بسم اهللا الرحمن الرحيم

Isolation, Identification and Antibiotic Sensitivity of Aerobic Bacteria

Associated with Respiratory Tract Infections of Chickens in Khartoum

North Area

BY

Myssarah Mohammed Saad Ahmed

B. Sc., Elnelein University (2004)

Supervisor: Dr. Abdelhafeez Hassan Nimir

A thesis submitted to the University of Khartoum in partial fulfillment of the

requirements for the degree of M. Sc. in Microbiology

Department of Microbiology,

Faculty of Veterinary Medicine,

University of Khartoum

April, 2010

II

DEDICATION

To my Parents

With Thankfulness

To my Brothers and Sister

With great love

To my Friends and Colleagues

With Best Wishes

III

ACKNOWLEDGEMENTS

First of all, my thanks and praise to almighty Allah, the most beneficent,

the merciful, for giving me health and strength to accomplish this work.

Then I wish to express my sincere gratitude to my supervisor Dr. A.

Hafeez Hassan Nimir for guidance, advice, kindness and support.

Also I wish to express my kind regards and thanks to Dr. Suliman

Mohammed Elhassan for his kind interest and unlimited help.

I am grateful to all members of Department of Microbiology for their

unlimited assistance.

Finally I am deeply indebted to my family and friends for their patience

continued encouragement and support throughout this work.

IV

LIST OF CONTENTS

Page

DEDICATION II

ACKNOWLEDGEMENTS III

LIST OF CONTENTS IV

LIST OF TABLES VIII

LIST OF FIGURES IX

ABSTRACT X

ARABIC ABSTRACT XI

INTRODUCTION 1

CHAPTER ONE LITERATURE REVIEW 3

1.1 Poultry respiratory system 4

1.2 Bacteria 4

1.2.1 Infection via the respiratory tract 4

1.2.2 Bacteria of the respiratory tract 4

1.2.4.1 Escherichia coli 4

1.2.4.2 Haemophilus 6

1.2.4.3 Pseudomonas 7

1.2.4.4 Pasteurella 8

1.2.4.5 Bordetella 9

1.2.2.6 Staphylococcus 9

1.2.4.6 Streptococci 10

1.2.4.7 Mycoplasmas 10

1.2.4.8 Chlamydia 11

1.2.4.10 Other bacteria species 11

V

1.3 Respiratory disease caused by fungal and viral agent 12

1.3.1 Aspergillosis 12

1.3.2 Newcastle disease 13

1.4 The avian immune system 14

1.5 The avian immune response 15

1.6 Antimicrobial agents 17

1.6.1 Antibiotics 17

1.6.2 Benefits of antimicrobial use 18

1.6.3 Disadvantages of antimicrobial use 19

CHAPTER TWO MATERIALS AND METHODS 22

2.1 Sterilization 22

2.2 Reagents and indicators 22

2.2.1 Reagents 22

2.2.1.1 Alpha-naphthol solution 22

2.2.1.2 Potassium hydroxide 23

2.2.1.3 Hydrogen peroxide 23

2.2.1.4 Methyl red 23

2.2.1.5 Tetra methyl-p-phenyl diamine dihydrochloride 23

2.2.1.6 Nitrate test reagent 23

2.2.1.7 Kovac’s reagent 23

2.2.2 Indicators 24

2.2.2.1 Andrade’s indicator 24

2.2.2.2 Bromothymol blue 24

2.2.2.3 Phenol red 24

2.2.2.4 Lead acetate paper 24

VI

2.2.2.5 Bromocresol purple (BDH) 24

2.3 Collection of blood for enriched media 24

2.4 Preparations of media 25

2.4.1 Nutrient broth (Oxoid CM 1) 25

2.4.2 Peptone water (Oxoid CM 9) 25

2.4.3 Peptone water sugars (Carbohydrate fermentation medium) 25

2.4.4 Nutrient agar slant 25

2.4.5 Glucose-phosphate medium (MR-VP test medium) 26

2.4.6 Nutrient agar (Oxoid CM 3) 26

2.4.7 Blood agar (Oxoid CM 55) 26

2.4.9. Chocolate agar medium 26

2.4.9 Diagnostic sensitivity test agar (Oxoid CM 261) 27

2.4.10 MacConkey agar medium (Oxoid CM 7) 27

2.4.11 Eosin Methyline Blue Agar – EMB 27

2.4.11 Motility medium - Cragie tube medium 27

2.4.12 Hugh and Liefsons (O/F) medium 28

2.4.13 Simmon citrate medium (Oxoid CM 155) 28

2.4.14 Urea agar medium 28

2.5 Collection of samples 29

2.5.1 Sampling and culture 29

2.5.1.1 Nostril 29

2.5.1.2 Trachea 29

2.5.1.3 Lung 29

2.5.1.4 Conjunctiva 29

2.6 Primary culturing 29

VII

2.7 Purification and preservation of culture 30

2.8 Microscopic examination 30

2.9 Identification of isolates 30

2.10 Biochemical methods for identification of isolated bacteria 31

2.10.1 Catalase test 31

2.10.2 Oxidase test 31

2.10.3 Oxidation fermentation (O/F) test 31

2.10.4 Motility test 31

2.10.5 Sugar fermentation test 32

2.10.6 Indole production test 32

2.10.7 Methyl red test 32

2.10.8 Voges-Proskure test 32

2.10.9 Citrate utilization test 32

2.10.10 Hydrogen sulphide production 33

2.10.11 Nitrate reduction test 33

2.10.12 Coagulase test 33

2.10.12.1 Slide coagulase test 33

2.10.12.2 Tube coagulase test 33

2.10.13 Novobiocin sensitivity test 33

2.11 Antibacterial sensitivity test 34

CHAPTER THREE RESULT 36

3.1.1 Bacterial isolated from nostrils 36

3.1.2 Bacteria isolated from conjuctiva 36

3.1.3 Bacteria isolates from trachea 37

3.1.4 Bacteria isolated from lung 37

VIII

3.2 Characters and biochemical reactions 38

3.3 Antibacterial sensitivity of the isolated bacteria 38

CHAPTER FOUR DISCUSSION 49

Conclusion and Recommendations 52

Conclusion 52

Recommendations 52

REFERENCES 53

LIST OF TABLES

Tables Page

1 Isolation frequency of aerobic bacteria from respiratory tract of infected chicken in Khartoum North area

39

2 Gram positive bacteria isolated from respiratory tract of infected chickens in Khartoum North area

40

3 Gram negative bacteria isolated from respiratory tract of infected chickens in Khartoum North area

41

4 Characters and biochemical reaction of gram-positive bacteria isolated from respiratory tract of infected chickens in Khartoum North area.

42

5 Characters and biochemical reactions of gram negative bacteria isolated from respiratory tract of infected chickens in Khartoum North area.

43

6 Antibacterial sensitivity of bacterial isolated from respiratory tract of infected chicken in Khartoum North area

44

7 The antibacterial sensitivity of Gram positive bacterial species isolated from respiratory of infected chickens in Khartoum North area.

45

8 The antibacterial sensitivity of Gram negative bacterial species isolated from respiratory of infected chickens in Khartoum North area.

46

IX

LIST OF FIGURES

Figure Page

1 Growth of E. coli on MacConkey's agar 47

2 Growth of E. coli on Eosin Methylene Blue Agar 47

3 Sensitivity test of Gram-positive bacteria 48

X

ABSTRACT

This study was carried out in Khartoum North area to isolate and identify the

bacteria associated with the respiratory tract infections of chickens.

Seventy eight samples were collected from five farms in Khartoum North

area one farm in each of Alkadaro, Umalgora, Almazallat and two farms in

Shambat. Samples were aseptically collected from different breeds of chickens

showing clear respiratory symptoms. The samples were nasal swabs, conjunctival

swabs, tracheal swabs and specimens from lungs. The sensitivity of bacteria

isolated from infected chicken to antibiotics was examined.

The collected samples showed bacterial growth in 64 (82.1 %) samples and

yielded 89 (114.1 %) isolates. Fifty two (66.7 %) of isolates were found to be

Gram positive bacteria and the remaining 37 (47.4 %) isolates were Gram negative

bacteria. The result of sensitivity test showed variable results, some showed high

sensitivity while others showed resistance. The most effective antibacterial drug

was Gentamicin (85.7%) but the lowest effective drug was Lincomycin (12.5%).

The result of this study described the bacterial respiratory diseases as the one

of the constrains to poultry production in Khartoum North area.

XI

ص األطروحةلخستم

ا بحرى ة الخرطومأجريت هذه الدراسة فى منطق ا التى له ى البكتري م التعرف عل وذلك بغرض عزل ومن ث

.الجهاز التنفسى فى الدجاج بإصاباتعالقة

أمفى وأخرى احد هذه المزارع تقع فى الكدرو شمالعينة من خمس مزارع فى منطقة الخرطوم 78جمعت

ه بوضوح القرى وآذلك واحده ف ى المظالت بينما تقع مزرعتان فى شمبات وذلك من دجاج مصاب تظهر علي

ة ،ملتحمة العين، المنخر العينات من أخذت .الجهاز التنفسى إصابة أعراض ة من الرئ ة وقطع . القصبة الهوائي

.البكتيرية التى عزلت األنواعلبعض ةيويالح تللمضادا اختبار الحساسية أجرى وأيضا

ات المجموعة و فى تاظهر العين ة والتى 64 )82.1(% نم ة 89(% 114.1) أنتجت عين ا . عزل 52 منه

.المتبقية سالبة الجرام (% 47.4)37عزلة موجبة الجرام بينما ال (66.7%)

ة ع ةيحساس أظهرت نتائج متنوعة فبعض االنواع أظهرت نتيجة اختبار الحساسية ة الي بعض اظهر مقاوم وال

ة للمضادات ول البكتيري ر إنلكن نستطيع الق ة هو الجنتامايسين أآث ا (%85.7)المضادات فعالي ا اقله بينم

. (%12.5)فعاليه آان اللينكومايسين

ذه الدراسة ائجنت از التنفسى آ أمراض وصفت ه ات حد أالجه ة الخرطوم ةناعص ل المعوق دواجن فى منطق ال

. بحرى

36

INTRODUCTION

Poultry are kept worldwide and they play a significant role in

economic cycle of the communities. This role is accelerated by the

comparative efficiency of poultry in conversion of cereal feed to protein and

to their adaptability to intensive management.

The value of poultry industry to the economic and social communities

is often reflected in the attention paid to the factor which may adversely

affect the industry. One of the most important factors affecting poultry

industry is diseases. They have devastating effects particularly in intensive

system of production.

Most of the important poultry diseases are of worldwide occurrence

(Gordan and Jordan, 1982) however some diseases are restricted to certain

areas due to the presence of vectors or other factors. The number of birds

kept in one unit and rearing of different age groups in the same farm may be

predisposing factors to the occurrence of the diseases and this may lead to a

heavy economic losses.

Diseases of the respiratory tract are often complex with anatomy,

management, environment and nutrition, all playing a role (Nighot et

al.,2002) and they are caused by wide range of pathogens of bacterial, viral,

mycoplasmal or fungal origins. They play a significant role in death and

losses in poultry industry. Any respiratory disease has a direct negative

impact on the commercial parameter of poultry industry like weight gain,

egg production or live ability and these causes considerable losses. Two

main factors contribute to the sensitivity of these diseases in chicken; these

are anatomy and physiology of the respiratory system and complex nature of

the respiratory disease. The clinical picture of the respiratory diseases is

37

usually complicated when other diseases are involved. The severity and

lesions of the respiratory diseases are sometime due mainly to the secondary

invaders.

Poultry industry in the Sudan showed a significant development in the

last decade. The number of large scale farming increased steadily, and the

industry became more specialized in its intensive form and it covered the

production of chicken in addition to meat and egg production.

This study was carried out:

1) To isolate and identify bacteria which associated with respiratory tract

infection of chickens.

2) To examine antibiotics susceptibility for some of bacterial isolates.

38

CHAPTER ONE

LITERATURE REVIEW

1.1 Poultry respiratory system:

The poultry respiratory system is composed of nostrils situated at the

base of the bill, leading to the nasal cavity, the larynx, trachea, syrinx, lung

and the air sacs (Getty, 1975). The main functions of the nasal cavity are

smelling, filtration of air borne particles and humidification of inspired air,

while the larynx main functions are prevention of foreign bodies’ entry,

opening of the air ways during inspiration, aiding in swallowing and

modulation of voice. The nasal cavity continues in to the long trachea, which

divides before entering in to the lung. The comparatively long trachea offers

pathogens easy access to an area where they can cause infection because of

the high volume and low respiratory frequency. The syrinx gives rise to the

left and right bronchi, each primary bronchus gives rise to four secondary

bronchi, and the secondary bronchi give off numerous para-bronchi where

gaseous exchange takes place. The lung in chicken is a flattened nearly

rectangular structure lining the roof of the cranial end of the celom (Getty,

1975). One of the important features of avian lungs is the efficient gas

exchange system which helps the bird to maintain oxygen pressure, even

during limited ventilation (Nighot et al., 2002).

The air sacs are found in the thorax and the anterior positions (Getty,

1975). During inspiration, the volume of the air sacs increases and the

pressure inside the air sacs decreases and vice versa during expiration. The

presence of the air sacs connected to para-bronchi and occupying most of the

inner body cavities are a crucial factor. A pathogen entering the nasal cavity

can travel through both thorax and abdomen to close proximity to the head

of femur bone.

39

1.2 Bacteria:

The bacteria are a group of single celled microorganisms with

prokaryotic configuration. The prokaryotic cells nuclear material is not

enveloped by a membrane. The bacterial cells are prokaryotes.

The bacteria have three architectural region; the appendages in the

form of flagella and pili; a cell envelope consisting of a capsule, cell wall

and plasma membrane; cytoplasmic region that contains the cell genome

(DNA) and ribosome and various sort of inclusions. Most of cellular

reaction incidental to life can be traced back to the activities of these

structural components (Quinn, 2002).

All animals have what is called a normal flora, it consists of bacteria

Mycoplasmas, viruses and fungi that live in or upon the normal animal

without producing disease, included in this normal flora are a number of

potential pathogens (Carter, 1986).

1.2.1 Infection via the respiratory tract:

The infection can be acquired by direct contact, as a result of

inhalation of contaminated air. The organisms are trapped on the moist

mucous membrane of the nasal pharynx and lower respiratory tract, so this is

the way that diseases enter in to the mucous membrane such as Pasteurella

(Tomas, 1983).

1.2.2 Bacteria of the respiratory tract:

The respiratory tract of poultry is infected by a wide range of bacteria,

these include the following:-

1.2.2.1 Escherichia coli:

Escherichia coli are a Gram-negative flagellated rod, motile, non

spore-formig bacteria (Sojka and Garnaghan, 1961). Species of this genus

are widely distributed in nature and constitute a part of digestive flora of

40

mammals and bird. Pathogenic E. coli differ from the non-pathogenic E. coli

by the presence of virulence factors organized in clusters in the chromosome

or plasmid. According to the large variation in DNA content and to the

difference in the distribution of genomic location (insertion site) of different

virulence determination (Puente and Finlay, 2001). Some serotype cause

specific disease in poultry known as colibacillosis which is a complex

syndrome characterized by multiple organ lesions with air sac sacculitis and

associated pericarditis, others cause disease under certain condition (Buxton

and Fraser, 1977), (Swayne et al. 1998) and some act as secondary invaders.

The organism adversely affects avian species through infection of blood,

respiratory tract and soft tissue. The organism has also been isolated from an

outbreak of respiratory disease (Chu, 1958) and from different sites of the

respiratory tract of normal chicken. Elnasri (1997) isolated the organism

from infra-orbital sinus and trachea. Secondary infection commonly occurs

as complication with Mycoplasma gallisepticum infection. E. coli infection

caused by a single agent occurs rarely. E. coli often infects respiratory tract

of bird concurrently with various combination of infectious bronchitis

viruses, Newcastle disease viruses, including vaccine strain; and

Mycoplasms. Transmissions can be through inhalation, contamination of

drinking water or feed, contamination of reproductive system and egg shell

surface (Zahida, 2004).

Escherichia coli associated with respiratory infection in chickens has

also been reported (Elsukhon et al., 2002). Tracheitis, exudative pneumonia,

pleuritis, air sacculitis, pericarditis, sinusitis characterize the infection (Canal

et al., 2005).

41

The primary routes of invasion by the organism are the respiratory

system and the gastrointestinal tract. Lisons occur firstly in the respiratory

tract through inhalation and in the mucus membranes in the case of intestinal

infection; local lesions develop into systemic infections (Zahida, 2004). The

symptoms vary with the different types of infections; in the acute septicemia

form mortality may begin suddenly and progress rapidly. The common signs

are restless with ruffled feathers indications of fever, also symptoms of

labouredly breathing, occasional coughing and rales (Sojka and Carnavan,

1961). Mouline (1983) found that E. coli was a predominant organism of the

tracheal flora. E. coli was isolated from the lung and air sac (Price et al.,

1975, Malokwa et al., 1987; and Rajashekar et al., 1998) and from sinues

(Eisa and Alnasri, 1985). Linzitto et al. (1988) isolated the organism from

cases of infectious coryza.

The Annual Reports of the Sudan Veterinary Service (1948-1958).

showed the presence of fowl coryza and E. coli infections since 1948. The

importance of this disease is due to difficulty of prevention and control

because of it's resistance to a wide range of antibiotics and due to the large

number of varying serogroups involved in field outbreaks (Abdellah, 2003).

1.2.2.2 Haemophilus:

De Bleich (1932) was the first to isolate the causative agent of

infectious coryza and named the organism Heamoglobinophillus coryza

gallinarum (Yamamoto, 1991; Linzitto et al., 1988).

Bacteriological studies indicated that a number of organisms are

associated with infectious coryza, these include Heamophillus avium,

Streptococcus, Staphylococcus, Escherichia coli, Pasteurella multocide,

Psedomonus aeruginosa, Pasteurella gallinarum and Mycoplasma

gallisepticum (Yamamoto and Mutsumoto, 1970; Kojiuchida et al., 1991).

42

Bacteriological examination of infectious coryza infected chickens in

Sudan shows the disease is caused by Haemophilus gallinarum (Shigidi,

1971). The disease is usually transmitted through drinking water

contaminated with infective nasal exudates (Page, 1962). Infection may also

occur by contact and by air-borne infected dust or droplet. Infectious coryza

is an acute or chronic disease of upper respiratory tract of poultry caused by

Haemophilus group of bacteria. These are heterogeneous group of small

Gram-negative, aerobic bacilli, non motile and non spore-forming (Gordan

and Jordan, 1982) requiring enriched media for culturing and growth. The

organism is classified according to the X factor (hemin) and V factor

(nicotinamide adenine dinucleotide) (Eliot and Lewis, 1934) also Narita et

al. (1978) and Black and Reid (1982) confirmed this finding. Two species

are named: Haemophilus gallinarum and Haemophilus paragalinarum

(required V factor). These two species are identical in growth characteristics

and ability to produce the diseases (Rimler, 1979).

All susceptible birds in a flock show clinical signs of the disease

within few weeks. They include depression, seromucoid nasal discharge,

conjunctivitis, facial oedema, swollen wattles and rales, appetite and

production are reduced, resulting in inferior food conversion ratio in broilers

and reduced egg production in layers. In avian host Haemophilus gallinarum

was involved in respiratory disease complex (Hafez, 2002).

1.2.2.3 Pseudomonas:

Gram-negative, rod shape, motile, aerobic and non spore-forming.

This organism is distributed widely in nature and found in soil and water.

Pseudomonas aeruginosa is associated with infection in man and animals

(Merchant and Packer, 1967). Pseudomonas aeruginosa is not only

responsible for embryonic mortality but also for mortality in chicken and

43

heavy losses of broilers (Valadae, 1961: Saad et al., 1981: Andreev et al.,

1982 and Bapat et al., 1985). The pathogenic effect of Pseudomonas

aeruginosa in chicken was reported by Markaryan (1975) and Mrden et al.

(1988) also this organism was isolated from infectious coryza cases by

Linzitto et al. (1988) and Elnasri (1997). Pseudomonas pyocyanae cause

septicemia in young chicks (Banerji and Ray 1969).

The species of this organism were significant in mixed infection

particularly with Streptococci and Staphylococci (Carter, 1986).

1.2.2.4 Pasteurella:

Avian pasteurellosis is an infectious disease caused by certain related

bacteria which are Pasteurella multocida, Yersinia pseudotuberculosis and

Pasteurella gallinarum.

Pasteurella is a Gram-negative, non motile, non spore-forming, rod

shape bacterium and shows bipolarity when stained with Gimsa (Jordan,

1986). All species of Pasteurella (exept Pasteurella urase) occurs as a

commensall in upper respiratory tract and digestive flora of animals (Carter,

1986).

Ibrahim (1995) isolated Pasteurella multocida from different species of

animals in Sudan on basis of their morphology, culture, biochemical

characteristics and serology. Pasteurella multocida and Pasteurella

galinarum were isolated by Linzitto et al., (1988).

The organism is pathogenic to a wide range of animal species. In

poultry Pasteurella causes an acute disease known as fowl cholera; the

infection may be acquired by contact, inhalation or ingestion Linzitto et al.,

(1988).

44

Respiratory infection is the most serious disease affecting poultry and

causes heavy economic losses in the poultry industry worldwide. In avian

host, several microorganisms of the genus Pasteurella (P. multocida,

P. gallinarum, P. haemolytica and P. anatipestifer are involved in

respiratory disease complex (Hafez, 2002).

1.2.2.5 Bordetella:

Bordetella is heterogenous group of Gram-negative, small, rod shaped

and oxidase-positive bacteria. The disease is of an upper respiratory tract,

named as Turkey coryza which affected bird and the milder form of the

disease affected the broilers (Jordan, 1986). It was first described in the USA

and reported in many countries. The causative agent is Bordetella avium.

There is considerable variation in virulence among strains, it is relatively

resistant to heat and can probably survive on farm premises and it is

susceptible to the common disinfectant at recommended concentrations and

to direct sunlight. The severity of the disease may be greatly influenced by

other pathogens, such as E. coli, Newcastle disease virus, Pasteurella

multocida, Mycoplasma gallicepticum as well as a number of management

faults such as overcrowding, excessive atmospheric ammonia, cold and high

humidity (Kersters et al., 1984). Hafiz (2002) demonstrated that Bordetella

avium was involved in respiratory disease complex and causes heavy

economic losses in the poultry industry worldwide.

1.2.2.6 Staphylococcus:

Staphylococci are spherical Gram-positive bacteria, non motile, non

spore-forming, non capsulated and usually arranged in grape-like irregular

clusters (Jawetz et al,1990,Geo et al.,1998). Staphylococci present in the

upper respiratory tract and on other epithelial surfaces of all warm blooded

animals, some strains are pathogenic others are nonpathogenic (Bibersein et

45

al., 1974). Pigmentation produced by staphylococcus is varying from white

to deep yellow (Jawets et al., 1990).Golden pigmentation is produced by

many strains especially with extended incubation (Songer, 2000). Coagulase

positive Staphylococcus produces colonies surrounded by yellow zones

while non pathogenic produces purple colonies (Saeed, 1995).

Staphylococcus can some time produce air sac infection but it is associated

with chronic arthritis, pyogenic infection and abscsses formation (Buxton

and Fraser, 1977). Linzitto et al., (1988) reported that Staphylococcus was

isolated from cases of infectious coryza. Elnasri (1997) isolated

staphylococcus from trachea and air sac. A study concerning in respiratory

diseases in commercial broiler flock. Bacteriologic examinations resulted in

the isolation of E. coli and Staphylococcus spp. (Georgiades et al., 2001).

1.2.2.7 Streptococci:

Streptococci are Gram-positive, non spore-forming cocci occurring in

pairs or chains (Jordan, 1986). They are usually found on the skin and

mucous membrane of the upper respiratory tract, the organism causes

pneumonia in chicken (Merchant and Packer. 1967). The organism was

isolated and considered one of the organisms associated with infectious

coryza.

1.2.2.8 Mycoplasmas:

Jordan (1986) stated that there are many species of the genus

mycoplasma, some of them are of economic importance to the poultry

industry. Such as Mycoplasma gallisepticum which causes disease in

chicken and turkey. Mycoplasma synoviae causes synovitis and respiratory

infection in chicken and turkey. Mycoplasma meleagridis causes respiratory

disease in turkey. Many species of Mycoplasmas are nonpathogenic.

Yamamoto and Matsumoto (1979) isolated Mycoplasma gallisepticum from

46

cases of infectious coryza. In the Sudan suspicion of Mycoplasma infection

in poultry was based on clinical manifestations and confirmed by serological

testing (Harbi et al., 1975; Elhassan et al., 1989). In eastern Sudan many

clinical cases of respiratory tract and joint infection were observed. The

prevalence of Mycoplasma synoviae and Mycoplasma gallisepticum in

poultry farms in eastern Sudan was proved serologically in flocks showing

clinical respiratory signs (Salim and Mohammed, 1993). Recently, it was

demonstrated using in vitro assays that the avian pathogen Mycoplasma

gallisepticum is able to invade nonphagocytic cells. It was also shown that

this mycoplasma can survive and multiply intracellularly for at least 48 h

and that this cell invasion capacity contributes to the systemic spread of M.

gallisepticum from the respiratory tract to the inner organs (Vogl et al.,

2008).

1.2.2.9 Chlamydia:

Chlamydiosis is a disease of man, birds, and other animals. The

Chlamydia form a well defined group of organism and with worldwide

distribution, they are associated with many different diseases in many

species of birds and animals. The infection can be source of serious

economic loss to the poultry industry .the signs of the disease are serous or

purulent exudates from eyes and nostrils accompanied by loss of appetite

and in-activity. A common feature is diarrhoea: respiratory distress and

hyperthermia are also observed. Egg production is severely affected and

drops rapidly (Jordan, 1986).

1.2.2.10 Other bacterial species:

One of most important bacteria is enterobacteria (Carter, 1986).

Enterobacteria are of worldwide distribution: many of them are part of the

normal flora of the intestinal tract. Some species are free living occurring on

47

the soil and water e.g. Yersinia pseudotuberculosis, Shigella and Klebsiella

spp mainly K. pneumoniae as causal agent of serious and sometimes fatal

infections in both man and animals has been seen at increasing rates in the

recent years. Elhassan and Elsanosi (2002) isolated K. pneumoniae

subspecies ozaenae from lung, intestines, liver, ovaries, and eyes of chicken.

Ornithobacterium rhinotracheal has recently been identified as a pathogen

causing respiratory tract infections in poultry and other birds (Vandamme et

al., 1994; Chin et al., 2003). Tracheitis, exudative pneumonia, pleuritis, air

sacculitis, pericarditis, sinusitis, characterize the infection (Zorman-Rojs et

al., 2000). Canal et al. (2005) isolated Ornithobacterium rhinotracheale

from chicken, turkeys, quails, ducks, geese, ostriches, guinea fowl,

pheasants, rooks and pigeons. There were reports of O. rhinotracheale

infections in the United States, Germany, South Africa, The Netherlands,

France, Israel, Belgium, Hungary, Japan, the United Kingdom, Turkey,

Canada, Jordan and Brazil (Travers, 1996; Joubert et al., 1999; Van Empel

and Hafez, 1999; Chin et al., 2003).

1.3 Respiratory disease caused by fungal and viral agents:

1.3.1 Aspergillosis:

Aspergillosis is a respiratory tract infection caused by members of the

genus Aspergillus, of which A. fumigatus is the primary species responsible

for infections in wild birds. Aspergillosis is not contagious, and it may be an

acute, rapidly fatal disease or a more chronic disease. Both forms of the

disease are commonly seen in free-ranging birds, but the acute form is

generally responsible for large-scale mortality events in adult birds and for

brooder pneumonia in hatching birds. Friend and Franson (1999-2001).

Fungi are of greatest importance in causing disease in poultry do so enter by

tissue invasion and damage or by producing toxin. The respiratory tract is

48

commonly infected by fungi. Infections are most frequently due to

Aspergillus species. Respiratory aspergillosis is common mismanagement

problem in commercial and backyard poultry. Organisms culture from

affected organ are Aspirgillus fumigatus, A. flavus, A. nigar, A. glaucus and

A. terreus. Chicken and duck are highly susceptible to infection.

Aspergilosis is produce by inhalation of spores from infected eggs that are

opened during incubation or hatching. It is also produced by inhalation of

spores from contaminate feed of poultry house litter. Airborne conidia come

to rest on conjunctiva, nasal, trachea, parabronchial and air sacs. Infected

poultry flocks exhibit a biphasic mortality pattern, also lesions are found in

the respiratory tract including particularly the trachea, bronchi, lung and air

sac (Jordan et al., 2001).

1.3.2 Newcastle disease:

Newcastle disease is highly contagious disease that affects chickens

and other birds. The virulence of some strains of the virus makes the disease

a serious problem in many countries (Zein et al., 2001). Newcastle disease

still constitutes a major hazard to the poultry industry in Sudan (Tabidi et al.,

1998). Elhussien et al. (1996) classified Newcastle as the most important

poultry disease and still remain the major killing disease. Outbreaks of the

disease have been reported regularly from different regions of the country

(Elhussien et al., 1996). The disease causes heavy losses due to death of

birds, drop of egg production and retardation of the growth. Acute

respiratory tract infections are of paramount importance in the poultry

industry. Avian influenza virus (AIV), infectious bronchitis virus (IBV),

Newcastle disease virus (NDV), avian pneumovirus (APV), and

Mycoplasma gallisepticum (MG) have been recognized as the most

important pathogens in poultry (Roussan et al., 2008).

49

1.4 The avian immune system:

The anatomical basis of the immune system of the chicken is the

lymphoid tissue which has both central and peripheral components. Central

parts consist of two structures which are the multilobed thymus and the

bursa of fabricius. The peripheral component is the lymphoid tissue which

include the spleen, caecal tonsils, bone marrow, and aggregates of lymphoid

cells in various organs and tissue. Avian lymphoid cells rapidly infiltrate

sites of antigenic stimulation throughout the body so that even in normal

birds lymphoid aggregates found in tissue such as the nasal passage and

upper respiratory tract, oesophagus and intestinal tract of the chicken

(Gordan and Jordan, 1982).The lymphoid foci which are normally found in

organs like the proventriclus and pancreas typically consist of diffuse

unencupsulated masses of small lymphocytes and germinal centers of

variable size consisting of B lymphocytes, dendritic cells and macrophages.

The main immunological function of the thymus and bursa is to generate the

lymphocytes (Gordan and Jordan, 1982). The bursa drive B lymphocytes

while the thymus drive T lymphocytes.

Antigen-specific protection of mucosae in the upper airway is

achieved mainly using the humeral immune system (Brandtzaeg, 1995;

Phalipon et al., 2002) through the production and secretion of polymeric IgA

and Ig M (Brandtzaeg et al., 1997). Secretary Ig performs immune exclusion

by inhibiting the uptake of soluble antigens and by blocking adhesion and

invasion of epithelia by micro-organisms (Avakian and Ley, 1993; Snoeck et

al., 2006). As discussed above, numerous B cells and ASC are present in the

head-associated lymphoid tissues and throughout the respiratory tract of

avian species, and Immunoglobulins have been detected. Secreted

immunoglobulins in the respiratory tract are produced by bursa-derived cells

50

(Lam and Lin, 1984), primarily of the Ig A isotype, although Ig G and Ig M

antibodies are also found (Russell, 1993).

Comparable results were obtained in M. gallisepticum-infected birds which

developed antigen-specific IgA, IgM and IgG responses in washings of the

upper and lower respiratory tract (Yagihashi and Tajima, 1986). Functional

in vitro assays demonstrated that these antibodies were protective but that

protection did not correlate with the IgA titres in the washing, indicating that

secreted IgM and IgG are also of relevance in mucosal defence (Avakian

and Ley, 1993). Intratracheal infection with M. gallisepticum induces the

accumulation of IgG, IgA, B cells and plasma cells in the lamina propria. In

contrast, vaccination prior to infection induced the formation of lymphoid

follicles and of strongly elevated numbers of antigen-specific ASC as

measured by an enzyme-linked immunospot (ELISPOT) technique. It also

leads to a significantly higher proportion of M. gallisepticum-specific IgA

and IgG antibodies in tracheal washings (Javed et al., 2005). While IgA is

most probably produced locally and secreted as a polymeric antibody, IgG

antibodies may be locally secreted or transudated from the serum as

suggested by several studies (Toro et al., 1993; Suresh and Arp, 1995; Javed

et al., 2005). Finally, induction of antigen- specific IgA and IgG antibodies

in tear fluid and lower respiratory tract lavage samples have been

demonstrated in IBV-vaccinated birds (Toro and Fernandez, 1994;

Thompson et al., 1997) and was induced equally well by antigen delivery

through ocular instillation, spray or drinking water application (Toro et al.,

1997).

1.5 The avian immune response:

The response to microbial infection involves interaction of both innate

and acquired immunity. The development of the immunity involves both

51

T&B cells and eliminating infection. However this varies from disease to

another, as an example the antibodies is the major protective factor in

Newcastle disease and influenza infection while in Marek's disease and fowl

pox; the cell mediated reactions are the most important. The avian

respiratory tract is lined with local lymphoid tissue throughout its length.

This protects the respiratory system by attempting to eliminate the pathogen,

as well as invoking a general immune system components take part in the

immune response. Various immunosuppressive agents hamper the

functioning of the immune mechanism, making the birds more susceptible to

respiratory challenge. The flocks suffering from immunosuppressant disease

never attain optimum immunity in spite of vaccination against various

diseases (Nighot et al., 2002).

Nutrition also has its effect; various dietary components play a role in

the immune response of birds. Generally a higher level of nutrients is

required to optimize the immune response than for growth, e.g. methionine,

vitamins C and K. Imbalance of sodium and chloride can affect broiler

immunity and high chloride levels may reduce immune response if sodium

levels are not raised accordingly. Selenium and vitamin E are important for

the protection and regeneration of tissues. As an integral part of biochemical

substances involved in tissue healing, zinc is an essential nutrient. Vitamin A

and C help to maintain epithelial integrity. The amino acid make-up of the

protein source also influences the immune response. The protein analysis

solely on nitrogen basis may not give correct idea about amino acid

components, the balancing of which is essential to develop an optimum

immune response.

In conclusion, respiratory disease is precipitated when the natural

defenses and immunity of the bird is challenged by infectious or non

52

infectious causes, which mostly accompany one another. Intensive poultry

farming puts additional pressure on the respiratory system, which therefore

needs protection from pathogenic agents (Nighot et al., 2002).

1.6 Antimicrobial agents:

Antimicrobial agents may be defined as those substances that interfere

with the growth and activity of microorganism generally, the term denotes

inhibition of microbial growth (biostatic) and or destruction (biocidal).Such

terms as antibacterial or antifungal are frequently employed to refer to

activities against specific group of microorganisms (Pelczar et al., 1977).

The modern era of antimicrobial effects began with the work of the

German physician Paul Ehrlich (1824 – 1915). In 1929, Flemings discovered

the powerful bactericidal activity of Penicillin, and Domagk's in 1935 the

synthetic chemicals sulfonamides with broad-spectrum activity.

In the early 1940, Penicillin was isolated, purified and injected into

experimental animals. The rapid isolation of streptomycin, chloramphenicol

and tetracycline soon followed and by 1950s these and several other

antibiotics were in clinical usage (Carter, 1986).

1.6.1 Antibiotics:

Antibiotics are low molecular weight substances that are produced as

a secondary metabolites by certain groups of microorganisms specially

streptomyces, bacillus and a few molds (pencillium and cephalosporium)

that are inhabitants of soils. Antibiotics may have a cidal (killing) effects or

a static (inhibitory) effects on a range of microbes. The ranges of bacteria or

other microorganisms that affected by a certain antibiotics is expressed as its

spectrum of action. Antibiotics effective against a wide range of Gram

positive and Gram negative bacteria are said to be broad spectrum, if

effective mainly against Gram-positive or Gram-negative bacteria they are

53

called narrow spectrum, if effective against a single organism or disease,

they are referred to as limited spectrum (Prescott et al., 2002).

I.6.2 Benefits of antimicrobial use:

Early warnings have been made since the 1940s regarding the

subtherapeutic use of antibiotics that might expose microbes to non-lethal

quantities of antibiotics, making them resistant (Fleming, 1945). The food-

animal industry is ostensibly ignoring this admonition by daily use of sub-

therapeutical antimicrobial doses in animal feeds. Although this common

practice is increasingly controversial, it is hard to disregard the potential

benefits of antibiotic use in food-animal production, for both animal and

human health. Antimicrobial prophylaxis prevents diseases and decreases

microbial competition for nutrients in the animal's GI tract. Furthermore,

sick animals are treated with adequate medication, reducing their microbial

load and thus, enhancing their growth performances and general health

status. This allows better economic returns to producers and increases

consumers confidence in consuming food products produced from those

animals. On the other hand, antibiotics significantly reduce the threat to

human health related to foodborne pathogens such as Campylobacter,

Salmonella, hemorrhagic Escherichia coli O157: H7, Listeria

monocytogenes and Clostridium (NRC, 1999).

Use of antibiotics to eliminate transferable bacteria from food

producing animals is a measure of food security that protects consumers

from exposure to life threatening diseases, thus preserving their good health

status. Most importantly, this measure also could reduce infiltration of

pathogenic bacteria into the environment, mainly through manure

application. Runoff water, especially after heavy rains, could contaminate

wells and other community water systems as happened in Walkerton in

54

2000. This deadly waterborne outbreak resulted from entry of Escherichia

coli O157:H7 and Campylobacter spp. from neighbouring farms into the

town water supply (Clark et al., 2003). Crops, fruit trees and other

commodities are commonly sprayed with antimicrobials to prevent

microbial spoilage, thus contributing in controlling environmental

contamination (Khachatourians, 1998).

The benefits for animal health and welfare and for human health are

undeniable. However, there are concerns arising about the appropriateness of

the use of antibiotics in food animal production. Some of these concerns

include antibiotic residues and the emergence of antibiotic resistant

pathogens (Khachatourians, 1998; Conly, 2002; Bywater, 2005).

I.6.3 Disadvantages of antimicrobial use:

Any drug has a connotation of toxicity, especially when it and/or its

metabolites and absorbed from the intestines. Antimicrobials used in food-

animal production are not an exception to this rule. Residues from drugs

used in food animal production could be ingested either directly through

animal tissues or products, or indirectly through the environment

(Samanidou and Evaggelopoulou, 2008). Antibiotic residue consumption

can induce adverse effects such as toxicities, allergies and infection by

disease-causing bacteria that are antibiotic-resistant. Antimicrobials and

their metabolites can concentrate in animal tissue leading to toxicities that

could be manifested by teratogenic or carcinogenic effects. Consequently,

the US FDA has strict regulations: (no proven carcinogen should be

considered suitable for use as a food additive in any amount) (Committee on

Drug Use in Food Animals Panel on Animal Health, Food Safety, and Public

Health, 1999); Canada also follows similar regulations (Health Canada

2002). Therefore antimicrobials, that are systemic and thus absorbed from

55

intestines in significant amounts (e.g. tetracycline, penicillin, erythromycin

and lincomycin), supplied to animals through feed or water, fall under

regulatory withdrawal periods. Non systemic antimicrobials, which are not

absorbed or very slightly absorbed from the intestine, do not require any

removal period and can be administered to animals until slaughter (e.g.

bacitracin, virginiamycin, bambermycin, neomycin, tylosin, novobiocin and

streptomycin) (Health Canada, 2002).

Allergic reactions are less frequently reported in the literature (Health

Canada, 2002). Few cases are related to the consumption of milk containing

penicillin residues although there is no enough evidence to prove that

antimicrobials used in animal food provoke allergic reactions in humans

(Borrie and Barret, 1961; Wicher et al., 1969; Barton, 2000). Heat treatment

during further processing could degrade the residue epitopes and reduce the

potential for allergic reactions (NRC, 1999). Although strict control exerted

by regulatory agencies strongly contributed to limit the risks of food-animal

tainting by antimicrobial residues, antibiotic resistant bacteria still could

contaminate animal carcasses and enter the food chain.

Numerous studies have eliminated any doubt that antibiotic-resistant

bacteria could be transferred from food animals to humans (FSIS 1997;

Wegener et al. 1999; Poppe et al., 2006). Commensal pathogens such as

Salmonella, entero-hemorrhagic E. coli could exchange or receive multiple

antibiotic resistance genes from non pathogenic carriers such as generic E.

coli. (Schwarz et al., 2006; Zhao et al., 2006). Young children, elderly and

immuno-compromised people are the most at risk during infection by such

pathogens. They pay for what they are usually not directly responsible for

and sometimes, they have to pay with their own lives. Seven pathogens were

recognized by the Center for Disease Control and Prevention (CDC) as the

56

most common cause of foodborne illnesses. Campylobacter is the most

frequently isolated foodborne bacterium (49.4%), followed by Salmonella

(27.4%), Shigella (15.7%), E. coli O157:H7 (4.2%), Yersinia (1.7%),

Listeria (1%) and Vibrio (0.6%). They constitute a serious health risk due to

the fact that they are easily transferable, thus making them difficult to

control (FSIS, 1997).

57

CHAPTER TWO

MATERIALS AND METHODS 2.1 Sterilization:

a. Flaming:

It was used to sterilize glass slides, cover slips, needles and scalpels.

b. Red heat:

It was used to sterile wire loop, points and searing spatulas by holding

them over Bunsen burner flame until they became red-hot.

c. Hot air oven:

It was used to sterilize glass wares such as test tubes, graduated

pipettes, flasks, forceps and cotton swabs. The holding period was one hour

and oven temperature was 160 ºC.

d. Steaming at 100 ºC:

Repeated steaming (Tyndallization) was used for sterilization of

sugars and media that could not be autoclaved without deteirment effect to

their constituents. It was carried out as described by Barrow and Feltham

(1993).

e. Moist heat (autoclave):

Autoclaving at 121ºC (15Ib/ inch2) for 15 minutes was used for

sterilization of media and plastic wares.

Autoclaving at 115ºC (10Ib/ inch2) for 10 minutes was used for

sterilization of some media such as sugars containing media.

2.2 Reagents and indicators:

2.2.1 Reagents:

2.2.1.1 Alpha-naphthol solution:

Alpha-naphthol is a product of British Drug House (BDH); London. This

58

reagent was prepared as 5% aqueous solution for Voges Proskauer (VP) test.

2.2.1.2 Potassium hydroxide:

It was used for Voges Proskauer test and prepared as 40 % aqueous

solution.

2.2.1.3 Hydrogen peroxide:

This reagent was obtained from Agropharm Limited Buckingham. It

was prepared as 3% aqueous solution, and it was used for catalase test.

2.2.1.4 Methyl red:

It was prepared by dissolving 0.04 g methyl red in 40 ml ethanol. The

volume was made to 100 ml with distilled water. It was used for methyl red

test (MR).

2.2.1.5 Tetra methyl-p-phenyl diamine dihydrochloride:

This was obtained from Hopkin and William; London. It was prepared

in a concentration of 3% aqueous solution and was used for oxidase test.

2.2.1.6 Nitrate test reagent:

Nitrate test reagent was consisting of two solutions which were

prepared according to Barrow and Feltham (1993). Solution A was

composed of 0.33% sulphanilic acid dissolved by gentle heating in 5N-acetic

acid. Solution B was composed of 0.6% dimethyl amine-alpha-

naphthylamine dissolved by gentle heating in 5N-acetic acid. It was used for

nitrate reduction test.

2.2.1.7 Kovac’s reagent:

This reagent composed of para-dimethylaminobenzaldehyde, amyl

alcohol and concentrated hydrochloric acid. It was prepared as described by

Barrow and Feltham (1993) by dissolving the aldehyde in the alcohol by

heating in water bath, it was then cooled and the acid was added carefully.

The reagent was stored at 4 ºC for later use in indole test.

59

2.2.2 Indicators:

2.2.2.1 Andrade’s indicator:

It composed of 5 g acid fuchin , 1 L distilled water and 150 ml N-

NaOH. The acid fuchin was dissolved in distilled water, and then the alkali

solution was added and mixed. They solution was allowed to stand at room

temperature for 24 h with frequent shaking until the color changed from red

to brown.

2.2.2.2 Bromothymol blue:

Bromothymol blue was obtained from BDH. The solution was

prepared by dissolving 0.2 g of bromothymol blue powder in 100 ml

distilled water.

2.2.2.3 Phenol red:

Phenol red was obtained from Hopkins and William ltd, London. It

was prepared as 0.2% aqueous solution.

2.2.2.4 Lead acetate paper:

Filter paper strips, 4-5 mm wide and 50-60 mm long were

impregnated in lead acetate saturated solution and then dried. It was used for

hydrogen sulphide test.

2.2.2.5 Bromocresol purple (BDH):

Bromocresol purple indicator was prepared by dissolving 0.2 g of the

powder in 100 ml distilled water.

2.3 Collection of blood for enriched media:

Blood for enriched media was collected aseptically into sterile flask

containing glass beads by veinopuncture of jugular vein of healthy sheep

kept for this purpose. The blood was defibrinated by shaking the flask after

collection. The defibrinated sheep blood was used for preparing blood agar

medium.

60

2.4 Preparations of media:

2.4.1 Nutrient broth (Oxoid CM 1):

The medium was prepared by adding 13 g of nutrient broth powder to

1 L of distilled water and well mixed. The pH was adjusted to 7.4. The

mixture was distributed in 5 ml volumes into clean bottles, and then

sterilized by autoclaving at 121ºC (15 Ib/inch2) for 15 minutes.

2.4.2 Peptone(Oxoid CM 9) water:

This medium was prepared by dissolving 10 g peptone and 5 g sodium

chloride in 1 L of distilled water. The mixture was distributed in 5 ml

volumes into clean bottles and sterilized by autoclaving at 121º C (15 Ib/

inch2) for 15 minutes.

2.4.3 Peptone water sugars (Carbohydrate fermentation medium):

Peptone water sugar medium was prepared according to Barrow and

Feltham (1993). It contained 900 ml peptone water, 10 ml Andrade’s

indicator, 15 g sugar and 90 ml distilled water. The pH of peptone water was

adjusting to 7.1-7.3 before the addition of Andrade’s indicator. The complete

medium was well mixed, then distributed in portions of 2 ml into clean test

tubes containing inverted Durham’s tube. The medium was autoclaving at

115 ºC (10 Ib/inch2) for 20 minutes. The carbohydrates examined were

glucose, sucrose, lactose, fructose, maltose, mannitol, xylose, sorbitol and

salicin.

2.4.4 Nutrient agar (Oxoid CM 3) slant:

This was prepared by adding 28 g of nutrient agar to 1 L of distilled

water and dissolved by boiling. The pH was adjusted to 7.4. The prepared

medium was distributed in 10 ml volume into clean bottles, sterilized by

autoclaving at 121 ºC (15 Ib/inch2) for 15 minutes and left to solidify in

inclined position.

61

2.4.5 Glucose-phosphate medium (MR-VP test medium):

This medium was prepared according to Barrow and Feltham (1993)

by adding 5 g peptone and 5 g phosphate buffer to 1 L distilled water, then

dissolved by steaming and filtered. The pH was adjusted to 7.5, 5 g of

glucose were added and then well mixed. The complete medium was

distributed into clean test tube in 10 ml amount. The medium was sterilized

by autoclaving at 115 ºC (10 Ib/inch2) for 15 minutes.

2.4.6 Nutrient agar (Oxoid CM 3):

This was prepared by adding 28 g of nutrient agar to 1 L of distilled

water and dissolved by boiling. The pH was adjusted to 7.4, and then

sterilized by autoclaving at 121ºC (15 Ib/inch2) for 15 minutes. The prepared

medium was distributed in 20 ml volume into sterile Petri dishes. The

poured plates were allowed to solidify on flat surface.

2.4.7 Blood agar (Oxoid CM 55):

This was prepared according to Barrow and Feltham (1993) by

suspending 40 g of blood agar base in 900 ml of distilled water and

dissolved by boiling. The mixture was sterilized by autoclaving at 121ºC (15

Ib/inch2) for 15 minutes and cooled down to about 50 ºC, then defibrinated

sheep blood was added aseptically to make a final concentration of 10%.

The prepared medium was mixed gently and distributed in 20 ml volumes

into sterile Petri dishes. The poured plates were allowed to solidify on

leveled surface.

2.4.9. Chocolate agar medium:

This medium was prepared according to Barrow and Feltham (1993)

by dissolving 40 g of blood agar base (Oxiod) in 1 L distilled water. The

medium was sterilized by autoclaving at 121 ºC (15 Ib/inch2), then cooled to

75-80 ºC in water bath and 5% sterile defibrinated sheep blood was added

62

with frequent mixing until the medium possessed a chocolate color. The

prepared medium was distributed in 20 ml amounts in sterile Petri dishes.

The poured plates were allowed to solidify on leveled surface.

2.4.9 Diagnostic sensitivity test agar (Oxoid CM 261):

This medium was prepared as described by Barrow and Feltham

(1993). It composed of peptone, veal infusion solid, dextrose, sodium

chloride, disodium phosphate, sodium acetate, adenine sulphate, guanine

hydrochloride, uricil, xanthine and ion agar. Forty grams of medium was

dissolved by boiling in 1 L of distilled water. The pH was adjusted to 7.4;

and sterilized by autoclaving at 121 ºC (15 Ib/inch2) for 15 minutes. The

sterilized medium was distributed in 20 ml volumes into sterile Petri dishes

.The poured plates were allowed to solidify on leveled surface.

2.4.10 MacConkey agar medium (Oxoid CM 7):

Fifty two grams of MacConkey’s agar were dissolved in 1 L distilled

water. The pH was adjust to 7.4, sterilized by autoclaving at 121 ºC (15

Ib/inch2) for 15 minutes and then distributed in 20 ml volumes into sterile

Petri dishes. The poured Petri dishes were allowed to solidify on flat surface.

2.4.11 Eosin Methyline Blue Agar – EMB:

Ten grams of peptone (Oxoid), 10 g of lactose, 2 g of dipotassium

hydrogen phosphate, 0.4 g of eosin, 0.065 g of methyline blue and 15 g of

agar No.3 (Oxoid) were added to 1 L of distilled water. The pH was adjust to

6.8, sterilized by autoclaving at 121 ºC (15 Ib/inch2) for 15 minutes and then

distributed in 20 ml volumes into sterile Petri dishes. The poured Petri

dishes were allowed to solidify on flat surface.

2.4.11 Motility medium - Cragie tube medium:

Thirteen grams of dehydrated nutrient broth (Oxoid CM 1) were

added to 5 g of Oxoid agar No.1 and dissolved in 1 L of distilled water. The

63

pH was adjusted to 7.4. The prepared medium was distributed in 5 ml

volumes into clean test tube which containing appropriate Cragie tubes and

then sterilized by autoclaving at 121 ºC (15 Ib/inch2) for 15 minutes.

2.4.12 Hugh and Liefsons (O/F) medium:

This medium was prepared as described by Barrow and Feltham

(1993). Two grams of peptone powder, 5 g of sodium chloride, 0.3 g of

potassium hypophosphate and 3 g of agar were added to 1 L of distilled

water. The pH was adjusted 7.1 and the indicator bromocresol purple was

added. The complete medium was distributed into test tubes in 5 ml amount.

The medium was sterilized by autoclaving at 115 ºC for 10 minutes.

2.4.13 Simmon citrate medium (Oxoid CM 155):

This medium contained sodium ammonium phosphate, ammonium

dihydrogen phosphate, magnesium sulphate, sodium citrate, sodium

chloride, bromothymol blue as indicator and agar NO.3 (Oxoid L 13). The

medium was obtained from Oxoid (Ltd). It was prepared according to

manufacture instruction by dissolving 17 g of powder in 1 L of distilled

water. The prepared medium was distributed in 10 ml volume into clean

bottles, sterilized by autoclaving at 121 ºC (15 Ib/inch2) for 15 minutes and

left to solidify in inclined position.

2.4.14 Urea agar medium:

This medium was obtained from Oxoid (Ltd). It contained peptone,

dextrose, disodium phosphate, sodium chloride, potassium dihydrogen

phosphate, agar and phenol red. It was prepared according to manufacture

instructions by dissolving 2.4 g in 95 ml of distilled water and dissolved by

boiling. The prepared medium was sterilized by autoclaving at 121º C for 15

minutes, cooled to 50 ºC, and then 5 ml of sterilized 40% urea solution

(Oxoid SR 20) were added under aseptic condition. The medium was

64

distributed in 5 ml volumes into sterile bottles and left to solidify in inclined

position.

2.5 Collection of samples:

A total of 78 samples were collected from sick chickens with clinical

symptoms of respiratory tract diseases. These symptoms include mucoid or

serous nasal discharge, sneezing, lacrimation, conjunctivitis and facial

swelling. The samples were collected from the common breeds raised in

Khartoum North area. All samples were collected from farms where

chickens are vaccinated against Newcastle disease and fowl pox.

2.5.1 Sampling and culture:

2.5.1.1 Nostril:

Sterile cotton wool swabs were used for sampling the nostril of live

chicken.

2.5.1.2 Trachea:

Sterile cotton wool swabs were used for taking samples from the

inside of trachea of recently slaughtered chicken.

2.5.1.3 Lung:

The lung of recently slaughtered chicken was cut into pieces with

sterile scalpel and small piece was taken by sterile forceps.

2.5.1.4 Conjunctiva:

The eyes of the dead chickens were opened with sterile forcep and

sterile cotton swab was used for sampling conjunctival sac.

2.6 Primary culturing:

Nasal, tracheal, conjunctival swabs and cut piece of lung were

inoculated into nutrient broth and incubated overnight at 37 ºC.

The collected samples which were inoculated into a nutrient broth and

incubated overnight at 37 ºC were subcultured onto a blood agar,

65

MacConkey’s agar.

The inoculated plates were incubated for 24-48 h at 37 ºC. The

colonies characteristics were observed. Smears were made from each type of

colony, stained by Gram’s Method and examined under light microscope for

cell morphology, cell arrangement and staining reaction.

2.7 Purification and preservation of culture:

Purification of culture was done by sub-culturing part of typical well

separated colony on the corresponding medium. The process was repeated

several times. The purity of the culture was checked by examining stained

smear. Pure culture was then inoculated into nutrient agar slant medium and

incubated overnight at 37 ºC. The pure culture was then stored at 4 ºC for

studying cultures and biochemical characteristics and sensitivity of the

isolates.

2.8 Microscopic examination:

Smears were made from each types of colony on primary cultures and

from purified colonies. Then fixed by heating and stained by Gram stain

method according to Barrow and Feltham (1993) and examined

microscopically under oil immersion lens. The smear was examined for cell

morphology, cell arrangement and stained reaction.

2.9 Identification of isolates:

The purified isolates were identified according to criteria described by

Barrow and Feltham (1993). This included staining reaction, cell

morphology, growth condition, colonial characteristics on different media,

haemolysis on blood agar and biochemical characteristics.

66

2.10 Biochemical methods for identification of isolated bacteria:

All biochemical tests were performed as described by Barrow and

Feltham (1993). They included:

2.10.1 Catalase test:

A drop of 3% H2O2 was placed on clean slide and colony of test

culture on nutrient agar was picked by glass rod and added to the drop of

H2O2. Positive reaction was indicated by evolution of gas (air bubbles).

2.10.2 Oxidase test:

Strip of filter paper was soaked in 1% solution of tetramethyl-p-

phenylene diamine dihydrocholoride and dried in hot air oven and then

placed on clean glass slide by sterile forceps. A fresh test culture put on the

filter paper strip. If a purple color developed within 5-10 second, the

reaction was considered positive

2.10.3 Oxidation fermentation (O/F) test:

Duplicate tubes of Hugh and Liefsons medium were inoculated by

stabbing with straight wire. One of the tubes was sealed by layer of sterile

soft paraffin oil to protect it from air; both inoculated tubes were incubated

at 37 ºC and examined daily for a period of fourteen days. Yellow color in

open tube indicated oxidative reaction, yellow color in both tubes indicated

fermentation reaction. Blue color in the open tube and green in the sealed

tube indicated production of alkali.

2.10.4 Motility test:

Motility medium was inoculated by stabbing with straight wire into

the center of the Cragie tube and then incubated at 37 °C for 24 h. The

organism was considered motile if there was turbidity in the medium in and

outside the Cragie tube while the growth of non motile organism confined

67

inside Cragie tube.

2.10.5 Sugar fermentation test:

Carbohydrate medium was inoculated with test culture then incubated

at 37 ºC and examined daily for 7 days. The acid production was indicated

by change in color to pink and gas production was indicated by presence of

empty space in Durham’s tubes.

2.10.6 Indole production test:

The test culture was inoculated into peptone water and incubated at 37

ºC for 48 h. One ml Kovac’s reagent was added to the tube. The appearance

of a pink color in the reagent layer within a minute indicated positive

reaction.

2.10.7 Methyl red test:

The test culture was inoculated into glucose phosphate medium and

then incubated at 37 ºC for 48 h. Two drops of methyl red reagent were

added and shaken well. Red color indicated positive reaction. Yellow or

orange color indicated negative reaction.

2.10.8 Voges-Proskure test:

The test culture was inoculated into glucose phosphate medium and

incubated at 37º C for 48 h. One ml of culture medium was transferred

aseptically into sterile test tubes and then 0.6 ml of 5% alpha-naphthol

solution was added, followed by 0.2 of 40% KOH aqueous solution. The test

tube was shaken well and kept at slant position for 1 h. Positive reaction was

indicated by strong red color.

2.10.9 Citrate utilization test:

The test culture was inoculated onto Simmon’s citrate medium, then

incubated at 37 ºC and examined daily for 7 days. Blue color indicated

positive reaction.

68

2.10.10 Hydrogen sulphide production:

A tube of peptone water was inoculated by tested organism and lead

acetate paper was inserted between the cotton plug and the tube ,then

incubated at 37 °C and examined daily for a week. Blacken of the paper

indicated H2S production.

2.10.11 Nitrate reduction test:

The nitrate broth was inoculated lightly and incubated for up to five

days and 1 ml of reagent (A) Sulphanilic acid was added followed by 1 ml of

reagent (B) α-naphthylene amine. A deep red colour indicated that nitrate

has been reduced. To the tubes not showing a red colour within 5 minutes,

powdered zinc was added and allowed to stand. Red colour indicated

negative reaction.

2.10.12 Coagulase test:

2.10.12.1 Slide coagulase test:

A colony of tested culture was placed on a clean glass slide,

emulsified in a drop of normal saline and then a loop-full of human plasma

was added to bacterial suspension. Appearance of coarse microscopically

visible clump was recorded as positive result.

2.10.12.2 Tube coagulase test:

To 0.5 ml of 1: 10 dilution human plasma in normal, 0.1 ml of an 18-

24 hours old broth culture of test organism was added, then incubated in

water bath at 37 °C and examined after 4-6 h for coagulation. Definite clot

formation indicated positive result.

2.10.13 Novobiocin sensitivity test:

The standard disc diffusion method was used to examine the

sensitivity of the test organism to Novobiocin. Five milligrams Novobiocin

69

sensitivity disc (Oxoid, LTd) was used. A plate of DST medium was dried in

oven at 40 °C for 20 minutes, and then 1 ml of diluted suspension of the test

organism was poured onto the surface of the medium in the plate. Excess

suspension was drawn off and the plate was allowed to dry at room

temperature for 30 minutes. The Novobiocin disc was gently applied on the

plate using sterile forceps. Then the plate was incubated at 37 °C for 24 h.

The test organism was reported as sensitive if there was a zone of growth

inhibition around the disc. The zone of growth inhibition around the disc

was measured in millimeters.

2.11 Antibacterial sensitivity test:

The sensitivity of isolates to antibacterial drugs agents was

determined by disc diffusion technique. The isolates were cultured in

peptone water and incubated at 37 °C for 2 h. Petri dishes containing

diagnostic sensitivity test (DST) agar medium were put in the incubator at

37 °C for 30 minutes to dry and then inoculated with 1 ml volume of the

culture. The inoculated culture was evenly distributed by rotation, the excess

inoculums were withdrawn by sterile microtitter pipette and the plate was

left to dry at room temperature for 15 minutes. Commercially prepared

antibiotic discs of Plasmatic laboratory were placed on surface of the

medium by sterile forceps and pressed gently to insure good contact with the

surface of the culture medium. The plates were then incubated at 37 °C for

24-48 h. The sensitivity of the isolates was examined to the following

antibacterial drugs: Ampicillin (20 mcg), Co-trimaxazole (25 mcg),

Cephalexin (30 mcg), Tetracycline (25 mcg), Cefotaxime (30 mcg),

Ciprofloxacin (5 mcg), pefloxacin (10 mcg), Ofloxacin (5 mcg), Cloxacillin

(1 mcg), Roxythromycin (15 mcg), Lincomycin (2 mcg), Gentamycin (10

mcg), Peperacillin\Tazobactam (100\10 mcg), Chloramphenicol (30 mcg),

70

Ceftizoxime (30 mcg) and Amikacin (30 mcg).

The test organism was considered sensitive if there was a zone of

inhibition of 10 mm or more a round the disc according to manufacture’s

instructions

71

CHAPTER THREE

RESULTS

3.1.1 Bacterial isolated from nostrils:

The number of samples collected from nostrils was 42.

Out of these, 34 (80.95%) samples gave positive growth and they yielded 55

(130.95%) isolates, while the remaining 8 (19.05%) samples did not show

any growth . The 55 (130.95%) bacteria comprised both Gram positive and

Gram negative bacteria. Thirty four (80.95%) isolates were found to be

Gram positive while the other 21 (50%) isolates were Gram negative. The 34

isolates of Gram positive bacteria were 7 (16.7%) Staphylococcus aureus, 4

(9.5%) Staphylococcus gallinarum, 2 (4.8%) Staphylococcus epedermidis 2

(4.8%) Staphylococcus auriclaris, 6 (14.3%) Bacillus cereus, 2 (4.8%)

Bacillus lentus, 1 (2.4%) Bacillus pantothenticus, 3 (7.1%) Micrococcus

roseus, 3 (7.1%) Micrococcus sedentarius, 2 (4.8%) Micrococcus lylae

and 2 (4.8%) Streptococcus lentus.

The 21 isolates of Gram negative bacteria were 6 (14.3%) E.coli, Fig

(1) & (2) demonstrated growth of E. coli on MacConkey’s agar & Eosin

Methyline blue agar medium consecutively. 3 (7.1%) Escherichia hermanii,

2 (4.8%) Klebseilla ozaenae, 1 (2.4%) Klebseilla pneumoniae, 1 (2.4%)

Klebseilla aerogenes, 3 (7.1%) Citrobacter koseri, 3 (7.1%) Acenitobacter

calcoaceticus and 2 (4.8%) Pseudomonas areuginosa.

3.1.2 Bacteria isolated from conjunctiva:

The samples collected from conjunctiva were 3. Two samples showed

a positive growth while the third was found negative. The 2 positive samples

gave three isolates. One (33.3%) isolate was Gram positive bacteria which

72

was Bacillus alvei.

The other two isolates were Gram negative bacteria, 1 (33.3%) was

Acenitobacter woffii and 1 (33.3%) was klebseilla ozaenae.

3.1.3 Bacteria isolates from trachea:

Twenty five samples were collected from trachea. Twenty two

samples showed positive growth while the other 3 samples were found

negative. The 22 samples gave 24 isolates. Thirteen isolates were Gram

positive bacteria, 2 (8 %) Staphylococcus aureus, 2 (8 %) Staphylococcus

epidermidis, 1 (7.7%) Staphylococcus chromogen, 1 (4 %) Staphylococcus

epedermidis, 1 (4 %) Bacillus cereus, 2 (8 %) Bacillus pantothenticus, 2 (8

%) Micrococcus roseus, 1 (4 %) Micrococcus lylae and 1 (4 %)

Streptococcus pneumoniae.

The other 11 isolates were Gram negative bacteria, 4 (16 %) isolates were

E.coli, 2 (8 %) isolates of Escherichia hermanii, 2 (8 %) Klebseilla

ozaenae, 2 (8 %) Citrobacter koseri and 1 (4 %) Acenitobacter woffii.

3.1.4 Bacteria isolated from lungs:

The 8 samples collected from lung gave 6 positive samples. The six

positive samples gave 7 isolates. Four isolates were Gram positive bacteria,

2 (25 %) isolates were Staphylococcus capitis, 1 (12.5%) Bacillus

thuringiensis and 1 (12.5%) Streptococcus pneumoniae.

The other 3 were Gram negative, 1 (12.5%) E.coli, 1 (12.5%) Citrobacter

koseri and 1 (12.5%) Pseudomonas maltophilia. Frequency of bacterial

isolates from infected organs is shown in table (1). Number & % of Gram

+ve & -ve isolates isolated from different organs are demonstrated in tables

(2) & (3).

73

3.2 Characters and biochemical reactions:

Characters and biochemical reactions of Gram-positive and Gram-negative

bacterial isolates showed in tables (4) & (5).

3.3 Antibacterial sensitivity of the isolated bacteria:

Table (6) summarized the number and percentage of sensitive isolates

to antibacterial examined. Fig. (3) demonstrated sensitivity test of Gram-

positive bacteria. The isolates were highly sensitive to Gentamicin (85.7%)

followed by Ofloxacin (78.6%), Chloramphenicol (75%), Co-Trimoxazole

(71.4%), Amikacin (66.7%), Piperacillin/Tazobactam (58.3%), pefloxacin

(50%), Cefotaxime (46.4%), Cloxacillin (43.75%) Ampicillin (42.9%),

Ceftizoxime (41.7%), Ciprofloxacin (32.1%), Cephalexin (31.25%),

Tetracycline (28.6%), Roxythromycin (18.75%) while the Lincomycin

(12.5%) showed the least inhibition to the growth. Detailed results of the No.

of the sensitive and resistant bacterial spp. Are shown in table 7 & 8.

74

Table (1) Isolation frequency of aerobic bacteria from respiratory tract of

infected chicken in Khartoum North area.

Samples source

No. of samples

examined

Total No. of isolates (%)

No. of Gram positive isolates (%)

No. of Gram negative isolates (%)

Nostrils 42

55 (130.95%) 34 (80.95%) 21 (50%)

Conjunctiva 3

3 (100%) 1 (33.33%) 2 (66.67%)

Trachea 25

24 (96%) 13 (52%) 11 (44%)

Lung 8

7 (87.5%) 4 (50%) 3 (37.5%)

75

Table (2) Gram positive bacteria isolated from respiratory tract of infected

chickens in Khartoum North area.

Bacterial species

Number (%) of isolates from:

Nostrils

n = 42

Conjunctiva

n = 3

Trachea

n = 25

Lung

n = 8

Staphylococcuc aureus 7 (16.7%) - 2 (8 %) -

Staphylococcus gallinarum 4 (9.5%) - 1 (4 %) -

Staphylococcus auriclaris 2 (4.8%) - - -

Staphylococcus epedermidis 2 (4.8%) 2 (8 %)

Staphylococcus chromogen - - 1 (4 %) -

Staphylococcus capitis - - - 2 (25 %)

Bacillus cereus 6 (14.3%) - 1 (4 %) -

Bacillus lentus 2 (4.8%) - - -

Bacillus alvei 1 (33.3%)

Bacillus pantothenticus 1 (2.4%) 2 (8 %)

Bacillus thuringiensis 1 (12.5%)

Micrococcus sedentarius 3 (7.1%)

Micrococcus roseus 3 (7.1%) 2 (8 %)

Micrococcus lylae 2 (4.8%) 1 (4 %)

Streptococcus pneumoniae - - 1 (4 %) 1 (12.5%)

Streptococcus lentus 2 (4.8%) - - -

76

Table (3) Gram negative bacteria isolated from respiratory tract of infected

chickens in Khartoum North area.

Bacterial species

Number (%) of isolates from

Nostrils

n = 42

Conjunctiva

n = 3

Trachea

n = 25

Lung

n = 8

Escherichia coli 6 (14.3%) - 4 (16 %) 1 (12.5%)

Escherichia hermanii 3 (7.1%) - 2 (8 %) -

Klebseilla ozaene 2 (4.8%) 1 (33.3%) 2 (8 %) -

Klebseilla pneumoniae 1 (2.4%) - - -

Klebseilla aerogenes 1 (2.4%) - - -

Pseudomonas aeruginosa 2 (4.8%) - - -

Pseudomonas maltophilia - - - 1 (12.5%)

Acenitobacter calcoaceticus 3 (7.1%) - - -

Acenitobacter woffii - 1 (33.3%) 1 (4 %)

Cetrobacter koseri 3 (7.1%) 2 (8 %) 1 (12.5%)

77

Table (4) Characters and biochemical reaction of Gram-positive

bacteria isolated from respiratory tract of infected chickens in

Khartoum North area.

Bacterial species Characters1 2 3 4 5 6 7 8 9 10 11 12 13 14

Staphylococcus aureus + + - F - NT + + + - + + + +

Staphylococcus gallinarum + + - F - NT - + - + + - + +

Staphylococcus auriclaris + + - F - NT - + - - - - - -

Staphylococcus epedermidis + + - F - NT - + + - - + + +

Staphylococcus chromogenes + + - F - NT - + + - + - + +

Bacillus cereus + + + - + C NT - - - - + - +

Bacillus lentus + + + F + C NT - - - - - - -

Bacillus alvei + + + - + C NT + - - - + -

Bacillus pantothenticus + + + F + T NT - - - - - - +

Bacillus thuringiensis + + + - + C NT - - - - + - +

Micrococcus sedentarius - + + O _ NT NT - - - NT - NT -

Micrococcus roseus + + + - - NT NT - NT NT NT - NT +

Micrococcus lylae - + + O - NT NT - NT NT NT - NT -

Streptococcus pneumonae + - - F - NT NT + + NT - - NT -

Streptococcus lentus + - - F - NT NT + NT + + NT -

+ = Positive reaction, _ = Negative reaction, F = Fermentative, O = Oxidative, NT =Not

tested, C = Central spore, T = Terminal spore.

1 = Glucose, 2= Catalase, 3 = Oxidase, 4 = O\F, 5 = Motility, 6 = Spore forming, 7 =

Coagulase, 8 = Sucrose, 9 = Lactose, 10 = Xylose, 11 = Manitol, 12 = VP, 13 = Urease,

14 = Nitrate.

78

Table (5) Characters and biochemical reactions of Gram negative bacteria

isolated from respiratory tract of infected chickens in Khartoum North area.

Bacterial

species

Characters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16E. coli + + _ F + + + + + + + _ _ _ + _

E.coli hermanii + + _ F + + + + + _ + _ _ _ + _

Klebseilla ozaenae + + _ F _ _ + + + + + _ _ _ _ _

Klebseilla aerogenes + + _ F _ + + + + + + + + _ _ +

Klebseilla

pneumonia

+ + _ F _ + + + + + + + _ _ _ +

Pseudomonas

aeruginosa

+ + + _ + _ _ _ _ _ + + _ _ + +

Pseudomonas

maltophilia

_ + + O + _ _ + _ _ _ _ _ _ _ _

Acinetobacter

calcoaceticus

_ + _ O _ _ + _ + _ + + _ NT _ +

Acinetobacter woffii _ + + O _ _ _ _ _ _ _ _ NT _ _ _

Citrobacter koseri + + _ F + + + + + + + + _ + + +

Keys: + = Positive reaction, _ = Negative reaction, F = Fermentative, O = Oxidative, NT

=Not tested.

1= Glucose, 2= Catalase, 3 = Oxidase, 4 = O\F, 5 = Motility, 6 = Sucrose, 7 = Lactose, 8

= Maltose, 9 = Salicin, 10 = Sorbitol, 11 = Xylose, 12 = Urease, 13 = VP , 14 = H2S,

15 = Indole, 16 = Citrate.

79

Table (6) Antibacterial sensitivity of bacterial isolated from respiratory

tract of infected chicken in Khartoum North area.

Antimicrobial drug

No. of bacterial

isolates

examined

No. of sensitive

(percentage)

isolates

No. of resistant

(percentage)

isolates

Ampicillin 28 12 (42.9%) 16 (57.1%)

Co-Trimoxazole 28 20 (71.4%) 8 (28.6%)

Cefotaxime 28 13 (46.4%) 15 (53.6%)

Piperacillin/Tazobactam 12 7 (58.3%) 5 (41.7%)

Chloramphenicol 12 9 (75%) 3 (25%)

Ciprofloxacin 28 9 (32.1%) 19 (67.9%)

Ceftizoxime 12 5 (41.7%) 7 (58.3%)

Tetracycline 28 8 (28.6%) 20 (71.4%)

Ofloxacin 28 22 (78.6%) 6 (21.4%)

Gentamicin 28 24 (85.7%) 4 (14.3%)

Amikacin 12 8 (66.7%) 4 (33.3%)

Pefloxacin 28 14 (50%) 14 (50%)

Cephalexin 16 5 (31.25%) 11 (68.75%)

Cloxacillin 16 7 (43.75%) 9 (56.25%)

Roxythromycin 16 3 (18.75%) 13 (81.25%)

Lincomycin 16 2 (12.5%) 14 (87.5%)

80

Table (7) The antibacterial sensitivity of Gram positive bacterial species isolated from respiratory of infected chickens in Khartoum North area.

Bacterial species

No. of examined isolates

No. of isolates inhibited by antibacterial drugsAS BA PR TE CF CP PF OF CX RF LM GM

Bacillus cereus 6 3(++)3(-)

3(++++)3(++)

2(++)4(+)

4(+++)2(-)

4(-)2(+)

6(+++) 4(++)2(-)

6(++) 4(++)2(-)

3(+++)3(-)

2(+)4(-)

6(+++)

Staphylococcus aureus

3 2(+)1(-)

3(-) 3(++) 1(++)2(-)

1(+)2(-)

3(-) 1(+)2(-)

1(++)2(-)

3 (-) 3(-) 3(-) 1(++)2(-)

Staphylococcus gallinarum

3 2(++)1(-)

2(+)1(-)

1(++)1(+)1(-)

3(-) 1(+)2(-)

3(-) 3(-) 1(+)2(-)

2(+)1(-)

3(-) 3(-) 2(+++)1(++)

Staphylococcus epedermidis

2 2(-) 1(+)1(-)

2(++) 2(-) 2(++) 2(++) 2(++) 2(++) 1(++)1(-)

2(-) 2(-) 2(+++)

Streptococcus lentus

2 2(++) 1(++)1(-)

2(+) 1(++)1(-)

2(-) 2(-) 1(+)1(-)

2(++) 2(-) 2 (-) 2 (-) 2(++)

(AS) Ampicillin/Sulbactam (20 mcg), (BA) Co-Trimoxazole (25mcg), (PR) Cephalexin (30mcg), (TE) Tetracycline (30 mcg), (CF) Cefotaxime

(30 mcg), (CP) Ciprofloxacin (5 mcg), (PF) Pefloxacin (10mcg), (OF) Ofloxacin (5mcg), (CX) Cloxacillin (1mcg), (RF) Roxythromycin

(15mcg), (LM) Lincomycin (2 mcg) and Gentamicin (10 mcg)

Inhibition zone: ( ++++) = 25 mm, (+++) = 20 mm, (++) = 15, (+) = 10 mm, (-) no inhibition.

81

Table (8) The antibacterial sensitivity of Gram negative bacterial species isolated from respiratory of infected

chickens in Khartoum North area.

Bacterial species

No. of examined isolates

No. of isolates inhibited by antibacterial drugsAS BA CF TZP CH CP CI TE OF GM AK PF

E. coli 7 2(+) 5(-)

3(+++) 4(++)

4(+) 3(-)

4(++) 3(-)

2(++++) 1(+++) 4(-)

1(+) 6(-)

3(++) 4(-)

3(+) 4(-)

4(+++) 1(++) 2(-)

7(++++) 3(+++) 1(++) 3(-)

3(++)4(-)

Citrobacter

koseri

2 2(-) 1(++++) 1(+++)

2(+) 2(+) 1(++) 1(-)

2(-) 2(++) 2(-) 1(+++) 1(++)

1(+) 1(-)

1(++) 1(-)

2(-)

Acinetobacter

woffii

2 1(+) 1(-)

2(-) 2(-) 2(+++) 2(++++) 2(-) 2(-) 2(-) 2(++)

2(++++) 1(+++) 1(++)

2(+++)

Klebsiella

pneumoniae

1 1(-) 1(+) 1(+) 1(-) 1(+++) 1(-) 1(-) 1(-) 1(++) 1(++++) 1(+) 1(-)

(AS) Ampicillin/Sulbactam (20 mcg), (BA) Co-Trimoxazole (25mcg), (CF) Cefotaxime (30 mcg) (TZP) Piperacilin/Tazobactam (100/10 mcg),

(CH) Chloramphenicol (30 mcg), (CP) Ciprofloxacin (5 mcg), (CI) Ceftizoxime (30 mcg), (TE) Tetracycline (30 mcg), (10mcg), (OF)

Ofloxacin (5mcg), Gentamicin (10 mcg) (AK) Amikacin (30 mcg), and (PF) Pefloxacin.

Inhibition zone: (++++) = 25 mm, (+++) = 20 mm, (++) = 15, (+) = 10 mm, (-) no inhibition.

82

Fig. 2 : Growth of E. coli on Eosin Methylene Blue Agar

Fig. 1 : Growth of E. coli on MacConkey's agar

83

Fig. 3 Sensitivity test of Gram-positive bacteria

84

CHAPTER FOUR

DISCUSSION

This study was carried out to isolate and identify aerobic

bacteria infecting respiratory tract of chickens in Khartoum North area.

Seventy eight samples were collected from infected chickens and cultured.

In this work the bacterial isolates were obtained from nostril, conjunctiva,

trachea and lung. Fourteen samples did not show any bacterial growth

despite of clear respiratory symptoms, this phenomenon may be due to

Mycoplasma or viral infections. Sixty four samples showed bacterial growth

and gave 89 isolates.

Pseudomonas species were isolated from nostrils of infected chickens.

This agrees with other studies which reported the isolation of Pseudomonas

species from nostrils of infected chickens (Valadae, 1961; Saad et al., 1981;

Andreev et al., 1982; Bapat et al., 1985 and Mrden, 1988). In sudan

Pseudomonas aeruginosa was isolated from cases of substantial deaths

among young chickens (Elnasry, 1997) and Mohamed et al. (1996).

In this study E. coli was isolated from nostrils, trachea and lung of

infected chickens. Several authors reported the isolation of E. coli from

respiratory tract of infected chickens (Price et al., 1957; Sojka et al., 1961;

Mac Martin, 1962; Khogali, 1970; Mouline, 1983; Eisa and Elnasry, 1985;

Mahgoub, 1986; Linzitto et al., 1988; Malokwa et al., 1987; and Elnasry,

1997). Also other study confirms isolation E. coli from lung (Hofstad et al.,

1978; Rajashekar et al., 1998; and Abdellah, 2003). Zahida (2004) described

the respiratory tract as the primary route of invasion by E. coli.

The lesions and symptoms of respiratory infection may be aggravated

when secondary E. coli invasion occurred. Sanikbi (1987) and Seethe (1988)

85

reported the complication of infectious coryza with E. coli which led to

chronic disease. History of recent Newcastle disease vaccination or

ammonia pollution was not ruled out as predisposing factors to E. coli

infection (Bakhiet et al., 1990). History of infectious bursal disease might

have had a role in E. coli infections (Pages et al., 1985). Nighot (2002)

reported that faulty management and lack of routine vaccination against

some viral diseases may lead to activation of commensally living bacterial

forming the normal flora challenges the natural immunity and defense

mechanism.

Staphylococcus species were isolated from nostril, trachea and lung of

infected chickens in this study also Staphylococcus species was isolated

from respiratory tract infection (Bibersein et al., 1974; Linzitto et al., 1988;

and Elnasry 1997).

In the present investigation Streptococcus species were isolated from

respiratory tract of infected chickens this is in agreement with the findings of

Linzitto et al., (1988).

The antibacterial sensitivity of the isolated bacteria obtained in this

study to antibacterial drugs was variable. In the present study, bacteria

isolated showed resistance to many antibiotic commonly used for treatment

of bacterial diseases in animals.

Members of enterbacteriaceae isolated in this study showed very high

resistance to Ampicillin and Tetracycline; this result is partially similar to

that reported by Mohamed (2005) who reported that, the enterbacteriaceae

species isolated from mastitic cows, goats milk, infected equine uterus and

infected humans were completely resistant to Ampicillin. E.coli isolated in

this study was found sensitive to Gentamicin (100%). This finding agrees

with that of Ahmed (2006), who reported that E. coli isolated from calf

86

feaces was completely sensitive to Gentamicin and in agreement with that of

Orden (2000) who found that E. coli strains isolated from diary calves

affected by neonatal diarrhea were susceptible (89-95%) to Gentamicin.

Staphylococcus epidermidis examined in this study showed high resistance

to Ampicillin, this finding agrees with Forbes et al. (1998) who reported that

Staphylococci are Gram-positive bacteria that most commonly produce B-

lactamase and approximately 90% or more of clinical isolates are resistant to

Pinicillin and Ampicillin as a result of the enzyme production. This

antibiotics resistance can be attributed to many factors, the extensive use of

antibiotics often without prescription from qualified veterinarians, the

animal owners usually use the drugs with subdosing and incomplete duration

of the treatment and they use one type of drug for a long period. Also use of

broad spectrum antibiotics without proper isolation of the causative agent

and drug sensitivity testing is a real cause of resistance.

87

Conclusions and Recommendations

Conclusions:

The result of the present study demonstrated that:-

1 Both Gram-positive and Gram-negative bacterial pathogens were

isolated from respiratory tract of infected chickens.

2 Mycoplasma and pathogens other than bacteria could be a cause of

respiratory tract infection of chicken as 14 (17.9%) of samples

collected from infected chickens did not show bacterial growth.

3 Respiratory tract bacterial infections could be an important constrain

in poultry industry in Khartoum state.

4 Many bacterial isolates were found sensitive to Gentamicin (85.7%),

Ofloxacin (78.6%) and Chloramphenicol (75%).

5 Antibiotic drug resistance may be due to use of the drugs with sub

dosing and incomplete duration of the treatment or extensive use of

these antibiotics also use one type of drug for a long period.

Recommendations:

From results and discussion of this study, the following

recommendations are suggested.

1 The high prevalence of E. coli associated with infection of chickens

needs further study.

2 The future studies should be considered to minimize the spread of

bacteria respiratory diseases.

3 The role of the others pathogens such as mycoplasmas and viruses

88

should be studied.

4 Sensitivity test of bacterial isolates should be conducted using more

variable types of antibacterial drugs to determine the most effective

drugs those kill or inhibit the bacterial growth.

89

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