Isolation of Aerobic Bacteria from Pneumonic Lung of Camels · a. Morphology and staining 44 b. Cultural characteristic 44 c. Biochemical reactions 44 4.4. Gram-negative bacteria

I

Isolation of Aerobic Bacteria from Pneumonic Lung of Camels

By

Asmhan Fadl Alla Mohammed Elhilali

B. Sc. (2003)

Faculty of Veterinary Medicine

University of Khartoum

Supervisor

D. Awadelkarim A. Ibrahim��

A thesis submitted to the University of Khartoum in partial fulfillment of the requirements for the Degree of M. Sc. in Microbiology

University of Khartoum

Faculty of Veterinary Medicine

Department of Microbiology

January, 2012

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II

Dedication

To my parents, my brothers and sisters

To all whom I love



First of all I would like to express my sincere gratitude and

thankfulness to my supervisor D. Awadelkarim A. Ibrahim, for

his help, guidance, suggestions and cooperation throughout this work.

My deepest thanks to

I am also grateful for all staff of the Department of

Microbiology, for providing all the necessary facilities for smooth

and effective execution

Finally my thanks are extended to my wonderful family for

their continuous encouragement and endless support.

III




My deepest thanks to prof. Sanousi for his great help.


providing all the necessary facilities for smooth

and effective execution of this work.


continuous encouragement and endless support.




his great help.


providing all the necessary facilities for smooth




IV

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�ŕƔŠƏƅƏƔũśƄŗ�ƏƇś��ƏƈƊ�ƑƆŷ�¿ƏŰţƅŔŔ�ŕƔũśƄŗƅ�Ɠž��řƊƔŷ��řƆƈŠ�Ɖƈ�ŚŕƊƔŷ��ŚŕœũƅŔƐƏœũƅŔ�ŖŕƎśƅƛŕŗ�řŗŕŰƈƅŔ��

¿Ūŷ�Ƈś�ŧƁƏ�ŧŧŷ��ƗŔ�ƐƏśŬƈ�ƑƆŷ�ŕƍŧƔŧţś�Ƈś�ƑśƅŔƏ�řƔũƔśƄŗ�řƅŪŷƏƊŗ�ŵŔŐ�ŚŕſŰƅŔ�ƇŔŧŦśŬ

ƙŔƏ�řƔŷũŪƈƅŔ�ŚŔũŕŗśŦ�řƔœŕƔƈƔƄƏƔŗƅŔƏŚƆƈŮ: ��

(12.7%) Coagulate- negative Staphylococci ϭ (8%) Staphylococcus aureusϭ

(17.8%) Streptococcus spp. ��ϭ(7.4%) Micrococcus spp.ϭ

(8.1%)Corynebacterium spp. �ϭ(14.1) Actinomyces spp. (15.5%) Bacillus spp.ϭ

(3%) Escherichia coli �ϭ (3%) Klebsiella pneumoniaϭ (3.7%) Aeromonas

hydrophila�ϭ (3.7�) Proteus mirabilissϭ (1.5�) Alcaligenes faecalisϭ(3.7%)

Kingella kingae.

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řųŻŕŲƅŔ�ŽƏũŴƅŔ�



V

Abstract

This study was carried out with the aim of identifying aerobic bacterial

species involved in lung lesions of one -humped adult camels

(Camelusdromedarius) slaughtered at Elsalam abattoir enterprise, Khartoum

State .The study was conducted from February to May 2011.

All camels were originated from various pastoral areas. A total of 100 camel

lungs were inspected at random during the study period, of which 60 (60%)

possessed gross pulmonary lesions. All pneumonic lungs with lesions were

processed for bacteriology and bacterial growth was only obtained from

52(81.5%) of the pneumonic lung samples.

A total of 135 bacterial species were isolated and identified to the species

level using cultural characteristics and biochemical tests. These included

coagulase- negative staphylococci (12.7%), Staphylococcus aureus(8%),

Streptococcus spp. (17.8%), Micrococcus spp. (7.4%) Corynebacteria

spp.(8.1%), Actinomyces spp. (14.1) Bacillus spp. (15.5%) Escherichia coli

(3%) Klebsiella pneumonia (3%), Aeromonas hydrophila(3.7%), Proteus

mirabilis (3.7%) Alcaligenes faecalis(1.5%) Kingella kingae(3.7%). Some of

these are potential pathogens that could induce respiratory diseases and other

isolates might induce Respiratory diseases under stressful conditions.



VI

Table of contents

Page DEDICATION

II

ACKNOWLEDGEMENTS

III

ARABIC ABSTRACT

IV

ABSTRACT

V

TABLE OF CONTENTS

VI

LIST OF TABLES

XVI

LIST OF FIGURES

XVII

Chapter one

1

Introduction

1

Chapter two 3

Literature Review

3

2. 1 Introduction 3

2.2. Anatomy of camels lung 3

2.3. Respiratory tract infections in camels 4

2.4. Common bacteria associated with pneumonia 5

2.4.1. Gram positive bacteria 5

2.4.1.1. Staphylococci 5

2.4.1.1.1. Staphylococcus aureus 5

2.4.1.2. Micrococci 6

2. 4.1.3. Streptococci 6



VII

2.4.1.3.1. Group B streptococci 7

a. Classification 7

b. Lance field's grouping

7

c. haemolysis production

7

2.4.1.4. Corynebacterium spp

7

2.4.1.5. Actinomycetes

8

2.4.1.6. Bacillus spp

8

2.4.2. Gram negative bacteria

9

2.4.2.1. Enterobacteriaceae

9

2.4.2.1.1. Escherichia coli

9

2.4.2.1.2. Klebsiella spp

9

2.4.2.1.3. Proteus spp

10

2.4.2.1.3.1. Proteus mirabilis

10

2.4.2.2. Aeromonas hydrophilia

11

2.4.2.3. Alcaligenes SPP

11

2.4.2.4. Kingella spp

11

2.5. The important bacterial respiratory diseases

11

2.5.1. Pasteurellosis

11

2.5.2. Tuberculosis

12

2.5.3. Mycoplasmosis

13

2.6. Bacteria isolated from infected respiratory tract of camels 13



VIII

2.7. Bacteria isolated from respiratory tract of apparently healthy camels

17

Chapter three

19

Materials and methods

19

3.1. Study area

19

3.2. Collection of samples

19

3.3. Isolation of organism

19

3.4. Sterilization

20

a. Flaming

20

b. Red heat

20

c. Hot air oven

20

d. Moist heat

20

3.5. Reagents and Indicators

20

3.5.1.1. Sodium Chloride

20

3.5.1.2 Alpha-naphthol solution

21

3.5.1.3. Potassium hydroxide

21

3.5.1.4. Hydrogen peroxide

21

3.5.1.5. Kovac's reagent

21

3.5.1.6. Methyl red Solution

21

3.5.1.7. Tetramethyl-p-phenylaminedihydrochloride

21

3.5.1.8. Nitrate test reagent

22



IX

3.5.2. Indicators

22

3.5.2.1 Andrade's indicator

22

3.5.2.2 Bromothymol blue

22

3.6. Preparation of Culture Media

22

3.6.1. Liquid media

23

3.6.1.1. Nutrient broth

23

3.6.1.2. Peptone water

23

3.6.1.3. Peptone water sugar

24

3.6.1.4. Glucose- Phosphate medium

24

3.6.1.5. Nitrate medium

25

3.6.1.6. Serum Water sugars

25

3.6.2. Semi solid media

25

3.6.2.1. Motility test medium- Cragie tube medium

25

3.6.2.2 Oxidation-fermentation medium

26

3.6.3. Solid media

27

3. 6. 3.1. Ammonium salt sugars

27

3.6.3.2. Bile Esculin Azide Agar

27

3.6.3.3. Collection of blood for enriched media

28

3.6.3.4. Blood agar

28

3.6.3.5. MacConkey's agar

29

3.6.3.6. Milk agar 29



X

3.6.3.7. Nutrient agar

30

3.6.3.8. Nutrient Gelatin

30

3.6.3.9. Simmons citrate agar

31

3.6.3.10. Starch

31

3.6.3.11. Urea agar Base

32

3.7. Culture media

33

3.7.1. Blood agar

33

3.7.2. MacConkey agar

33

3.8. Isolation

33

3.9. Purification

33

3.10. Preservation

33

3.11. Microscopic Examination

34

3.12. Biochemical Tests

34

3.12.1. Sugar fermentation test

34

3. 12.2. Oxidase test

34

3.12.3. Catalase test

34

3.12.4. Coagulase test

35

3.12.5. The oxidation- fermentation test

35

``3.12.6. Indole production test

35

3.12.7. Methyl red test

36



XI

3.12.8. Voges- Proskauer test

36

3.12.9. Nitrate reduction

36

3.12.10. Urease activity test

37

3.12.11. Citrate utilization

37

3.12.12. Aesculin hydrolysis

37

3.12.13. Hydrogen sulphide (H2s) production

37

3.12.14. Ammonium salt sugar test

37

3.12.15. Gelatin hydrolysis

38

3.12.16. Mobility test

38

3.12.17. Digestion of casein

38

3.12.18. Starch hydrolysis

38

Chapter four

39

Result

39

4.1. Postmortem findings

39

4.2. Isolation of organisms

39

4.3. Gram- positive bacteria

39

4.31. Staphylococcus spp.

40

a. Morphology and staining

40

b. Cultural characteristic

40

1. Blood agar medium

40

2. Nutrient agar 40



XII

3. Manitol salt agar

40

c. Biochemical reactions

41

4.3.2 Streptococcus spp.

41


41


41


41

b. Biochemical reaction

41

4.3.3.Corynebacterium spp.

42


42


42


42

2. Nutrient agar

42


42

4. 3. 4. Actinomyces spp

42


42


43


43

4.3.5. Bacillus spp

43


43


43

c. Biochemical reactions 43



XIII

4.3.6. Micrococcus spp

44


44


44


44

4.4. Gram-negative bacteria

44

4.4.1. Enterobacteriaceae

44

4.4.1.1. Escherichia coli

44


44


45

1. Blood agar

45

2. MacConkey agar

45

3. Nutrient agar

45


45

4. 4. 1. 2. Klebsiella pneumoniae

45


45


46

1. MacConkey agar

46

2. Blood agar

46


46

4. 4. 1. 3. Proteus mirabilis 46 a. Morphology and staining

46



XIV


46

1. Blood agar

46

2. Nutrient agar

46

d. Biochemical reactions

47

4. 4. 2. Aeromonas hydrophila

47


47


47

1. Blood agar

47

2. MacConkey agar

47


47

4. 4. 3. Kingella kingae

47


47


48


48

4. 4.4. Alcaligenes faecalis

48


48


48

1. Blood agar

48

2. Nutrient agar

48


48



XV

Chapter five

66

Discussion

66

Conclusions

72

Recommendations

73

References 74



XVI

List of Tables

Table Page

1 Types of bacteria isolated from pneumonic lungs 49

2 Staphylococci isolated from pneumonic lungs 51

3 Micrococci isolated from pneumonic lungs of camels 52

4 streptococci isolated from pneumonic lungs of camels 53

5 Actinomyces isolated from pneumonic lungs of camels 54

6 Corynebacteria spp. isolated from pneumonic lungs of

camels

55

7 Bacillus spp. isolated from pneumonic lungs of camels 56

8 Gram negative bacteria isolated from pneumonic lungs

of camels

57

9 Characters and Biochemical reactions of isolated Gram

positive cocci

58

10 Characters and Biochemical reactions of isolated

Corynebacterium spp.

59

11 Characters and Biochemical reactions of isolated

Actinomyces spp.

60

12 Characters and biochemical reactions of isolated

Enterobacteriae.

61

13 Characters and biochemical reactions of isolated

Aeromonas hydrophila

61



XVII

List of figures

No. of Figure Title of figure Page

Figure 1 Gram stained smear showing Actinomyces 64

Figure 2 Growth of Actinomyces in blood agar

65



1

CHAPTER 1

INTRODUCTION

Camels are the most capable animal species in utilizing marginal

areas and in survival and production under harsh environmental

conditions (Knoess, 1977; Gauthier-Pilters and Dagg, 1981; Hjort and

Hussein, 1986; Abbas and Tilley, 1990 and Schwartz, 1992). Many

pastoral groups and communities in the Sudan and diverse eco-zones

throughout the world are depending on camels for their livelihood.

Camels were, and still are, valued for carrying baggage, hair, hides

and as well as the best food providers in the arid areas (Sweet, 1965).

Sudan and Somalia have 70% of the total Africa camels and 55% of that

of the world camel’s population (Wilson, 1984). Moreover, increased

frequencies of drought recurrence, shrinkage and deterioration of the

rangeland by desert encroachment together with increasing aridity are the

major governing factor for the movement of dromedary camels into the

savannah belt. Thus contact between camels and other farm animals has

been increasingly noticeable from the early eighties of the last century.

Camel is a comparatively sturdy animal and is less susceptible to many

of the diseases that affect other livestock species in the same areas

(Schwartz and Dioli, 1992 and Dirie and Abdurhaman, 2003). However,

it is apparent that little is known about the diseases from which it suffers.

Camel of Sudan has suffered from various diseases like trypanosomosis,

camel pox, mange, respiratory disease complex, hemorrhagic septicemia,

pustular dermatitis, dermatomycosis, gastrointestinal parasites and acute

plant poisoning (Agab and Abbas, 1999).



2

Flora of respiratory tract of apparently healthy and infected camels

was reported by different authors (Farrag et al., 1953; Shigidi, 1973;

Chauchan et al., 1986; Alani et al., 1990 and Mustafa, 1992).

Respiratory tract infections are of common occurrence in various species

of domestic and farm animals (Mohamed and Abdelsalam, 2008).

Viruses, bacteria, fungi and parasites have been incriminated as the main

causative agents of pneumonia in mammals (Jubb et al., 19

93 and Cortan et al., 1999). These agents may represent risk to camels,

other livestock and even human population (Abou, 2000; Ogusan et al.,

2000; Bardonn et al., 2002 and Teshom et al., 2003).

Few studies were conducted on the extent of respiratory problems of

camels compared to other livestock species. Respiratory diseases have

been a major threat to the camel population. Respiratory disease outbreak

in camels that is characterized by sudden death has occurred in Ethiopia

(Awol et al., 2011). The most important predisposing factors for camel

diseases are sudden climatic changes, poor management practices,

exposure to various diseases, traveling and low grade nutrition (Schwartz

and Dioli, 1992 and Abubakar et at., 2008). Despite the versatile uses in

the pastoralists' areas, camel has been largely neglected by international

agencies and local governments regarding to improvement of its health

and productivity (Bekele, 1999). Yet there is a need to identify the causes

of respiratory diseases in camels in order to design better control

strategies.

This work was done to identify the etiology of aerobic bacteria

associated with pneumonic lungs of camels slaughtered in Elsalam

abattoir in Khartoum State.



3

Chapter 2

Literature Review

2.1. Introduction:

Pneumonia is an inflammation of pulmonary parenchyma usually

accompanied by inflammation of bronchioles and often with pleurisy

(Blood, Radostitis and Henderson, 1983). It is manifested clinically by

increase in respiratory rate, cough and abnormal breathing sounds. Most

of the bacterial agents of pneumonia are considered to be of aerogenous

orign reaching the lungs through the upper respiratory tract. Infection of

the latter causes inflammation, either acute or chronic (Blood and

Henderson, 1974).

The response of the respiratory tract to disease is determined largely

by the structural and functional complexity of the system. Most of the

diseases of the respiratory system are caused by damaging agents that

arrive either air borne (aerogenous) or blood borne (haematogenous). Air

borne infectious agents commonly cause respiratory diseases and in some

intensive systems of animal husbandry they constitute the most important

cause of morbidity and mortality.

2.2. Anatomy of camel Lungs:

Camel lungs are soft and spongy and characterized by absence of

lobulation and fissures and resemble those of horses. The right lung

consists of cranial lobe, caudal lobe, and accessory lobe while it lacks the

middle lobe. The left lung consists of cranial and caudal lobes only. The

tracheal bronchus, which aerates the apical lobe, is also found. The apical

lobe of the right lung takes its bronchus directly from the trachea.



4

Histologically, the alveolar wall is relatively thick. The type-1

pneumocytes are elongated, flattened cells with densely stained nuclei.

The type -11 pneumocytes are large cuboidal or round cells having large,

round nuclei with prominent nucleolus and vacuolated cytoplasm. The

interlobular septa are prominent. They divide the lung into lobules. Large

amounts of connective tissue are present between lobules which are

composed of collagen and elastin. (Alani, 2004)�

Camel pleura are characteristic and consist of visceral and parietal

layers occupying normally by a small amount of pleural fluid. The pleural

cavities are completely separated. The line of pleural reflection for left

lung extends from the first to twelfth rib on the dorsal aspect while the

cupulae epleurae of the right side extend beyond the first rib. The

phringes, they were once called, or extensions of pleura are at the

periphery of both lungs. These are composed of collagenous and elastic

fibers. They contain blood capillaries, lymph vessels, arterioles, venules

and group of inflammatory cell (Alani, 2004).

2.3. Respiratory tract infections in camels:

The normal flora of upper respiratory tract is important in that it

prevent adherence and colonization by invading bacteria (Jubb and

Kennedy, 1985). Respiratory infection in camels may be due to a variety

of microorganisms that are present in respiratory tract as commensals and

play pathogenic role when the general immune response of the host is

lowered (Shigidi, 1972).

Bacteria can affect respiratory tract of camels causing rhinitis,

pharyngitis, trachitis, bronchitis, pneumonia and lung abscesses.

Exudation and consolidation of lungs characterize bacterial pneumonia

(Jubb and Kennedy, 1985). Haematogenous infection by bacteria results



5

in a varying number of septic foci, which may enlarge to form lung

abscesses (Blood and Henderson, 1983). Pneumonia occurs when these

abscesses rupture inducing a secondary bronchopneumonia.

Arcanobacterium pyogenes and Staphylococcus aureus were isolated

from Lung abscesses (Abubakr et al., 2010).

2.4. Common bacteria associated with pneumonia:

2.4.1. Gram-positive bacteria:

2.4.1.1. Staphylococci

The staphylococci are Gram-positive spherical cells, usually arranged

in grape- like irregular clusters although organisms may appear as single

cells, pairs or short chains (Murray et al., 1998). Staphylococci are non-

motile and do not form spores. They grow readily on most bacteriological

media under aerobic conditions. They grow most rapidly at 37°�C but form

pigment best at room temperature (20- 25°�C).

2.4.1.1.1. Staphylococcus aureus:

Staphylococcus aureus is non- motile, non sporing, aerobic and

facultative anaerobic bacteria, it is catalase positive (Barrow and Feltham,

1993). It is able to grow in a medium containing salt such as manitol salt

agar, which used as a selective medium for isolation of staphylococci that

ferment manitol and colonies appear yellow to cream 1.2 mm in diameter.

The pathogenic Staphylococcus aureus often hemolyse blood, coagulase

plasma and produce a variety of extra- cellular enzymes and toxins.

Coagulase is in extra- cellular protein, which binds to prothrombin to

form a complex called staphylothrombin (Todar, 2002).

Coagulase is a traditional marker for identifying Staphylococcus



6

aureus in the clinical microbiology laboratory. However, there is no

overwhelming evidence that coagulase is a virulence factor, although it is

reasonable to speculate that the bacteria could protect themselves from

phagocytes and immune defenses by causing localized clotting (Todar,

2002).

2.4.1.2. Micrococci

Micrococcus is Gram-positive cocci that are 0.5 to 3.5 micrometers

in diameter and usually arranged in tetrads or irregular clusters. Defining

characteristics of Micrococcus are the ability to aerobically produce acid

from glucose, glycerol. They hydrolyse aesculin and arginine and reduce

nitrate to nitrite (Smith et al. 1999). They attack sugars oxidatively or not

at all (Barrow and Feltham, 1993). Micrococcus species are part of

normal flora of the nasopharynx of domestic animals, they are frequently

found in soil, water, dust, air and skin and on articles of daily use (Carter,

1986). Occasional strains were associated with disease (Barrow and

Feltham, 1993).

2.4.1.3. Streptococci

They are Gram-positive cocci, usually non-motile, smaller than

staphylococci in diameter, aerobic and facultatively anaerobic, catalase-

negative, oxidase negative.They attack sugars fermentatively without gas

production and some may produce haemolysis (Barrow and Feltham,

1993).



7

2.4.1.3.1. Group B streptococci

a. Classification

b. Lance field’s groupings

Serologically, streptococci are classified on the basis of a cell surface

carbohydrate antigen in the cell wall called component C, into serotypes

A to T (Carter et al., 1985 and David Green et al., 2002).

c. Haemolysis production

ß�- haemolytic streptococci produce a clear zone of haemolysis on

sheep blood agar, whereas a�-haemolytic streptococci produce partial

haemolysis with zone of greenish discoloration, ?�- haemolytic

streptococci produce no hemolysis on blood agar (Carter et al., 1985).

Many isolates, however, are only weakly haemolytic and anaerobic

incubation often increases the extent of haemolysis with such strains

(Barrow and Feltham, 1993). Streptococcus pyogenes secretes different

toxins and enzymes that are associated with its virulence. These include

streptolysin O which is oxygen labile, hylouridase which increases tissue

permeability and leucocidin which destroys leucocytes (Barrow and

Feltham, 1993).

2.4.1.4. Corynebacterium spp.:

They are Gram-positive, catalase positive, non-spore-forming, non-

motile, rod-shaped bacteria that are straight or slightly curved (Collins

and Cummins 1986). Both toxin and non-toxin producing strains of C.

dephtheriae can adhere to and colonize the mucosal tissue of the upper

respiratory tract (Jensen and Warigh, 1998). The bacteria group together

in a characteristic way, which has been described as the form of a "V",



8

"palisades", or "Chinese letters". They may also appear elliptical. They

are aerobic or facultatively anaerobic.

2.4.1.5. Actinomycetes:

Actinomycetes appear as Gram-positive bacilli (Hall, 2006) which

comprise a group of branching unicellular microorganisms. They produce

branching mycelium which may be of two kinds substrate mycelium and

aerial mycelium. Members of the actinomycetes, which live in marine

environment, are poorly understood and only few reports are available

pertaining to actinomycetes from mangroves (Siva Kumar, 2001;

Vikineswari et al., 1997; Rathana Karla and Lakshmanaperumalsamy,

1978). Actinomyces pyogenes, normally commensal bacterium, resides on

mucous membranes of cattle, sheep, swine, and other economically

important animals (Carter et al., 1991).

2.4.1.6. Bacillus spp.: Bacillus species are Gram-positive rods often arranged in pairs or

chains with rounded or square ends and usually have a single endospore.

The endospores are generally oval and are very resistant to adverse

conditions. They have different stabilities and degrees of resistance to

heat, radiation, chemicals, desiccation, and other hostile conditions.

Sporulation is not repressed by exposure to air (Holt et al., 1994).

Bacillus spp. can be broadly divided in three groups based on the

morphology of the spore (Berkeley et al., 1997). The genus Bacillus

currently comprises more than in excess of 60 species, commonly found

in the environment and as laboratory contaminants (Konemann et al.,

1997).



9

2.4.2. Gram-negative bacteria:

2.4.2.1. Enterobacteriaceae:

Members of this family are Gram negative bacilli. They are non-

motile or motile with peritrichous flagella and do not form spores. All

members where growing aerobically and are facultative anaerobes on a

variety of non-selective media (blood agar) and selective media

(MacConkey’s agar medium). Enterobacteriaceae have simple nutritional

requirements, ferment glucose, reduce nitrate. They are catalase- positive

and oxidase- negative (Murray et al., 1998).

2.4.2.1.1. Escherichia coli:

It is Gram-negative rods methyl red positive and 80% of strains are

motile. It can grow on simple media containing glucose as the sole carbon

source (Geo et al., 1998). E. coli is Gram-negative rods that may form

chains under unfavorable conditions (exposure to penicillin). Capsules or

microcapsules are produced by many strains.

2.4.2.1.2. Klebsiella pneumoniae :

It is Gram negative bacillus with a very thick mucoid capsule that

inhibits phagocytosis (Mac Sween and Whaly, 1992). Klebsiella

pneumoniae is present in the respiratory tract causes a small proportion of

bacterial pneumonias (Geo et al., 1995). It is opportunistic pathogen; it

may produce pyogenic lesions like abscesses, infection of wound or

respiratory tract (Satish, 1995). It is the main species of medical

importance . Four sub-species of Klebsiella pneumoniae are recognized

K .pneumoniae sub sp pneumoniae, K. pneumoniae sub-sp. aerogenes, K

.pneumoniae sub-sp ozaenae, K. pneumoniae sub-sp. rhinoscleromatis

(Cheesbrough, 2000).



10

2.4.2.1.3. Proteus species

They are Gram-negative rods, motile, aerobic and facultatively

anaerobic, catalase positive, oxidase negative. They attack sugars

attacked fermentatively usually with gas production and hydrolyze urea

and gelatin (Barrow and Feltham, 1993).

Proteus cultures have a distinctive smell (fishy smell) with swarming

on non-inhibitory solid media such as nutrient agar and blood agar. Most

strains swarms with periodic cycles of migration producing concentric

zones, or spread in a uniform film over moist surfaces of nutrient media.

Proteus species are free living saprophytes in soil, vegetation, water and

sewage, and are found in the intestine in many healthy persons. These

organisms occur naturally in the environment of animals and man and

particularly in the intestines, animal manure, human sewage, soil and

water.

2.4.2.1.3.1. Proteus mirabilis:

It is the main species of medical importance. Many strains of P.

mirabilis produce bacteriocins (proticins) which have a lethal action

against other strains.

Senior (1977) described a highly discriminating method of typing

strains by determination of their proticin production and sensitivity. The

method was made more discriminating when it was used in combination

with O serotyping (Senior, 1977).



11

2.4.2.2. Aeromonas hydrophila:

It is Gram- negative rod, motile and non-motile, aerobic and

facultatively anaerobic, catalase positive, oxidase positive. It attack

sugars fermentatively and gas may be produced (Barrow and Feltham,

1993). It is opportunistic pathogen of fish, reptile and rarely mammals

(Quinn et al., 2002). Camels might acquire it from contaminated drink

water.

2.4.2.3. Alcaligenes spp.:

Gram- negative rods, motile, aerobic, catalse- positive, oxidase-

positive do not produce acid from sugars even in suitable media (some

strains produce acid from ethanol), (Barrow and Feltham, 1993).

2.4.2.4. Kingella spp.:

They are Gram- negative short rods, non-motile, aerobic or

facultatively anaerobic. catalas- negative and oxidase- positive they attack

sugars by fermentation in suitable media and do not produce pigment or

hydrolyse arginine. (Barrow and Feltham, 1993).

2.5. The Important bacterial Respiratory diseases:

2.5.1. Pasteurellosis:

Acute infection with Pasteurella multocida causes hemorrhagic

septicemia in camels. The disease was reported in Africa, India, and

USSR (Macgrane and Higgins, 1985). They described three forms of the

disease. The per- acute form, which is associated with sudden death. The

acute form, which is characterized by fever, anorexia, edematous swells

in pharyngeal and prescapular region. She camels sometimes abort and



12

death occurrs within 2-5 days. The third form of the disease shows

bloody diarrhea as the predominant symptom.

In Ethiopia, Mannheimia haemolytica was identified as the most

important organism involved in an outbreak of camel respiratory disease

(Mersie, 1997).

In Pakistan, an outbreak of a respiratory disease occurred in the

dromedary population of Greater Cholistan desert. The organism isolated

from representative clinical and morbid specimens was Pasteurella

multocida sub spp.multocida. The duration of outbreak was more than a

month (Fraz Munir Khan, 2012).

2.5.2. Tuberculosis:

The disease was first reported in Egypt by Littlewood in (1888). This

was followed by the findings of Archibald (1910) who isolated acid-fast

organisms from lung lesions resembling miliary tuberculosis. The disease

was apparently rare among the camels of nomads, whereas it tended to

occur among camels kept for farming and in close proximity to cattle.

The incidence of camel tuberculosis in Egypt along with typing of the

causal agent was reported by El-Afifi et al. (1953)

Camel tuberculosis in India was first mentioned by Lingard, (1906)

and then by Leese, (1908) who considered it to be comparatively rare,

except in old camels where the usual pulmonary form and occasionally a

generalized form. The disease was reported in Ethiopia by Richard,

(1975) and a spontaneous case of camel tuberculosis in Somaliland has

been described by Pellegrini, (1942-1945). It was characterized by

progressive debility, coughing and death within six months.



13

2.5.3. Mycoplasmosis:

There is very little in the literature with regards to the role played by

mycoplasmas in the etiology of pneumonia in camels.

Elfaki et al. (2002) isolated M. arginini along with several other

bacteria from camel lungs with lesions suggestive of chronic interstitial

pneumonia.

In Egypt, Abeer El-metwally et al. (2010), detected M. arginine from

lung, uterus, cervix, vagina and mammary gland of she camels. Isolation

rate of mycoplasma from lung was 6%.

2.6. Bacteria Isolated From Infected Respiratory Tract of

Camels:

In different regions of Sudan, Mustafa (1992) studied pneumonia in

camels. The examination was carried clinically, bacteriologically and

histopathologically. Speciemens were collected, including nasal swabs,

tracheal swabs and pneumonic lungs. Bacteria isolated were Bacillus

spp., Staphylococcus aureus, Streptococcus pyogenes, Actinomyces.

pyogenes, coagulase- negative staphylococci, diplococci, Klebsiella

pneumoniae, E. coli, diphteroid and mixed flora. The most severe lung

lesions were found to be caused by Actinomyces. pyogenes,

Staphylococcus aureus, Streptococcus pyogenes and Klebsiella

pneumoniae. Also, Tigani et al. (2006). carried out bacteriological and

pathological studies in Central region of the Sudan (Tamboul abattoir)

and Western region (Nyala abattoir) to investigate the condemnation

causes of camel lungs. Camel lungs were inspected at post-mortem in

both areas. The survey revealed various causes of camel’s lung

condemnation with slight variations between the two areas of survey. The



14

microorganisms isolated from Pneumonic cases were Staphylococcus

spp., Corynebacterium spp., Streptococcus spp., Bacillus spp.,

Pneumococcus spp., Enterobacteria spp., Micrococcus spp.,

Haemophilus spp., Actinomyces spp., Pasteurella spp. and

Pseudomonas spp.

Another study of Respiratory infections of the camels (Camelus

dromedarius) in Sudan by Nasr, (2003) has been carried out in camels

brought to Tamboul Market -Butana region, East Central Sudan- for

slaughter. Specimens were collected and they consisted of nasal swabs,

tracheal swabs, lung tissue and bronchial lymph nodes. Bacteria isolated

from nasal cavity in order of abundance, were: Coagulase- negative

staphylococci, Bacillus spp., E. coli, diphteroids, alpha-hemolytic

streptococci, coagulase- positive staphylococci, Actinomyces. pyogenes,

Klebsiella pneumoniae, Alcaligenes faecalis, Streptococcus pyogenes,

Micrococcus luteus, Diplococcus pneumoniae, Bordetella

bronchiseptica, Mannhemia haemolytica, Haemophillus somnus, mixed

flora, Micrococcus kristinae and Pasteurella multocida. Bacteria

isolated from trachea were: Bacillus spp., coagulase- negative

staphylococci, diphteroids, E. coli, Actinomyces pyogenes, Bordetella

bronchiseptica, Alcaligenes faecalis, coagulase- positive staphylococci,

alpha haemolytic streptococci, Klebsiella pneumoniae, Diplococcus

pneumoniae, Micrococcus luteus, Micrococcus kristinae, Haemophillus

somnus and mixed flora. Bacteria isolated from lung tissue were: A.

pyogenes, Bacillus spp., coagulase- negative staphylococci, diphteroids,

coagulase -positive staphylococci, Streptococcus pyogenes, Klebsiella

pneumoniae, alpha-hemolytic streptococci, Mannheimia haemolytica,

Diplococcus pneumoniae and E. coli. Bacteria isolated from bronchial

lymph nodes were: Coagulase-negative staphylococci, Bacillus spp.,



15

diphteroids, coagulase-positive staphylococci, A. pyogenes, alpha-

hemolytic streptococci, Streptococcus pyogenes, Corynebacterium equi,

Pasteurella multocida, Mannheimia haemolytica Klebsiella pneumoniae

and Citrobacter koseri. In this study Haemophillus somnus, Micrococcus

spp. and Bordetella bronchiseptica were isolated for the first time from

respiratory tract of camels.

In Egypt, Farrag et al. (1953) studied pneumonia in camels

slaughtered at Cairo abattoir. Specimens taken were parts of lungs

showing different stages of pneumonia. Bacteria isolated were

Corynebacterium pyogenes, alpha-hemolytic streptococci, Alcaligenes

faecalis, and B. pyocyaneus. Mahamoud et al. (1988) carried out

bacteriological and pathological examination of affected camel lungs in

Egypt. Microorganisms isolated were Klebsiella spp., Proteus vulgaris,

Staphylococcus aureus, Streptococcus pyogenes, E. coli and proteus

rettergi. Another study was carried out in Egypt by Ibtsam (2006). She

investigated bacterial pneumonia in camels. The study included camels

of both sexes, 9 month-2 years old. Bacteria isolated were Streptococcus

pneumoniae, Staphylococcus aureus, E. coli, Mannheimia haemolytica

and Streptococcus pyogenes.

In 1973, Arora and Karla described a case of bronchopneumonia in a

camel in India. The bacteria isolated from affected lungs were

Diplococcus and Klebsiella pneumoniae.

Respiratory infection of camels in Iraq was studied by Alani, (1990).

The isolates from nasal cavity were Pasteurella multocida,

Staphylococcus spp., A. pyogenes, E. coli and Mannheimia haemolytica

The isolates from infected lungs were Pasteurella multocida, Neisseria

spp., Pseudomonas spp., Staphylococcus spp. and A. pyogenes.



16

Moalin and Zessin, (1990) studied Diseases of camels in Somali

Microorganisms isolated from infected lungs were Staphylococcus spp.,

Psudomonas aeroginosa and Citrobacter freundii.

Aldoughaym et al. (1999) studied etiology of pneumonia in camels.

Specimens including nasal swabs, tracheal swabs and pneumonic lung

tissue were collected. Bacteria isolated were Staphlococcus auerus, A.

pyogenes, Streptococcus pyogenes, Coagulase-negative staphylococcus,

Klebsiella pneumoniae, Diplococcus pneumoniae, E. coli, diphteriods

and mixed organisms. The percentage of isolation of Staph.aureus, A.

pyogenes, Streptococcus pyogenes and Klebsiella pneumoniae from

pneumonic tissue was the highest as compared with that from nasal cavity

and the trachea. In contrast the isolation of Bacillus spp., coagulase

negative staphylococcus and diphtheriods from pneumonic lung tissue

was markedly less as compared to that isolated from nasal cavity and

trachea. The isolation percentage of E. coli and Diplococcus pneumoniae

was nearly similar from pneumonic lung tissue, nasal cavity and trachea.

Al-Tarazi (2001) carried out bacteriological and pathological study

on pneumonia in the One-Humped Camels in Jordan. Specimens taken

were lung tissues of slaughtered camels and bacteria isolated from

pneumonic lungs were E. coli, Klebsiella spp., Staphylococcus aureus,

Pseudomonas aeruginosa, A. pyogenes, Mannheimia haemolytica,

Hemolytic streptococci, Proteus spp. and Bacillus spp.

Abubakar et al. (2008) studied camel pneumonia epidemiology and

bacterial flora in normal and diseased lungs of camels in Kano and

Sokoto main abattoirs in northwestern of Nigeria, Their findings in

normal lungs were Staphlococcus aureus, Corynebacterium spp.,

Arcanobacterium pyogenes, Streptococcus spp., Micrococcus spp.,



17

Klebsiella pneumoniae, Bacillus spp., Proteus spp., Pasteurella

multocida and Mannheimia haemolytica. Bacteria isolated from

pneumonic lungs were Staphlococcus aureus, Corynebacterium spp.,

Streptococcus spp., Micrococcus spp., Klebsiella pneumoniae, Bacillus

spp., Proteus spp., Pasteurella multocida and Mannhemia haemolytica.

In Ethiopia Bacteriological study on pulmonary lesions of camels

was carried out. Bacteria isolated were E. coli, Flavobacterium spp.,

Rhodococcus equi, Bordetella bronchoseptica, Aeromonas hydrophila,

Neisseria spp., Streptococcus agalactiae, Staphlococcus aureus,

Pasteurella trehalosi, Pasteurella anatipestifer, Pseudomonas

aeruginosa, and Micrococcus spp. ( Nesibu et al., 2010).

2.7. Bacteria Isolated From Respiratory Tract of Apparently

Healthy Camels:

In Sudan, Shigidi (1972) studied aerobic flora of respiratory tract of

apparently healthy camels. Specimens collected were nasal swabs, lung

tissues and bronchial lymph nodes. Organisms isolated were Bacillus

spp., coagulase-negative staphylococci, diphteroids, Aspergillus spp., A.

pyogenes, alpha- hemolytic Streptococci, Streptomyces spp., coagulase-

positive staphylococcci, E. coli and Enterobacter aerogenes

Bacterial flora of upper respiratory tract of apparently healthy camels

was studied in India. Specimens taken were nasal swabs and bacteria

isolated were coagulase-negative staphylococci, E. coli, Klebsiella

pneumoniae, A. pyogenes, coagulase-positive staphylococci,

Corynebacterium equi, beta-hemolytic streptococci, hemolytic

Diplococci, Anthracoids and Corynebacterium hemolytica (Chauhan et

al., 1986).



18

In 1999, Elhendi studied nasal microflora of upper respiratory tract of

apparently healthy camels under two conditions in Saudi Arabia. First

group contained apparently healthy camels were presented from slaughter

house and second group contained apparently healthy camels of a local

herd of Research Unit at King Faisal University Farm. All camels of both

groups were sampled for a period of six months at appropriate intervals.

Bacteria isolated in first group were Staphlococcus auerus, E. coli,

Streptococcus spp. Bacteria isolated in second group were Staphlococcs

auerus, E. coli, Streptococcus spp., Klebsiella spp and Corynebacterium

spp.

In Iran Azizollah et al., (2008) studied the aerobic bacteria of the

respiratory passageways flora of apparently healthy camels. Specimens

were taken from the nasal cavity, trachea, tonsils and lungs. Bacteria

isolated were staphylococci, followed by Neisseria spp., Bacillus spp. and

streptococci. The majority of the isolates colonized all the anatomical

sites investigated with the exception of E. coli and, streptococci, which

were absent from the lungs, and Klebsiella which was absent from the

nasal tract. Gram-positive bacteria were dominant in this environment,

followed by Neisseria spp., E. coli and Klebsiella spp.



19

Chapter three

Materials and Methods

3.1. Study area:

Specimens were collected from a total of 100 lungs of slaughtered one

humped adult camels of both sexes, they were examined for pneumonic

lesion at Elsalam abattoir about 20 kilometers western of Khartoum. The

study was conducted from February to May 2011.

3.2. Collection of Samples:

Immediately after slaughter, lung tissue of surface area greater than 10

x 10 cm2 showing pneumonic lesions was collected using sterile forceps

and scissors or scalpel blade. Specimens were then transported to the

veterinary laboratory using ice box.

3.3. Isolation of organism:

Immediately after arrival the surface of tissue specimens was burned

by hot spatula and aspirates were taken from the periphery of the lesion.

Aspirates were inoculated onto appropriate primary media (blood agar

and MacConkey’s agar). The cultures were incubated at 37°C for 24 to 72

h depending on the type of bacteria. Bacterial colonies were characterized

by their color, size, edge, surface, consistency, odor, transparency, and

emulsification. Single colonies were taken and smears were prepared for

Gram’s reaction. Finally, identification of bacteria to the species level

was performed using various biochemical tests.



20

3.4. Sterilization:

a. Flaming: It was used to sterilize glass slides, needles, scalpels, points of

scissors and mouth of culture tubes by passing them through Bunsen

burner flame without allowing it to become red hot. ��b. Red heat: It was used to sterilize loop wires, points of forceps and searing

spatulas by holding them over Bunsen burner flame until became red-

hot. ��

c. Hot air oven:

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

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

one hour and oven temperature was 160 °�C. ��

e. Moist heat (Autoclave):

Autoclaving at 121 °�C (151b/inch²) for 15 minutes was used for

sterilization of media and plastic wares. Autoclaving at 115 °�C

(101b/inch²) for 10 minutes was used for sterilization of sugars. ��

3.5. Reagents and Indicators:

3.5.1.1 Reagents:

Most reagents were obtained from British Drug House (BDH).

3.5.1.2. Sodium Chloride/ normal saline

Normal, physiological, or isotonic saline was prepared as described in

Oxide Manual by dissolving 8.5 g of sodium chloride in 1 liter of distilled

water to obtain 0.85% concentration.



21

3.5.1.2. Alpha-naphthol solution (Hopkin and Williams):

This solution was prepared as 5% aqueous solution for Voges-Proskaur

(VP) test.

3.5.1.3. Potassium hydroxide (Hopkins and Williams):

This reagent was prepared as 40% solution for Voges-Proskaur (VP) test.

3.5.1.4. Hydrogen peroxide (H2O2) (BDH):

This reagent was prepared as 3% aqueous solution and used for catalase

test.

3.5.1.5. Kovac's reagent:

This reagent was made of 5 g para- dimethyl amino benzaldehyde, 75ml

amyl alcohol and 25 ml concentrated hydrochloride acid. The aldehyde

was dissolved in alcohol gently in a water bath at temperature of 50-55°�C,

then cooled and the acid was added with care. The reagent was protected

from light and stored at 4°�C for indole test.

3.5.1.6. Methyl red Solution (BDH):

This solution was prepared by dissolving0.04 g of methyl red in 40 ml

ethanol and the volume was made to 100 ml with distilled water. It was

used for methyl red test.

3.5.1.7.Tetramethyl-p-phenylamine dihydrochloride

(Hopkin and Williams)

This reagent was prepared as 1% aqueous solution and it was used for

oxidase test.



22

3.5.1.8. Nitrate test reagent:

Nitrate test reagent was consisted of two solutions which were prepared

according to Barrow and Feltham (1993). Solution A composed of 0.33%

sulphanilic acid was dissolved by gentle heating in 5N- acetic acid.

Solution B composed of 0.6% dimethyl amine-alpha-naphthylamine

dissolved by gentle heating in 5N- acetic acid.

3.5.2. Indicators:

3.5.2.1. Andrade's indicator:

It was composed of 5 g of acid fuchsin, 1 liter of distilled water, and

150 ml of N-NaOH. The acid fuchsin was dissolved in distilled water,

and then the alkali solution was added, mixed and allowed to stand at

room temperature for 24 hour with frequent shaking until the color

changed from red to brown.

3.5.2.2. Bromothymol blue:

It was obtained from (BDH). The solution was prepared by dissolving

0.2 g of the powder in 100ml distilled water.

3.6. Preparation of Culture Media:

All media were dispensed under aseptic conditions in a laminar air flow

cabinet type 11 (Prettl R., Germany) provided with fan, ultra violet lamp

and flame . Media were either prepared from the original ingredients or

obtained in a dehydrated form.



23

3.6.1. Liquid media

3.6.1.1. Nutrient broth (Biomark Code No. B274)

FORMULA

1- Peptic digest of animal tissue …….………5g

2- Beef extract …………………………..1.50 g

3- Sodium chloride …………………………5 g

4- Yeast extract ………………………….1.50 g

pH 7.4 ± 0.2

Thirteen grams of Nutrient broth were added to one liter of distilled

water, mixed well and distributed in 3 ml amounts into clean test tubes,

then sterilized by autoclaving at 121°�C for 15 minutes.

3.6.1.2. Peptone water (HIMEDIA Code No. M 028)

FORMULA :

1- Peptic digest of animal tissue ………...10.0 g

2- Sodium chloride ………………………5.0 g

pH 7.2 ± 0.2

15 g were suspended in 1000 ml distilled water, mixed well, dispensed

into tubes and sterilized by autoclaving at 121°�C for 15 minutes.



24

3.6.1.3. Peptone water sugar (Barrow and Feltham, 1993):

FORMULA

1- Peptone water ……………… 10 g

2- Carbohydrates ………………10 g

3- Andrade's indicator...….…….10 g

4- Sugar ……………………..…10 g

Solid ingredients were dissolved in 1000 ml distilled water. The pH was

adjusted to 7.1- 7.3 before the addition of andrade's indicator. The

complete medium was mixed, distributed into 2 ml volumes into test

tubes containing Darham's tube (for gas trapping in case of glucose) and

sterilized by autoclaving at 115°�C for 10 minutes.

3.6.1.4. Glucose- Phosphate medium (MR-VP test medium)

(Oxoid Code NO. 43)

FORMULA

1- Peptone (Oxoid L49) ….………...…5 g

2- Dextrose ……………….…………...5 g

3- Phosphate buffer (k2HO4)……….…5 g

pH 7.5

15 g of the powder were added to 1 liter of distilled water. Mixed well,

distributed into final containers and sterilized by autoclaving at 121°�C for

15 minutes.



25

3.6.1.5. Nitrate medium (Barrow and Feltham, 1993):

FORMULA

1- Potassium nitrate …………………………0.2 g

2- Peptone …………………………….……..5.0 g

Solid ingredients were dissolved into 1000 ml distilled water. The

medium mixture was distributed in 5 ml amounts in test tubes and then

autoclaved for 15 minutes at 121°�C.

3.6.1.6. Serum Water sugars (Barrow and Feltham, 1993):

FORMULA

1- Peptone …………………………….……. 4 g

2- Na2HPO2………..………………….…...0.8 g

3- Distilled water ………………..………. 800 ml

4- Sterile Serum ………………..…...…… 200 ml

5- Bromcresol purple, 02% aq. Sol. ….....….10 ml

Dissolve the peptone and phosphate in the water, steam at 100°�C for 15

min and filter. Add the serum and steam for a further 15 min. adjust pH to

7.5- 7.8 and add the indicator. Sterilize at 115°�C for 10 min.



26

3.6.2. Semi solid media:

3.6.2.1 Motility test medium- Cragie tube medium (Barrow

and Feltham, 1993)

FORMULA :

1- Nutrient broth (Oxoid) …….….13 g

2- Agar (Oxoid agar No. 1)………..5 g

13 g of dehydrated nutrient broth, were added to 5 g of agar and were

dissolved in 1 liter of distilled water. The pH was adjusted to 7.4. The

medium was dispended in volumes of 5 ml into 20 ml capacity test tubes

containing Cragie tube. The medium was then sterilized by autoclaving in

121°�C for 15 minutes.

3.6.2.2. Oxidation-fermentation medium (Barrow and

Feltham, 1993)

FORMULA

1- Peptone ………………………………………….2.0 g

2- Sodium Chloride ………………………………...5.0 g

3- Di- potassium hydrogen orthophosphate ………..0.3 g

4- Dextrose ……………………….……………….10.0 g

5- Agar (Oxoid agar No. 1) …….………………...…3.0 g

6- Bromothymole blue …………….………………15 ml

pH 7.1



27

The solid ingredients were dissolved in 1000 ml distilled water and the

pH was adjusted before the indicator and agar were added. The medium

was then dispensed in tubes and sterilized by autoclaving at 121°�C for 15

min.

3.6.3. Solid media:

3.6.3.1. Ammonium salt sugars (ASS) (Barrow and Feltham,

1993):

FORMULA

1- (NH4)2HPO4 ……………………….1.0 g

2- KCL ………………………………....0.2 g

3- MgSO4.7H2O ………….……………0.2 g

4- Yeast extract ………………..……….0.2 g

5- Agar ………………………………...20.0 g

6- Distilled water ………………...……10.0 g

7- Bromocrysol purple 0.2%………… 0.04ml.

Solids were added to distilled water and dissolved by steaming then

indicator was added before autoclaving at 115°�C for 20 minutes. The

basal medium was allowed to cool to about 60°�C and then appropriate

carbohydrate was added as sterilized solution to give final concentration

1%, mixed and distributed aseptically into sterile tubes that allowed to

solidify in an inclined position so that the medium set in slope and butt.



28

3.6.3.2. Bile Esculin Azide Agar (Barrow and Feltham,

1993):

FORMULA

1- Ox bile, dehydrated ………………………40.0 g

2- Esculin ..................................................…...1.0 g

3- Ferric citrate …………….…………….…...0.5 g

4- Sodium azide ……………………….…..…0.2 g

5- Agar …………………………….….….…15.0 g

6- Nutrient broth …………………….….….1000 g

All ingredients except esculin were dissolved by heating. Allowed to

cool and then esculin was added. The medium was then distributed in

screw capped bottles, sterilized at 115°�C for 20 minutes and allowed to

set as slopes.

3.6.3.2.Collection of blood for enriched media:

Blood for enriched media was collected aseptically into sterile flasks

containing glass beads by venipuncture of jugular vein of a healthy sheep

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

flask containing the glass beads.

3.6.3.4. Blood agar ( Scharlau Code NO. 01-352)

Blood agar base

FORMULA

1- Meat extract ……………………………10.0 g



29

2- Tryptone ………………………….….....10.0 g

3- Sodium chloride ………………….………5.0 g

4- Agar …………………………………….15.0 g

pH 7.3± 0.2

Forty grams of blood agar base were suspended in one liter of distilled

water, dissolved by boiling mixed and sterilized by autoclaving at 121°�C

for 15 minutes. Then cooled to about 50°�C and defibrinated sheep blood

(10%) was added, mixed gently and 20 ml of complete medium were

poured into each sterile petri dish. The plates were allowed to solidify at

room temperature on a leveled surface.

3.6.3.5. MacConkey's agar (Scharlau Code No. 01-118)

FORMULA

1- Peptone ……………………….20.0 g

2- Lactose ……………………..…...10.0 g

3- Bile salt ……..……………….…...5.0 g

4- Sodium chloride …….……………5.0 g

5- Neutral red ……………………..0.03 g

6- Crystal violet …………………...0.001 g

7- Agar …………………...……..….15.0 g

pH 7.1

51.5 g of the powder, were suspended in 1 liter of distilled water,

brought to boiling until dissolved completely then sterilized by

autoclaving at 121°�C for 15 minutes. The medium was then cooled to

about 50 °�C and aseptically poured into sterile petri dishes in 20 ml

amounts.



30

3.6.3.6. Milk agar (Barrow and Feltham, 1993):

FORMULA

1- Skim milk ……………………….500 ml

2- Nutrient Agar, double- strength….500 ml

The skim-milk was sterilized by heating at 115°�C for 10 minutes. Cooled

to about 50°�C and add to the double strength Nutrient agar which melted

and cooled to 50- 55°�C. The mixture distributed in petri dishes.

3.6.3.7. Nutrient agar (Scharlau Code No. 01-144)

FORMULA

1- Peptone…………………………. 5 g

2- Meat Extract .………………….. .3 g

3- Agar ……………………………15 g

pH 7.0 ± 0.2

23 g nutrient agar and 5 g of sodium chloride were suspended in 1 liter

of distilled water, brought to boiling until dissolved completely then

sterilized by autoclaving at 121°�C for 15 minutes. The medium was then

cooled to about 50°�C and aseptically distributed in 20 ml amount per

sterile petri dish.

3.6.3.8.Nutrient Gelatin (Oxoid):

FORMULA

1- Beef extract ………………….3 g



31

2- Peptone ……………………..15 g

3- Gelatin ………….………….120 g

4- Distilled water….….……1000 ml

pH 6.8 ±0.2

An amount of 128 grams of Nutrient gelatin powder were added to one

liter of distilled water, boiled to dissolve, mixed well, then poured into

sterile Bijou bottles in 2ml amount and sterilized by autoclaving at 121°�C

for 15 minutes.

3.6.3.9. Simmons citrate agar (Scharlau Code No. 01-177)

FORMULA

1- Magnesium sulfate …………….…………..0.2 g

2- Manoammonium dihydrogen phosphate ….1.0 g

3- Dipotassium phosphate ………………...…..1.0 g

4- Sodium citrate …………………….………..2.0 g

5- Sodium chloride …………….…………...…5.0 g

6- Bacto- agar ………….…………………….15.0 g

7- Brom thymol blue …………………….….0.08 g

24.2 g were suspended in 1000 ml distilled water and heated to boiling

until dissolved completely and the pH was adjusted to 7.0. The medium

was then dispended into Bijou bottles in portions of 5 ml each and

sterilized in the autoclave for 15 minutes at 121°�C. Then the medium was

left to solidify in slope position.



32

3.6.3.10. Starch agar (Barrow and Feltham, 1993)

FORMULA

1- Potato starch …………….… 10 g

2- Distilled water …..….……..50 ml

3- Nutrient agar ………...….1000 ml

The starch was suspended with water to a smooth cream, and added to

the molten nutrient agar. Then Mixed, and sterilized at 115°�C for 10

minutes. Finally it was distributed into petri dishes.

3.6.3.11. Urea agar Base (OxoidCode No. CM53)

FORMULA

1- Peptone (Oxoid L37)……………………….….1.0 g

2- Dextrose ………………………………….……1.0 g

3- Sodium chloride ………………………………5.0 g

4- Disodium phosphate .…………………………..1.2 g

5- Potassium dihydrogen phosphte ………………0.8 g

6- Phenol red ……………..….…………………0.012 g

7- Agar No.3 (Oxoid L13)…….…………….……15.0 g

pH 6.8

2.4 g were suspended in 95 ml of distilled water, brought to boiling

until dissolved completely. The medium was then sterilized by

autoclaving at 115°�C for 20 min, cooled to 50°�C and aseptically 5 ml

sterilized distilled water to which 2 g of urea extra pure were dissolved,



33

were dissolved, and were added to it. Mixed well and distribution in 7 ml

amounts into sterilized containers and allowed to set in slope position.

3.7. Culture media:

3.7.1. Blood agar:

It was used as an enriched, non- inhibiting medium for primary

isolation of bacteria and for determination of colonial morphology and

hemolytic activity of streptococci, staphylococci and other organisms.

3.7.2. MacConkey’s agar:

This medium was used in this study for isolation of coliform bacteria.

3.8. Isolation:

Lung tissue aspirates were inoculated onto 10% defibrinated sheep

blood agar and MacConkey's agar and incubated aerobically at 37°�c for

48 h as described by Barrow and Feltham (1993).

3.9. Purification:

All bacteria isolated were purified by subculturing from single well

separated colony on separate nutrient agar plates or blood agar plates for

those which do not grow on nutrient agar. Then examined for purity

microscopically as described later.

3.10. Preservation:

The isolates were preserved for further studies to determine their cultural

and biochemical characterizations. Preservation was made by sub

culturing the purified isolates on fresh sheep blood agar plates weakly.

Cultures were kept at 4°�C.



34

3.11 Microscopic Examination:

Smears were made from purified colonies, fixed by heating and stained

by Gram stain method (Barrow and Feltham, 1993). Then examined

microscopically for purity and morphology characterization of bacteria.

3.12. Biochemical Tests:

3.12.1. Sugar fermentation test:

The tests were carried out as described by Barrow and Feltham (1993).

The peptone water sugar was inoculated with organism under the test,

organism incubated at 37°�C and then examined daily for 7 days. Acid

production was indicated by appearance of reddish color, while gas

production was indicated by presence of an empty space in the inverted

Durham's tubes.

3.12. 2. Oxidase test:

The method of Barrow and Feltham (1993) was followed. Strips of filter

paper was soaked in 1% solution of tetramethyl- p-phenylenediamine

dihydrochloride and dried in hot air oven and then placed on a clean glass

slide with sterile forceps. A fresh young test culture on nutrient agar was

picked off with sterile glass rod and rubbed on the filter paper strips. If a

purple color developed within 5-10 seconds, the reaction was considered

positive.

3.12.3. Catalase test:

The test was carried out as described by Barrow and Feltham (1993). A

drop of 3% aqueous solution of hydrogen peroxide was placed in a clean

glass slide. A colony of test culture on nutrient agar was picked off and



35

put on the drop of hydrogen peroxide. Evolution of gas and appearance of

bubbles indicated positive test.

3.12.4. Coagulase test:

The test was performed as described by Barrow and Feltham (1993).

0.1ml of 18-24 h old broth culture of the tested organism was added to

0.5 ml of 1:10 dilution of human plasma in saline, then incubated at 37 °�C

and examined after 2-24 hrs for coagulation. Definite clot formation

indicated a positive result.

3.12.5. The oxidation- fermentation (O/F) test:

The test was carried out as described by Barrow and Feltham (1993).

Duplicate test tubes OD Hugh and Leifeson's medium were inoculated by

the test organism with straight wire. To one of the test tube a layer of

sterile melted soft paraffin oil was added to a depth of 3cm above the

medium to seal it from air. The inoculated tubes were incubated at 37°�C

and examined daily for fourteen days. Yellow color in the open tube only

indicated oxidation of glucose, yellow color in both tubes showed

fermentation reaction and blue or green color in open tube and green

color in the sealed tube indicated production of alkali.

3.12.6. Indole production test:

Indole production test was carried out as described by Barrow and

Feltham (1993). The test organism was inoculated into peptone water and

incubated at 37c for 48h. One milliliter of the Kovac's reagent was run

down along side of the test tube. Appearance of pink color within a

minute indicated positive reaction.



36

3.12.7. Methyl red (MR) test:

Methyl red test was carried out as described by Barrow and Feltham

(1993). The test organism was inoculated into glucose phosphate medium

(MR-VP medium) then incubated at 37c for 48h. Two drops of methyl

red reagent were added, shaken well and examined. Appearance of red

color indicated positive reaction, whereas orange or yellow color

indicated a negative reaction.

3.12.8. Voges- Proskauer (VP) test:

The test was performed as described by Barrow and Feltham (1993). The

test culture was inoculated into glucose phosphate medium (MR-VP

medium) then incubated at 37°�C for 48h. 0.6 ml of 5% alpha-naphthol

solution and 0.2 ml of 4% potassium hydroxide were added and shaken

well. The tube was sloped and examined after 15 min and 1hr. A positive

reaction was indicated by strong red color.

3.12.9. Nitrate reduction:

Nitrate broth was inoculated with the test organism, incubated at 37°�C for

2 up to five days. Then1 ml of nitrate reagent solution A was added

followed by one ml of nitrate reagent solution B. Positive reaction was

indicated by the development of red colour. To tubes not showing red

colour within five minutes, powdered zinc was added up to 5 gram/ml of

culture and allow standing. The development of red colour meant nitrate

was not reduced by the bacteria (i.e. negative) while absence of red

colour meant the nitrate was reduced to nitrite and then further reduced to

nitrogen.



37

3.12.10. Urease activity test:

The test was carried out as described by Barrow and Feltham (1993). The

test organism was inoculated heavily onto a slope of urea agar medium

and then incubated at 37°�C for two days. Appearance of red color

indicated a positive reaction.

3.12.11. Citrate utilization:


test culture was inoculated as a single streak over the surface of a slope of

Simmons' citrate medium and examined daily for 7 days. Growth of the

organism and chang of color indicated a positive test.

3.12.12 Aesculin hydrolysis:

The procedure of Barrow and Feltham (1993) was followed. The test

organism was inoculated into Aesculin broth, incubated at 37°�C and

examined daily for up to five days. Blackening indicated a positive

reaction. Alternatively, aesculin agar was incubated at 37°�C. Blackening

in and around the bacterial colonies indicated a positive reaction.

3.12.13 Hydrogen sulphide (H2S) production:

The method of Barrow and Feltham (1993) was followed. The test culture

was inoculated into nutrient broth; filter paper impregnated with 10%

lead acetate solution was placed in the neck of the tube and incubated. A

positive reaction was indicated by blacking of the tip of the filter paper.

3.12.14 Ammonium salt sugar test:


test organism was inoculated onto a slope of ASS medium and incubated



38

at 37°�Cfor up to 7 days. The medium was examined on alternative days

for growth and acid production.

3.12.15. Gelatin hydrolysis:

Gelatin hydrolysis test was carried out as described by Barrow and

Feltham (1993). The test culture was stabbed into nutrient gelatin and was

incubated at 37°�C for up to 14 days. The inoculated tube was placed in a

refrigerator for 2h and examined every 2-3 days. Liquefaction of gelatin

indicated a positive test.

3.12.16. Mobility test:


tube of motility medium was stabbed by inoculum to depth of about 5mm

and incubated at 37°�C for 18 hrs. Motile organisms migrated through the

medium, which became turbid while the growth of non-motile organisms

was confined to the stab inoculums.

3.12.17. Digestion of casein:

Plates of Casein Agar was inoculated and examined at intervals for up to

14 days for clearing of the medium around the bacterial growth.

3.12.18. Starch hydrolysis:

Starch agar was inoculated lightly and incubated at 30°�C for 5 days. The

plates were hooded with lugol's iodine solution. The medium remained

blue where starch had not been hydrolysed; clear colorless zones

indicated hydrolysis.



39

Chapter 4

Results

4.1. Postmortem findings:

Post-mortem inspection revealed various types of pneumonia, red and

grey hepatization, consolidation and pulmonary abscesses in pneumonic

lungs.

From a total of 100 camel lungs inspected randomly at post-mortem

only 60 (60%) lungs were found to have pneumonic lesions. From the

sixty lung samples cultured for bacteriological examination, only 52

(86.7%) specimens gave bacterial growth on blood agar.

4.2. Isolation of Organisms:

The 52 lung tissue specimens yielded bacterial colonies and among

these 81. 5% (n =110) were Gram-positive and 18, 5% (n = 25) were

Gram-negative bacteria.

4.3. Gram- positive bacteria:

Isolated organisms included Staphylococcus spp. (18.5%),

Streptococcus spp. (17.8%), Bacillus spp. (15.5%), Corynebacterium spp.

(8.1%), Micrococcus spp. (7.4%), and Actinomyces spp. (14.1%)

(Table1).

Staphylococci represented the highest percentage of the total Gram

positive bacteria isolated 25 (18.5%). They were identified as- coagulase-

negative staphylococci this comprised 12.7 % of the total Gram positive

isolates and 56% of the genus Staphylococcus. Coagulase-positive

Staphylococci represented 10% of the total Gram positive isolates and

44% of the genus staphylococci.



40

4.3.1. Staphylococcus spp.

The criteria used for the identification of the isolates were as follows:

a. Morphology and staining: The organism appeared, in stained smear as Gram positive cocci,

arranged in clusters. Staphylococci were non- motile.

b. Cultural characteristics:

1. Blood agar medium:

On primary isolation, the culture was incubated at 37 °�C for 24 hours.

Colonies produced were circular, smooth, opaque, low convex, soft and

easily to be emulsified when touch by the wire loop. They formed a zone

of hemolysis. Different strains of Staphylococcus aureus colonies with

grey and cream colourless colonies and beta hemolysis.

2. Nutrient agar:

Within 24 hours incubation, Staphylococcus aureus produced

round, smooth and low convex colonies.

3. Manitol salt agar:

After 24 hours incubation at 37°�C, colonies were round, smooth

and low convex. Most strains fermented mannitol and produced yellow

colour in colonies.



41

c. Biochemical reaction: All examined isolates were catalase positive and usually oxidase

negative. Aerobic and facultatively anaerobic. Attacked sugars by

fermentation and produced acid with no gas from glucose, sucrose,

lactose and fructose but not from xylose and raffinose. (Table 9).

4.3.2. Streptococcus spp.:

These represented 24(17.8%) from the total isolates.

a. Morphology and staining:

Isolates of this organism were Gram-positive cocci, non-motile, non-

capsulated, non-sporing, arranged in chains and some strains were

capsulated.



After 24 hours incubation at 37°�C, streptococcus pyogenes colonies

were always dew-drop like surrounded with a narrow zone of complete

haemolysis. Whereas Streptococcus viridans colonies were surrounded

by an area of partial haemolysis and green-brown colour, it was described

as alpha- haemolytic streptococci (a�-H.S).

c. Biochemical reaction: The isolates of streptococci isolated were catalase and oxidase-

negative and aerobic and facultatively anaerobic. Indole and hydrogen

sulphide were not produced and nitrate was not reduced. They attacked

carbohydrates fermentatively and produced acid with no gas from

glucose, sucrose, and trehalose but they did not attack sorbitol or

arabinose (Table 9 ).



42

4.3.3. Corynebacterium spp.:


They were Gram-positive small rods, exhibiting a tendency to form

clumps and palisade arrangement simulating Chinese letters. The

organism was non –motile.



On primary isolation, the cultures were incubated at 37°�C, for 48 hours.

Growth was in the form of small tiny colonies (one mm in diameter).

They were white, opaque and flat with matt surface.

2. Nutrient agar: Growth was obtained after 48 hours incubation and the colonies

appeared small and splashed.

c. Biochemical reactions: All isolates tested were positive for catalase and negative for oxidase.

They attacked carbohydrates fermentatively without gas from glucose

and maltose but did not attacked lactose and manitol. (Table 10)



43

4.3.4. Actinomyces spp.:

a. Morphology and staining: They were Gram positive, short to filamentous rods with branching.

Non- motile.

b. Cultural characteristics: Actinomyces spp. required enriched culture and growth was enhanced

by an atmosphere with 6 - 10% added carbon dioxide. The optimum

growth temperature was 37°C. Colonies appeared after 3 - 7 days of

incubation. They were described often as ‘breadcrumb’colonies. Colonies

were powdery appearance with convex, concave or flat surface and color

ranged from white, gray to pinkish and yellowish.

c. Biochemical reactions: All examined isolates were negative for catalase and oxidase, indole

and aesculin. They were variable in gelatin liquefaction. They fermed

sugars and produced acid with no gas from lactose and maltose. They did

not attack sorbitol, sucrose and manitol. (Table 11).

4.3.5. Bacillus spp.:


The isolates were Gram-positive rods that grew aerobically on nutrient

agar and formed resistant endospores. Most were motile.


The isolates grew cans occur on all the ordinary media. In broth

media, a surface scum formed with or without turbidity.

c. Biochemical reactions: All isolated were oxidase variable and catalase positive. attacked

glucose and cellobiose but not attacked glactose, raffinose and xylose.



44

4.3.6. Micrococcus spp.:


The isolates were Gram- positive cocci resembling staphylococci

morphologically. They were pigmented. They were non –motile.

b. Cultural characteristics: The isolates grew on ordinary media and usually halotolerant (5%

NaCl). Optimum growth temperature was 25- 37°C and colonies were

usually pigmented with yellow or red shade.

c. Biochemical reactions: All isolates were catalase positive and usually oxidase positive. They

did not reduce nitrate but attacked sugars oxidatively or not at all. They

were variable in sugars fermentation.

4.4. Gram-negative bacteria:

Gram- negative bacteria isolated were Klebsiella pneumoniae (3%),

Escherichia coli (3%), Aeromonas hydrophila (3.7), proteus

mirabilis(3.7), Kingella kingae (3.7) and Alkaligenes faecalis (1.5). as

shown in ( Table1).

4.4.1. Enterobacteriaceae

They included Eshericha coli, Klebsiella Pneumoniae and Proteus

Mirabilis.



45

4.4.1.1. Escherichia coli:


Isolates of E. coli were Gram-negative rods, occurred singly, long and

thin. Thin were motile and non-spore forming.


1. Blood agar:

The isolates formed colonies after 24 hours incubation at 37°�C

were white to yellowish, moist, glistening, opaque and circular with an

entire edge. Some isolates of E. coli were haemolytic.

2. MacConkey’s agar:

The isolates formed colonies after 24 hours incubation at 37°�C

were smooth, shiny, large (2-4 mm in diameter) and pink in colour.

(Lactose fermenting).

3. Nutrient agar:

Colonies formed after 24 hours incubation at 37°�C, were small,

White and low convex.

d. Biochemical reactions: All isolates were catalase and indole positive, reduced nitrate,

hydrolyzed aesculin and were MR positive. They were oxidase

negative.they did not produce acetoin or hydrogen sulphide or

liquefy gelatin .They produced acid with gas due to fermentation of

glucose, lactose, fructose, maltose, xylose and manitol. (Table 12).



46

4.4.1.2 Klebsiella pneumoniae:

a. Morphology and staining: Isolates of Klebsiella pneumoniae were Gram-negative rods,

capsulated, short and thick. They were non-motile.


1. MacConkey agar:

Colonies formed after 24 hours incubation at 37°�C, were large,

(2-4mm in diameter), mucoid and pink in colour (Lactose fermenting).

2. Blood agar:

Colonies formed after24 hours incubation at 37°�C, were large, mucoid

and grey-white in colours.

c. Biochemical reactions: All examined isolates were catalase positive and oxidase negative.

They were indole and MR negative. They utilized citrate and urease.

They prduced and acid with gas due to fermentation of glucose, sucrose,

lactose, fructose, maltose, xylose and manitol. (Table 12)

4.4.1.3. Proteus mirabilis:

a. Morphology and staining: Isolates of Proteus mirabilis were Gram-negative rods. They were

motile.


1. Blood agar:

After 24 hours incubation at 37°�C. They showed swarming

appearance. Most strains produced fishy smell and wavy culture.



47

2. Nutrient agar:

Growth was similar to that shown on blood agar.

Biochemical reactions: All examined isolates were indole and oxidase-negative; they were

catalase and MR positive. They produced hydrogen sulphide and acid

with gas due to fermentation of glucose, trehalose and xylose. (Table 12).

4.4.2. Aeromonas hydrophila:

a. Morphology and staining: Isolates of Aeromonas hydrophila were Gram-negative rods, motile but

some were non-motile.


1. Blood agar:

Colonies formed after 24 hours incubation at 37°�C, were large,

greyish, white, translucent, moist, circular and flat with either a� or ß�

haemolysis. They turned into light green with age.

2. MacConkey’s agar:

Colonies formed after 24 hours incubation at 37°�C were non-

lactose fermenting.

c. Biochemical reactions: All examined isolates were catalase and oxidase positive, reduced nitrate

and were indole positive. They produced acid with gas due to

fermentation of glucose, sucrose, lactose, maltose (Table13).



48

4.4.3. Kingella kingae:

a. Morphology and staining: The isolates were Gram-negative rods or coccobacilli occurring in pairs

or chains and non- motile.


Colonies formed after48 hours incubation at 37°�C, were

smooth,convex and haemolytic.

c. Biochemical reactions:

All examined isolates were oxidase positive, catalase negative.

Urease negative. They attacked carbohydrates fermentatively and

produced acid with no gas.

4.4.4 Alcaligenes Faecalis:

a. Morphology and staining: The isolates were Gram- negative rods, motile, and motile.

b. Cultural characteristic:

1. Blood agar: After 24 h, colonies were usually 1.0-1.5mm in diameter flat, often

with a characteristic spreading periphery.

2. Nutrient agar: Colonies formed were circular, non-pigmented to grayish-white,

translucent to opaque, flat to convex, usually smooth and with margin

that was usually entire.



49

c. Biochemical reactions: The isolates were oxidase and catalase positive. They did not produce

Indole or utilize carbohydrates they produce alkali from several organic

salts and amides.



50

Table 1: Types of bacteria isolated from pneumonic lungs of camels

Isolated organism

Number of isolates

Percentage %

Staph spp.

25

18.5

Strepto spp.

24

17.8

Bacillus spp.

21

15.5

Actinomyces spp.

19

14.1

Micrococcus spp.

10

7.4

A. hydrophyla

5

3.7

K. kingae

5

3.7

P. mirabilis

5

3.7



51

Continue table 1

Isolated organism

Number of isolates

Percentage%

K. pneumonia

4

3

E.coli

4

3

P. mirabilis

5

3.7

A. faecalis

2

1.5

Total

135

100



52

Table 2: Staphylococci isolated from pneumonic lungs of camels

Staph spp.

No. of isolates

% from Staph. spp.

% from total isolates

S. aureus

11

44

8.1

S. epidermides

8

32

5.9

S. chromogen

6

24

4.4

Total

25

100

18.5



53

Table 3: Micrococci isolated from pneumonic lungs of camels.

Micrococcus spp.

No. of Isolates

% from Micrococcus spp.

% from isolates

M. kristinae

6

60

4.4

M. luteus

4

40

3

Total

10

100

7.4



54

Table 4: Streptococci isolated from pneumonic lungs of Camels

Strepto.spp.

No. of isolates

% from Strepto.spp

%from total isolates

S. pneumonia

9

37.5

6.7

S. equi

8

33.3

6

S. anginosus

7

29.2

5.2

Total

24

100

17.8



55

Table 5: Actinomyces spp. isolated from pneumonic lungs of camels

Actinomyces spp.

No. of Isolates

% from A. spp


A.israelii

9

47.4

6.7

A. pyogenes

7

36.8

5.2

A. odontolyticus

3

15.8

2.2

Total

19

100

14.1



56

Table (6): Corynebacterium spp. isolated from pneumonic lungs of Camels

Corynebacteria spp.

No of isolates

% From Corynbacterium

spp.


C. ulcerans’

5

45.5

3.7

C. pseudotuberculosis

6

54.5

4.4



57

Table (7): Bacillus spp. isolated from pneumonic lungs of Camels

Species of isolate

No. of isolates % from Gram positive bacteria


B. lentus

10

47.6

7.4

B. thuringiensis

5

23.8

4.4

B.mycoides

6

28.6

3.7

Total

110

100

15.5



58

Table (8): Gram negative bacteria isolated from pneumonic lung of camels

Species of isolate Number of isolates

% from gram negative bacteria


K. hydrophila

5

20

3.7

K. kingae

5

20

3.7

P.mirabilis

5

20

3.7

K. pneumonia

4

16

3

E .coli

4

16

3

A. faecalis

2

8

1.5

Total

25

100

18.5



59

Table (9): Characters and Biochemical properties of isolated Gram positive cocci

Test genus Staphylococcus Micrococcus Streptococcus

Growth in air + + +

Anaerobic growth + - -

Motility - - -

Oxidase - + +

Catalase + + +

Acid from glucose + - +

O/F F O F

V: variable +ve: Positive -ve: Negative O: Oxidative F: Fermentative



60

Table (10): Characters and Biochemical reactions of isolated

Corynebactria spp.


Test species C. pseudotuberculosis C. ulcerans

Catalase + +

Oxidase - -

O/F F F

Acid from: Glucose + +

Lactose + -

Manitol - -

Gelatin - +

Urease + +

VP - -

Aesculin hydrolysis - -

Sucrose + -

Starch + +



61

Table (11): Characters and Biochemical reactions of isolated

Actinomyces spp.

Tests Actinomyces pyogenes

Actinomyces israelii

Actinomyces odontolyticus

Growth in air + w +

Anaerobic growth

+ + +

Motility - - -

Catalase - - -

O/F F F F

Acid from glucose

+ + +

Lactose - + +

Maltose + + -

Manitole - + +

Raffinose - + -

Sucrose - + +

V: variable +ve: Positive -ve: Negative O: Oxidative F: Fermentative w:

weak



62

Table (12): Characters and biochemical reactions of isolated

Enterobacteriae.

Tests species Klebseilla pneumoniae

E.coli Proteus mirabilis

Growth in air + + +

Motility - + +

O/F F F F

Oxidase - - -

Catalase + + +

Growth on macConkey agar

+ + +

Gelatin liquefaction

- - -

H2S production - - +

Citrate utilization + - +

Indole - + -

MR + + -

VP - - -



63

Table 12 Continued:

Test Klebseilla pneumoniae

E.coli Proteus mirabilis

Urea hydrolysis + - +

Acid from glucose(gas)

+ + +

Sucrose + - -

Raffinose + - -

Manitole + + -

Maltose + + -

Salicin + + -

Trehalose + + +

Xylose + + +




64

Table (13): Characters and biochemical reactions of isolated


Test


Gram reaction G-ve rods

Motility +

Catalase +

Oxidase +

O-F F

Gas from glucose +

Indole -

VP +

Simmon’s

utilization

+

Urease -

Acid from

Lactose +

Sucrose +

Salicin +

Xylose +



65

Fig: Gram stained smear showing Actinomyces.



66

Fig : Growth of Actinomyces in blood agar



67

Chapter 5

Discussion

In holy Quran it is said "Do they not look at the Camels, how they are

made? And at the Sky, how it is raised high? And at the Mountains, how

they are fixed firm? And at the Earth, how it is spread out. In this honored

verses, the Creator, who knows the secrets of his creatures, advises

people to think and contemplate in creating camels as a creature that

witnesses Allah's Glory, power and planning.

The normal flora of the upper respiratory tract is important as they

prevent adherence and colonization by pathogenic organisms (Jubb and

Kennedy, 1985). There are large numbers of microorganisms that are

present in the respiratory tract of camels as commensals and play

pathogenic role when general immune responses of the host are lowered

(Shigidi, 1973).

This study assessed the types of aerobic bacteria involved in different

types of pulmonary lesions encountered in camel lungs slaughtered at

Elsalam abattoir, in which a relatively high number of bacterial isolates

(135) was isolated from 52 pneumonic lungs. The remaining 8 lung

tissues with observable lesions, failed to yield microorganisms on

primary culture, these infections may be probably due to the involvement

of other pathogens such as anaerobic bacteria, virus, Mycoplasma and

fungi. Lopéz (2001) reported that normal lungs were ideally supposed to

be sterile as the pulmonary macrophages continue to scavenge microbial

agents in the lungs there by making the lung free from pathogens as a

defensive mechanism



68

The isolated organisms included Gram positive and

Gram negative bacteria which were Staphylococcus spp.,

Streptococcus spp., Micrococcus spp., Corynebacteria

spp., Actinomyces spp., Bacillus spp., Escherichia spp.,

Klebsiella spp., Aeromonas spp., Proteus spp., Alcaligenes

spp. and Kingella spp.

Gram positive bacteria represented the majority of the

bacterial isolates which was similar to that observed by

different workers (Farrag et al., 1953; Shigidi, 1972;

Chauchan et al., 1986; Alani, 1989 and Mustafa, 1992).

Staphylococcus spp. represented 18.5% of the total Gram positive

bacteria isolated, most of them were identified as Coagulase negative

staphylococci (CNS) they were the commonest bacteria isolated from

10.3% of lungs Lower than those of Tigani et al. (2006) who recovered

them from 27.9% of pneumonic lungs of camels but considerably higher

than the results of Al- Doughaym et al. (1999) and Al-Tarazi (2001) who

recovered them from 6.5% and 4% respectively. It was suggested that

they exist as part of the normal micro flora of respiratory tract of camels

(Shigidi, 1972 and Chauchan 1987). Coagulase positive staphylococci

were recovered from 8.1% of lungs lower than the results of Al-

Doughaym et al. (1999) and Al-Tarazi (2001) who isolated them from

24.8% and 10.7% of lungs respectively. Also they had been reported in

pneumonic camels by different workers (Chauchan et al., 1987;

Mahmoud et al., 1988 and Moalin and Zessin, 1990). Staph aureus were

isolated from pneumonic cases in other studies (Farrag et al., 1953;

Ghawi, 1978 and Elmaghauri et al., 1986).



69

On the other hand, the frequency of isolation of Streptococcus spp.

was 20.7%. It was similar to that reported by Nesibu Awol (2011) who

isolated them from 19.3%. In contrast Azizollah et al. (2009) and Zubair

et al. (2004) reported a frequency of 4.5% and 7% only respectively.

Streptococcus pyogenes was reported to occur in pneumonic camels by

various authors, among these (Farrag et al., 1953, Elmaghauri et al.

(1986) and Mahmoud et al. (1988).

Buxton and Fraser, (1977) indicated that Streptococcus spp. are widely

distributed in nature and live as commensals in the respiratory tract of

many species of domestic animals.

In this study Corynebacterium spp. were isolated from pneumonic

lungs this was in agreement with the previous findings of (Farrag et al.,

1953; Ghawi1978; Elmaghauri et al., 1986; Moalin and Zessin 1990).

Other Gram-positive bacteria incriminated in pneumonic lungs were

Arcanobacterium pyogenes 5.2% which were isolated from pulmonary

abscesses and this was similar to that reported in Iraq. The same organism

was reported in cases of pneumonia by various authors (Farrag et al.,

1953; Shigidi, 1972; Chauchan et al., 1987; Mahmoud et al., 1988; Alani

et al., 1989 and Mustafa, 1992).

A. pyogenes has been previously reported in camels as a respiratory

pathogen. The organism enters the blood stream and causes septic

arthritis, lesions and abscesses in various organs and tissues, mainly in

the lungs Tolle et al., (1983). Further more A. pyogenes is often isolated

from the abscesses in the lungs of ruminants, pigs and sometimes people

(Hinto, 1972 and Hommez et al., 1991). The same organism was reported

in cases of pneumonia by various authors (Farrag et al., 1953; Shigidi,



70

1972; Chauchan et al., 1987; Mahmoud et al., 1988; Alani et al.1989 and

Mustafa, 1992).

Moreover, the frequency of isolation of Aeromonas hydrophila was

3.5% which was similar to the previous report of Nesibu et al. (2011).

There are no previous reports of isolation of this organism in the Sudan,

and this is the first time to be isolated from camels.

The presence of E. coli is not uncommon, this could be due to

inhalation of dry faeces or from water containers as it is a normal resident

of intestinal tract. In this study four isolates 3% were identified, this is

much lower than the results of Al-Tarazi, (2001) and Nesibu et al. (2004).

This pathogen was isolated by (Elmaghauri et al. (1986); Omer, (1987);

Mahmoud et al. (1988) and Al- Ani, 1989).

Klebsiella pneumoniae was microorganisms commonly isolated from

pneumonic lesions in camels (Zubair et al., 2004; El-Tigani et al., 2004

and Kane et al., 2005). In this study they were isolated at a rate of 3%.

The same organism was stated by Arora and Karla, (1973); Ghawi,

(1978); Mahmoud et al.,1988; Zubair et al., 2004; El-Tigani et al.,

2004 and Kane et al., 2005).

Despite the fact that Micrococcus species are considered non-

pathogenic (Quinn et al., 1999). Its isolation from pneumonic lung

specimens of animals was reported by various workers (Al-Tarazi, 2001;

Nasr, 2003; Kane et al., 2005 and Tigani et al., 2006). It could have a role

in the development of respiratory infections. In this study Micrococcus

spp. was isolated from 7.4% of lungs so these findings supported those of

Nasr (2003) who isolated the same organism for the first time in the

Sudan.



71

Bacillus spp. were isolated by Farrag et al. (1953); Shigidi, 1987;

Omer (1987) and Mustafa, 1992). Bacillus spp. isolated during this study

are comparable to those reported from apparently healthy camels

(Shigidi, 1973 and Chauhan et al. 1987). Thus a variety of

microorganisms may reside the respiratory tract which under suitable

predisposing conditions could invade the different parts of the tract and

cause pathological lesions. In this study Bacillus and Proteus isolates

were isolated from mixed infection of pneumonic lungs. Such isolates

were previously mentioned by others and were considered less pathogenic

(AL-Doughaym et al., 1999 and Mohamed and Farah, 1994).

Alcaligenes faecalis was isolated from 1.5% of lungs, it is considered

to be alkaline tolerant as indicated in the result. Farrag et al. (1953) also

isolated the same organism and he suggested their entrance through blood

or lymph or they might be a contaminant during slaughter.

Pasteurella spp. and Mannheimia hemolytica were not isolated in this

study and this is in agreement with the reports from Egypt, where

Mannheimia haemolytica was not isolated from any pathological lesions

of pneumonic lungs (Mahmoud et al., 1988).

In the previous studies, different bacterial species were isolated from

pneumonic lung specimens of camels. In addition, Azizollah et al. (2009)

isolated various bacterial species from lung lesions of apparently healthy

camels. Variation in the frequencies isolation of different bacteria could

be due to variation in sample size, sample collection and processing,

types of pneumonia or geographical areas.

Al-Tarazi, (2001) reported that, interstitial pneumonia that affected

young camels was caused by Klebsiella spp. and E. coli. Our isolates



72

Klebsiella spp. and E. coli isolated from pneumonic lungs of camel in this

study could probably be involved in induction of these pneumonias.

Lung abscesses are caused by pyogenic organisms. The majority of

bacterial pneumonia cases had multifactorial causes.



73

Conclusions

1- In this study aerobic bacteria isolated and identified included

Staphylococcus spp., Micrococcus spp., Streptococcus spp.,

Actinomyces spp., Corynebacterium spp., Bacillus spp., Klebsiella

spp., Escherichia spp., Aeromonas spp., Kingella spp., Proteus spp.

and Alcaligenes spp.

2- Some of these bacteria reside the lungs normally and under stress

conditions they might become pathogenic.

3- Arcanobacterium pyogenes were associated with pulmonary

abscesses.

4- In this study Aeromonas hydrophila was isolated from lesions of

camel lungs for the first time in Sudan.



74

Recommendations

1- It is highly needed to determine the pulmonary pathologies and

associated bacterial agents and possibly normal bacterial flora of

respiratory tract of camels in Sudan.

2- The role of A. hydrophila in pneumonia and respiratory tract infection

in camels has not previously been studied or documented thus it requires

further studies.



75

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Documents

Isolation of Aerobic Bacteria from Pneumonic Lung of Camels · a. Morphology and staining 44 b. Cultural characteristic 44 c. Biochemical reactions 44 4.4. Gram-negative bacteria