isolated 58 Salmonellae which comprise 3.9% of total isolates. In another study Ezdihar (1996) examined 610 samples from poultry in the Sudan and isolated 45 Salmonellae which counted for 7.4% of the total isolates. The later study showed higher isolation rate compared to the finding of this study and that may be due to the large difference in the number of samples collected in both studies. Hiba (2007) examined 102 samples from sick chickens in Khartoum state and isolated three Salmonella which counted (2.9%).

Salmonella was isolated only from samples obtained from a farm of layers in Al-Halfaya and from a farm of broilers in Shambat. It was not isolated however, from animal production research center farms or from a farm in Al-Zakiab area. This finding did not indicate that Salmonella was not present in these areas, but might be due to the small number of collected samples. On the other hand it confirms the presence of Salmonella contamination in farms from which Salmonellae were isolated.

The higher isolation rate was obtained from a farm of layers in Al- Halfaya, with the fact that all samples were collected from open system farms; this can be due to poor hygiene in this farm.

Among the examined samples, the highest rate of isolation was obtained from litter samples (three isolates) then water samples (one isolate).

This finding indicates a high shedding of Salmonella from the intestinal tracts of birds in this farm. S. enteritidis is the most important serovar in poultry flocks and recently it was of high

60 occurrence worldwide (Pitol et al, 1991). Phillips and Optiz (1995) showed that S.enteritidis could attach to granulose cells in the preovulatory membrane and subsequently infect the ovum during the ovulation. On the other hand, S. enteritidis had the ability to penetrate eggs through the shell pores and causes egg contamination.

In the present study three isolates of S. enteritidis were recovered, our finding confirmed previous records (Mamon et al., 1992; Hiba., 2007) that S.enteritidis was detected in Khartoum state. As long as the Sudan depends on importation of chickens it could have been come with infected imported flocks.

From the view point of public health, human salmonellosis was reported to increase recently in France and United States of America due to S. enteritidis (Barrow et al., 2003). It was reported to cause food poisoning due to consumption of under cooked egg dishes (Quinn, 2002). Isolation of this bacterium from some farms in Khartoum state represents a real threat to the public health.

S. arizonae was widely distributed in nature in a variety of avian, mammalian and reptile species (Cambre et al., 1980). The variety of infection sources in the nature will expose hen flocks to infection. S. arizonae was reported to cause arizonae infection in chickens (Carter and Wise, 2004).

The antimicrobial sensitivity test was carried out for salmonella isolates. All strains of Salmonella were found sensitive to chloramphenicol, ceftizoxime, amikacin and resistant to ambicillin\sulbactam, piperacillin\tazobactam, tetracycline and gentamycin. Also all isolates were found sensitive to co-trimoxazole

61 except one isolate of S. enteritidis; two isolates of S. enteritidis were found sensitive to cefotaxime while S. arizonae and the other isolate of S. enteritidis were moderately sensitive; S. arizonae and two isolates of S. enteritidis were showed resistance to ciprofloxacin while the other isolate of S. enteritidis was moderately sensitive.

Resistance to gentamycin has been reported by Lee et al., (1993) which determined 10% resistance to this agent from 105 Salmonella isolates. Also there was an increasing development of quinolones resistance all over the world (Fey et al., 2004). Treatment failure due to a reduced susceptibility to ciprofloxacin in Salmonella is now well established (Aarestrup et al., 2003).

In general, Salmonella is the most important agent implicated in outbreaks in food-borne diseases around the world (Lacey, 1993). Effective control or eradication programs for salmonellosis depend on good management system, identification of carrier birds and accurate medication.

Conclusion

In search for Salmonellae in poultry environment and feed in open system farms for layers and broilers in Khartoum North, only four isolates of Salmonella were recovered from litter samples and drinking water sample while no Salmonella isolates were recovered from feed samples.

Three isolates belong to S. enteritidis while the other was S. arizonae.

Isolation of three S. enteritidis isolates out of 80 samples is consider a high isolation rate and may represent a real threat for public health for this organism implicated in serious health problems.

All isolates were found sensitive to chloramphenicol, amikacin and ceftizoxime while resistant to tetracycline, gentamycin, ambicillin\sulbactam and piperacillin\tazobactam.

Recommendation:

1- Further studies are needed to investigate the relation between contamination of poultry environment with Salmonellae and Salmonella infections in poultry and threat to public health.

2- Application of quick procedures (e.g. PCR) is needed to trace sources of contamination.

3- Drug resistance among Salmonella bacilli has emerged worldwide, there for we strongly recommended for more prudent uses of antimicrobial agents in both medical and veterinary fields.

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