experimental pathogenesis study of infectious …

i

EXPERIMENTAL PATHOGENESIS STUDY OF INFECTIOUS CORYZA IN CHICKS BY LOCAL ISOLATE OF

Avibacterium paragallinarum

A Thesis

Submitted to Bangladesh Agricultural University, Mymensingh

In partial Fulfillment of the Requirements for the Degree of

Master of Science in

Pathology

By

MOHAMMAD ALI

Roll No.: 11Vet Path JJ 04 M Registration No.: 31947, Session: 2005-06

Department of Pathology Bangladesh Agricultural University

Mymensingh

May, 2012

ii

EXPERIMENTAL PATHOGENESIS STUDY OF INFECTIOUS CORYZA IN CHICKS BY LOCAL ISOLATE OF

Avibacterium paragallinarum

A Thesis

Submitted to Bangladesh Agricultural University, Mymensingh

In partial Fulfillment of the Requirements for the Degree of

Master of Science in

Pathology

By

MOHAMMAD ALI

Approved as to Style and Contents by

(Prof. Dr. Md. Abu Hadi Noor Ali Khan) (Prof. Dr. Md. Mokbul Hossain) Co-Supervisor Supervisor

(Prof. Dr. Priya Mohan Das) Chairman, BOS & Head Department of Pathology

May, 2012

iii

ACKNOWLEDGEMENTS

All panegyrics are due to the Almighty Allah, the Supreme Authority of the Universe, Who has

kindly enabled the author to conduct the research and thesis work successfully for the degree of

Master of Science in Pathology.

The author would like to express his heartfelt gratitude, indebtedness and profound respect to his

honorable teacher and research supervisor Professor Dr. Md. Mokbul Hossain, Department of

Pathology, BAU, Mymensingh for his generosity, scholastic guidance, invaluable advice,

suggestions, constructive criticism, untiring help and constant inspiration throughout the course of

this research work and immense help in preparing the thesis manuscript.

The author wishes to convey his profound respect and sincere gratitude to his honorable teacher

and research co-supervisor Professor Dr. Md. Abu Hadi Noor Ali Khan, Department of Pathology,

BAU, Mymensingh, for his affectionate encouragement, constructive criticism, kind co-operation,

necessary correction and instruction to complete this manuscript.

It is a great opportunity for the author to express his gratefulness, sincere appreciation, high

indebtedness and deep respect to Professor Dr. Priya Mohan Das, Head, Department of

Pathology for his valuable suggestion, encouragement and help throughout the research period and

preparation of the thesis.

The author would like to express his immense indebtedness to Professor Dr. Md. Iqbal Hossain,

Professor Dr. Md. Abdul Baki, Professor Dr. Md. Rafiqul Islam, Professor Dr. Md. Habibur

Rahman, Professor Dr. A. S. Mahfuzul Bari, Professor Dr. Emdadul Haque Chowdhury, DR.

Rokshana Parvin, DR. Jahan Ara Begum, DR. Mohammed Nooruzzaman and DR Munmun

Pervin Department of Pathology, Bangladesh Agricultural University, Mymensingh, for giving

encouragement, advice and facilitating the lab equipments and reagents to conduct the research

work.

iv

The author also wishes to express his gratefulness and sincere appreciation to all PhD and MS

students of Department of Pathology, Bangladesh Agricultural University, Mymensingh and DR.

Md. Zubaed Hossain, MS student, Department of Physiology, Bangladesh Agricultural

University, Mymensingh for their inspirations and assistances during the course of the research.

The author would like to express his cheerful acknowledgements to the sweet surroundings of well

wisher specially DR. Sankar, DR. Mehedi, DR. Mamun, DR. Harun, DR. Tarek, DR. Shuvo,

DR. Sujon, DR. Sulaiman and DR. Saleha Akter for their kind cooperation throughout the whole

research period.

The author expresses his thanks to all technicians and staff especially Md. Idris Ali and Md.

Raihan, Department of Pathology for their assistance.

The author gratefully acknowledges to his beloved elder sisters Most. Angumanara Begum and

Most. Samina Nargis, younger brother Md. Morshedul Alam and brother-in-law Md. Samsul

Alam for their marvelous sacrifices, inspiration and blessing throughout his life.

Finally indebtedness is due to his beloved father Md. Balel Uddin Sarker and mother

Most. Momotaz Begum for their sacrifices, inspiration, cooperation and blessing to get him to this

position.

The Author

May, 2012

v

ABSTRACT

This research work was undertaken to study the experimental pathogenesis of

infectious coryza by a local isolate of Avibacterium paragallinarum in broiler chicks

in Bangladeah. For this purpose, 24 chicks of 14 days of age were grouped into two

(A and B), each group containing 12 birds. Chicks of group A were inoculated with 1

ml of 2 days old nutrient broth and were kept as control group while group B were

inoculated with 1 ml of 2 days old culture broth of Avibacterium paragallinarum. To

study the pathology, 4 birds from each group were sacrificed on day 3, 5 and 7 of post

inoculation. Sacrificed birds of group A did not reveal any significant clinical sign and

lesion. Chicks of group B showed mild nasal discharge, conjunctivitis, depression and

inability to move. The gross lesions of the chicks of group B included mucus in nasal

passage, conjunctivitis, swelling of sinuses and face and congested lungs. The

microscopic lesions in this group were acanthosis and congested blood vessels of

nasal passage, pneumonic lesion of lung, focal hepatitis of liver and fatty change and

lipid nodules in macrophages of heart which were progressively prominent on day 7

of bacterial inoculation. Avibacterium paragallinarum was reisolated from day 7 of

post inoculation (PI) from nasal passage of chicks in which lesions were prominent.

The proposed experimental pathogenesis might be inoculation of A. paragallinarum

through nasal passage it produced rhinitis following reached to the different organs via

blood and finally revealed lesions. The lesions that found in this experiment (rhinitis

in association with focal hepatitis, fatty change in heart with lipid granuloma,

progressive pneumonic lesions) are not normally present in adult and young birds. In

this study there was no lesion in control group (inoculated without A.

paragallinarum). But in comparison with control group time dependently severity of

lesions was found in different organs in experimental inoculated group (inoculated

with A. paragallinarum). This may be a new finding of this disease. However, it needs

further investigation.

vi

CONTENTS

CHAPTER TITLE PAGE NO.

ACKNOWLEDGEMENTS iii-iv

ABSTRACT v

LIST OF CONTENTS vi-ix

LIST OF TABLES x

LIST OF FIGURE xi

LIST OF ABBREVIATIONS AND SYMBOLS xii

CHAPTER I INTRODUCTION 1-2

CHAPTER II REVIEW OF LITERATURE 3-21

2.1. History 3

2.2. Economic Significance 3-4

2.3. Public Health Significance 4

2.4. Etiology 4

2.5. Pathobiology and Epizootiology 5-10

2.5.1. Incidence and Distribution 5

2.5.2. Natural and Experimental Hosts 5

2.5.3. Age of Host Most Commonly Affected 5

2.5.4. Transmission, Carriers, and Vectors 5-6

2.5.5. Incubation Period 6

2.5.6. Pathogenicity 6-7

2.5.7. Virulence Factors 6-7

2.5.8. Clinical Signs 7

2.5.9. Morbidity and Mortality 8

2.5.10. Pathology 8-9

2.5.11. Immunity 9-10

2.6. Morphology and Staining 10

2.7. Growth Requirements 10-11

2.8. Colony Morphology 11

vii

CONTENTS (Contd.)


2.9. Biochemical Properties 11-12

2.10. Susceptibility to Chemical and Physical Agents 12-14

2.11. Strain Classification 14-15

2.12. Immunogenicity or Protective Characteristics 16-17

2.13. Molecular Techniques 17-18

2.14. Diagnosis 18-20

2.14.1. Isolation and Identification of Causative Agent 18-20

2.14.2. Serology 20-21

CHAPTER III MATERIALS AND METHODS 22-36

3.1. Preparation of experimental house 22

3.2. Chicks 22

3.3. Feed 22

3.4. Experimental Pathogenesis Study 22-36

3.4.1. Groupings 22

3.4.2. Inoculation of Bacteria 23

3.4.3. Post-mortem of chicks and sample collection 23-24

3.4.4. Histopathology 24-28

3.4.4.1. Processing of tracheal tissue 24-25

3.4.4.2. Processing of nasal passage tissue 25

3.4.4.3. Preparation of decalcifying solution 26

3.4.4.4. Preparation of stains 26-27

3.4.4.4.1. Preparation of Harris Hematoxylin solution 26

3.4.4.4.2. Preparation of eosin solution 26-27

3.4.4.5. Routine Hematoxyrlin eosin staining procedure 27

3.4.4.6. Photomicrography 28

3.4.5. Reisolation of Avibacterium paragallinarum in

Bacteriological Media

28-30

viii

CONTENTS (Contd.)


3.4.5.1. Preparation of various bacteriological culture media

and different liquid solution

28

3.4.5.1.1. Nutrient broth 28

3.4.5.1.2. Nutrient agar 28-29

3.4.5.1.3. Blood agar 29

3.4.5.2. Isolation and identification of organisms 30

3.4.5.2.1. Isolation and identification of Staphylococcus

aureus

30

3.4.5.2.1.1 Primary culture of Staphylococcus aureus 30

3.4.5.2.1.2. Isolation of Staphylococcus aureus in pure culture 30

3.4.5.2.2. Isolation and identification of Avibacterium

paragallinarum

30

3.4.5.2.2.1. Primary culture of A. paragallinarum 30

3.4.5.2.2.2. Isolation of A. paragallinarum in pure culture 30

3.4.5.3. Study of colony morphology for identification 30

3.4.6. Staining 31-32

3.4.6.1. Preparation of Gram staining solution 31-32

3.4.6.2. Microscopic study of the suspected colonies 32

3.4.7. Biochemical studies for the identification of

organisms

32-36

3.4.7.1. Reagents for biochemical test 33

3.4.7.2. Sugars 33

3.4.7.3. Carbohydrate fermentation test 33-34

3.4.7.4. Indole test 35

3.4.7.5. Methyl-Red & Voges-Proskauer (MR-VP) test 35-36

3.4.8. Enzyme activity test 36

3.4.8.1. Catalase test 36

ix

CONTENTS (Contd.)


CHAPTER IV RESULTS 37-45

4.1. Clinical findings of chickens 37

4.2. Gross study 37

4.3. Histopathological study 38

4.4. Reisolation of Avibacterium paragallinarum on day 7 39-40

4.4.1. Results of Gram's stain 39

4.4.2. Results of biochemical tests 40

4.4.3. Results of sugar fermentation test 40

4.4.4. Results of other biochemical tests 40

4.5. Enzymatic activity test 41

4.5.1. Catalase activity test 41

CHAPTER V DISCUSSION 46-47

CHAPTER VI SUMMARY AND CONCLUSION 48-49

REFERENCES 50-68

x

LIST OF TABLES

TABLE TITLE PAGE

Table 1 Differential tests for the avian haemophili 13

Table 2 No. of slaughtered chicks for sample collection on different days

24

Table 3 Results of gross study 38

Table 4 Results of histopathological studies of group A (inoculated

with nutrient broth) 38

Table 5 Results of histopathological studies of group B (inoculated

with A. paragallinarum) 39

Table 6 Results of biochemical characteristics of A. paragallinarum 40

xi

LIST OF FIGURES

FIGURE TITLE PAGE Fig. 1 Intranasal inoculation of A. paragallinarum on day 14 of age 23 Fig. 2 Depression of chicks of group B( inoculation with A. paragallinarum ) on

day 7 of post inoculation 42

Fig. 3 A chick of group B ( inoculation with A. paragallinarum ) with conjunctivitis and mild facial edema on day 7 of post inoculation

42

Fig. 4 Severely congested lung of a chicken of group B (inoculation with A. paragallinarum ) with on day 7 of post inoculation.

42

Fig. 5 Mild tracheal haemorrhage of a chicken of group B ( inoculation with A. paragallinarum ) on day 3 of post inoculation

Fig. 6 Staphylococcus aureus produces golden yellow color colony on manitol salt agar media.

43

Fig. 7 Fermentation of glucose, sucrose, mannitol, maltose with production of only acid and no galactose by A. paragallinarum.

43

Fig. 8 A. paragallinarum showing gram negative rod shaped bacilli (Gram's staining. x830)

43

Fig. 9 A. paragallinarum produce smooth iridescent colonies with no hemolysis on blood agar media

43

Fig. 10 Nasal passage of group A on day 7 of post inoculation showing no lesion.

44

Fig. 11 Nasal passage of group B (inoculation with A. paragallinarum ) on day 7 of post inoculation showing acanthosis.

44

Fig. 12 Nasal passage of group B ( inoculation with A. paragallinarum ) on day 5 of post inoculation showing presence of reactive leukocytes

44

Fig. 13 Nasal passage of group B ( inoculation with A. paragallinarum ) on day 7 of post inoculation showing congestion of blood vessels and acanthosis

44

Fig. 14 Section of lung of a chicken of group A showing almost no lesion (±) 44 Fig. 15 Lung of group B (inoculation with A. paragallinarum ) on day 3 of post

inoculation showing mild pneumonic lesion. (+). 44

Fig. 16 Lung of group B ( inoculation with A. paragallinarum ) on day 5 of post inoculation showing moderate pneumonic lesion (++)

45

Fig. 17 Lung of group B ( inoculation with A. paragallinarum ) on day 7 of post inoculation showing severe pneumonic lesion (+++)

45

Fig. 18 Fatty change, lipid nodules in macrophages and micronodules in heart on day 5 of post inoculation

45

Fig. 19 Fatty change, lipid nodules in macrophages and micronodules in heart on day 7 of post inoculation

45

Fig. 20 Liver of group A on day 7 of post inoculation 45 Fig. 21 Liver of group B ( inoculation with A. paragallinarum ) on day 7 of post

inoculation showing focal hepatitis 45

xii

LIST OF ABBREVIATION AND SYMBOLS

A. : Avibacterium Av. : Avibacterium

BAU : Bangladesh Agricultural University BCRDV : Baby chick Ranikhet disease vaccine

cm : Centimeter Co. : Company

dl : Deciliter et al. : Associate

etc. : Etcetera Fig. : Figure

H. : Haemophilus IC : Infectious Coryza

Kcal : Kilocalorie kg : Kilogram g : Gram

Ltd. : Limited Max. : Maximum Min. : Minimum No. : Number

PI : Post inoculation rpm : Rotation per minute µm : Micrometer ºC : Degree Celsius ± : Plus minus

NAD : Nicotinamide adenine dinucleotide ELISA : Enzyme linked immunosorbent assy MR : Methyl red VP : Voges proskauer

1

CHAPTER I

INTRODUCTION

There is no denying fact that nowadays the poultry sub-sector is crucial in the context

of agricultural growth and improvement of diet for the people in Bangladesh. This

sub-sector is particularly important in the sense that it is a significant source for the

supply of protein and nutrition in a household’s nutritional intake. At present chicken

contributes 51% of total meat production of the country (Raha, 2007). It is an

attractive economic activity as well, especially to women and the poorer sections.

Poultry farms in Bangladesh have witnessed a rapid growth in recent times. Poultry

industry is an emerging agribusiness started practically during 1980s in Bangladesh

(Huque, 2001). The poultry sector in Bangladesh is very important for the reduction of

poverty and creation of employment opportunities. Many people are directly

dependent on this industry for their livelihood. A total of 5 million people are engaged

in this sector (Saleque, 2006). About 110,800 different size poultry farms have been

established in the country (Anon, 2006). Per capita annual consumption of meat is

5.99 kg against the universal standard is 80 kg per head (Raha, 2007).

Though there is satisfactory growth of poultry industry in Bangladesh it also faces

some constraints. Among the constraints, emerging and reemerging diseases play a

pivotal role for the development of this sector. Of all the emerging and reemerging

diseases, there are lots of diseases that can affect the upper respiratory tact of chickens

resulting low production of meat and egg.

Among the respiratory diseases infectious coryza is an acute respiratory disease in

chickens. The disease has worldwide economic recognition and causes infection in

both broiler and layer flocks. The disease has a low mortality rate but leads to a drop

in egg production of up to 40 % in layer hens and increased culling in broilers and

thus poses significant financial liability to chicken farmers (Mouahid et al., 1989).

The morbidity and mortality rate of broilers is 10-30% and 0.5 to 2%, respectively

(Ibrahim et al., 2004). The disease is caused by gram negative bacteria Avibacterium

2

paragallinarum (previously called Haemophilus paragallinarum) which are classified

into three serovars (A, B and C) using a slide agglutination test (Page, 1962).

Clinically, the disease is characterized by rapid onset and high morbidity in flock,

decreased of feed consumption, decreased egg production/growth, oculonasal

conjunctivitis, edema of the face, respiratory noise, swollen infraorbital sinus, and

exudates in the conjuncivital sac (Eaves et al., 1989). Respiratory sign of infectious

coryza persists for a few weeks if complicated by Fowl pox, Mycoplasma

gallisepticum, Newcastle Disease, Infectious Bronchitis, Pasteurellosis and Infectious

laryngotracheitis (Yamamoto, 1972; Sandoval et al., 1994). So, certainly it has a huge

negative impact in poultry industry.

Clinically and grossly, the disease was diagnosed as infectious coryza in Bangladesh

(Talha et al., 2001) but the causal agent was not identified. The confirmatory

diagnosis of the disease in poultry of Bangladesh is inevitable. Pathological study

(gross and microscopic) on the disease, isolation and identification of the causal agent;

and study of pathogenesis by local isolate of this bacterium in experimental bird will

enrich the knowledge to identify the disease rapidly and that will reflect the

prevention and control measures of the disease. To my knowledge, experimental

pathogenesis study by local isolate of the bacteria was not performed.

Therefore, the present investigation was undertaken with the following objective:

To study the pathogenesis of the disease in experimental chicks with local isolate of

Avibacterium paragallinarum.

3

CHAPTER II

REVIEW OF LITERATURE

There is no doubt that infectious coryza (IC) caused by Avibacterium paragallinarum

(formerly known as Haemophilus paragallinarum) has a great negative impact in

poultry industry of Bangladesh. A few relevant published information on the

experiment have been reviewed in the following paragraphs.

2.1. History

As early as 1920, Beach believed that IC was a distinct clinical entity. The etiologic

agent eluded identification for a number of years, because the disease was often

masked in mixed infections and with fowl pox in particular. In 1932, De Blieck

isolated the causative agent and named it Bacillus hemoglobinophilus coryzae

gallinarum.

2.2. Economic Significance

The greatest economic losses result from poor growth performance in growing birds

and marked reduction (10-40%) in egg production. The disease can have significant

impact in meat chickens. In California, two cases of infectious coryza, one

complicated by the presence of Mycoplasma synoviae, caused increased

condemnations, mainly due to airsacculitis, which varied from 8.0—15% (Droual et

al., 1990). In Alabama, an infectious coryza outbreak in broilers, which was not

complicated by any other disease agent, caused a condemnation rate of 69.8%,

virtually all due to airsacculitis (Hoerr et al., 1994). When the disease occurs in

chicken flocks in developing countries, the added presence of other pathogens and

stress factors can result in disease outbreaks that are associated with greater economic

losses than those reported in high health flocks in developed countries. In China,

outbreaks of infectious coryza have been associated with morbidities of 20-50% and

mortalities of 5-20% (Chen et al., 1993). In Morocco, outbreaks on 10 layer farms

caused egg drops that ranged from 17-41% and mortalities of 0.7-10% (Mouahid et

al., 1989). A study of village chickens in Thailand has shown that the most common

4

cause of death in chickens less than 2 months old and those more than six months old

was infectious coryza (Thitisak et al., 1988). It was only in chickens that were

between 2 and 6 months of age that other diseases, such as Newcastle disease and

fowl cholera, killed more chickens than infectious coryza (Thitisak et al., 1988).

Overall, considerable evidence shows that infectious coryza outbreaks can have a

much greater impact in developing countries than in developed countries.

2.3. Public Health Significance

The disease is limited primarily to chickens and has no public health significance.

2.4. Etiology

Based on studies conducted during the 1930s, the causative agent of IC was classified

as H. gallinarum because of its requirement for both X-(hemin) and V-(nicotinamide

adenine dinucleotide—NAD) factors for growth (Eliot et al., 1934; Schalm et al.,

1936). Since 1962, however, Page, 1962 and others (Narita et al., 1978; Rimler, 1979;

Hinz, 1980) have found that all isolates recovered from cases of IC required only the

V-factor for growth. This led to the proposal and general acceptance of a new species,

H. paragallinarum (Zinnemann, and Biberstein, 1974), for organisms requiring only

the V-factor. H. gallinarum and H. paragallinarum are identical in all other growth

characteristics and diseaseproducing potential (Rimler, 1979). These observations, in

addition to the apparent abrupt change in the X-factor requirement of all isolates

recovered worldwide since 1962, have led some workers to question the validity of

tests used by earlier workers in classifying their isolates as H. gallinarum (Rimler,

1979). Indeed, it has been suggested that the early descriptions of the causative agent

of IC as an X- and V-factor—dependent organism were incorrect (Blackall, and

Yamamoto, 1989). More recently, V-factor independent isolates of H. paragallinarum

have been recovered from chickens with coryza in South Africa (Horner et al., 1992;

Bragg et al., 1993). Thus, it is apparent that classification of hemophili based strictly

on in vitro growth factor requirements may be misleading, as suggested by Kilian and

Biberstein (Kilian et al., 1984).

5

2.5. Pathobiology and Epizootiology

2.5.1. Incidence and Distribution

Infectious coryza occurs whereever chickens are raised. The disease is a common

problem in the intensive chicken industry; significant problems have been reported in

California, southeastern United States, and most recently in the northeastern regions

of the United States. The disease has also been reported in other, less intensive

situations. As an example, infectious coryza has been a problem in kampung (village)

chickens in Indonesia (Poernomo et al., 2000).

2.5.2. Natural and Experimental Hosts

The chicken is the natural host for H. paragallinarum. Several reports indicate that the

village chickens of Asia are as susceptible to infectious coryza as normal commercial

breeds (Zaini and Kanameda, 1991; Poernomo et al., 2000). Although there have been

reports of IC due to H. paragallinarum in a number of bird species other than

chickens, reviewed by Yamamoto (1991)), these reports need to be interpreted

carefully. As a range of hemophilic organisms, none of which are H. paragallinarum,

have been described in birds other than chickens (Grebe and Hinz. 1975; Piechulla et

al., 1985; Devriese et al., 1988), only those studies that involve detailed bacteriology

can be regarded as definitive proof of the presence of H. paragallinarum in birds other

than chickens. The following species are refractory to experimental infection: turkey,

pigeon, sparrow, duck, crow, rabbit, guinea pig, and mouse (Yamamoto, 1972;

Yamamoto, 1978).

2.5.3. Age of Host Most Commonly Affected

All ages are susceptible (Yamamoto, 1991), but the disease is usually less severe in

juvenile birds. The incubation period is shortened, and the course of the disease tends

to be longer in mature birds, especially hens with active egg production.

2.5.4. Transmission, Carriers, and Vectors

Chronic or healthy carrier birds have long been recognized as the main reservoir of

infection. The application of molecular fingerprinting techniques has confirmed the

6

role of carrier birds in the spread of IC (Blackall et al., 1990). Infectious coryza seems

to occur most frequently in fall and winter, although such seasonal patterns may be

coincidental to management practices (e.g., introduction of susceptible replacement

pullets onto farms where IC is present). On farms where multiple-age groups are

brooded and raised, spread of the disease to successive age groups usually occurs

within 1-6 weeks after such birds are moved from the brooder house to growing cages

near older groups of infected birds (Clark and Godfrey, 1961). Infectious coryza is not

an egg-transmitted disease. Whereas the sparrow could not be implicated as a vector,

epidemiologic studies suggested that the organism may be introduced onto isolated

ranches by the airborne route (Yamamoto, and Clark, 1966).

2.5.5. Incubation Period

The characteristic feature is a coryza of short incubation that develops within 24-48

hours after inoculation of chickens with either culture or exudate. The latter will more

consistently induce disease (Rimler, 1979). Susceptible birds exposed by contact to

infected cases may show signs of the disease within 24-72 hours. In the absence of a

concurrent infection, IC usually runs its course within 2-3 weeks.

2.5.6. Pathogenicity

As a general observation, the pathogenicity of H. paragallinarum can vary according

to both the growth conditions and passage history of the isolate and the state of the

host. Some specific evidence of variation in pathogenicity exists amongst H.

paragallinarum isolates. Yamaguchi et al. (1990) found that one of four strains of H.

paragallinarum serovar B failed to produce clinical signs. Horner et al. (1995) have

suggested that the NAD-independent isolates may cause airsacculitis more commonly

than the classic NAD-dependent H. paragallinarum isolates.

2.5.7. Virulence Factors

A range of factors has been associated with the pathogenicity of H. paragallinarum.

Considerable attention has been paid to HA antigens. In both Page serovar A and C,

mutants lacking HA activity have been used to demonstrate that the HA antigen plays

7

a key role in colonization (Sawata and Kume, 1983; Yamaguchi et al., 1993). The

capsule has also been associated with colonization and has been suggested to be the

key factor in the lesions associated with IC (Sawata and Kume, 1983; Sawata et al.,

1985). The capsule of H. paragallinarum has been shown to protect the organism

against the bactericidal activity of normal chicken serum (Sawata et al., 1984). It has

been suggested that a toxin released from capsular organisms during in vivo

multiplication was responsible for the clinical disease (Kume et al., 1984). H.

paragallinarum can acquire iron from chicken and turkey transferrin, suggesting that

iron sequestration may not be an adequate host defense mechanism (Ogunnariwo et

al., 1992). In contrast, two strains of H. avium were unable to acquire iron from these

transferrins, despite apparently having the same receptor proteins (Ogunnariwo et al.,

1992). Crude polysaccharide extracted from H. paragallinarum is toxic to chickens

and may be responsible for the toxic signs that may follow the administration of

bacterin (Iritani et al., 1981). The role, if any, of this component in the natural

occurrence of the disease is unknown.

2.5.8. Clinical Signs

The most prominent features are an acute inflammation of the upper respiratory tract

including involvement of nasal passage and sinuses with a serous to mucoid nasal

discharge, facial edema, and conjunctivitis facial edema. Swollen wattles may be

evident, particularly in males. Rales may be heard in birds with infection of the lower

respiratory tract. A swollen head-like syndrome associated with H. paragallinarum

has been reported in broilers in the absence of avian pneumovirus, but in the presence

or absence of other bacterial pathogens such as M. synoviae and M. gallisepticum

(Droual et al., 1990; Sandoval et al., 1994). Arthritis and septicemia have been

reported in broiler and layer flocks, respectively, in which the presence of other

pathogens has contributed to the disease complex (Sandoval et al., 1994). Birds may

have diarrhea, and feed and water consumption usually is decreased; in growing birds,

this means an increased number of culls; and in laying flocks, this means a reduction

in egg production (10—40%). A foul odor may be detected in flocks in which the

disease has become chronic and complicated with other bacteria.

8

2.5.9. Morbidity and Mortality

IC is usually characterized by low mortality and high morbidity. Variations in age and

breed may influence the clinical picture (Blackall, 1983). Complicating factors such as

poor housing, parasitism, and inadequate nutrition may add to severity and duration of

the disease.

When complicated with other diseases such as fowl pox, infectious bronchitis,

laryngotracheitis, Mycoplasma gallisepticum infection, and pasteurellosis, IC is

usually more severe and prolonged, with resulting increased mortality (Sandoval et

al., 1994; Yamamoto, 1972).

2.5.10. Pathology

Gross

H. paragallinarum produces an acute catarrhal inflammation of mucous membranes

of nasal passages and sinuses. Frequently, a catarrhal conjunctivitis and subcutaneous

edema of face and wattles occur. Typically, pneumonia and airsacculitis are rarely

present; however, reports of outbreaks in broilers have indicated significant levels of

condemnations (up to 69.8%) due to airsacculitis, even in the absence of any other

recognized viral or bacterial pathogens (Droual et al., 1990; Hoerr et al., 1994).

Microscopic

Fujiwara and Konno (1965) studied the histopathologic response of chickens from 12

hours to 3 months after intranasal inoculation. Essential changes in the nasal cavity,

infraorbital sinuses, and trachea consisted of sloughing, disintegration, hyperplasia of

mucosal and glandular epithelia, and edema and hyperemia with heterophil infiltration

in the tunica propria of the mucous membranes. Pathologic changes first observed at

20 hours reached maximum severity by 7-10 days, with subsequent repair occurring

within 14-21 days. In birds with involvement of the lower respiratory tract, acute

catarrhal ronchopneumonia was observed, with heterophils and cell debris filling the

lumen of secondary and tertiary bronchi; epithelial cells of air capillaries were swollen

and showed hyperplasia. Catarrhal inflammation of air sacs was characterized by

9

swelling and hyperplasia of the cells, with abundant heterophil infiltration. In addition,

a pronounced infiltration of mast cells was observed in the lamina propria of the

mucous membrane of the nasal cavity (Sawata et al., 1985). The products of mast

cells, heterophils, and macrophages may be responsible for the severe vascular

changes and cell damage leading to coryza. A dissecting fibrinopurulent cellulitis

similar to that seen in chronic fowl cholera has been reported in broiler and layer

chickens (Droual et al., 1990).

2.5.11. Immunity

Chickens that have recovered from active infection possess varying degrees of

immunity to reexposure. Pullets that have experienced IC during their growing period

are generally protected against a later drop in egg production. Resistance to

reexposure among individual birds may develop as early as 2 weeks after initial

exposure by the intrasinus route (Sato and Shifrine, 1964). Experimentally infected

chickens develop a crossserovar (Page scheme) immunity (Rimler and Davis, 1977).

In contrast, as discussed earlier, bacterins provide only serovar-specific immunity

(Blackall and Reid, 1987; Kume et al., 1980; Rimler et al., 1977). This suggests that

cross-protective antigens are expressed in vivo that are either not expressed or

expressed at very low levels in vitro.

The protective antigens of H. paragallinarum have not been definitively identified. It

has been suggested that the capsule of H. paragallinarum contains protective antigens

(Sawata et al., 1984). Using both a Page serovar A and C strains, a crude

polysaccharide extract was shown to provide serovar-specific protection (Iritani et al.,

1981). Considerable attention has been paid to the role of HA antigens as protective

antigens. It has been long noted that for Page serovar A organisms, a close correlation

exists between HI titer and both protection (Otsuki and Iritani, 1974; Kume et al.,

1980) and nasal clearance of the challenge organism (Kume et al., 1984) in vaccinated

chickens. Purified HA antigen from a Page serovar A organism has been shown to be

protective (Iritani et al., 1980). Takagi and colleagues have shown that a monoclonal

antibody specific for the HA of Page serovar A provides passive tection and that the

10

HA antigen purified by use of this antibody is also protective (Takagi et al., 1991;

Takagi et al., 1992).

Based on studies conducted to date, considerable evidence shows that the protective

antigens of H. paragallinarum are surface located. The antigens implicated have been

the antigens detected during Page serotyping, HA antigens, and some component or

components of the polysaccharide content of the cell. It seems probable that a number

of different antigens (outer-membrane proteins, polysaccharides, lipopolysaccharides)

are all likely to be involved.

2.6. Morphology and Staining

H. paragallinarum is a gram-negative nonmotile bacterium. In 24-hour cultures, it

ppears as short rods or coccobacilli 1-3 mm in length and 0.4-0.8 mm in width, with a

tendency for filament formation. A capsule may be demonstrated in virulent strains

(Hinz, 1973; Sawata et al., 1980). The organism undergoes degeneration within 48-60

hours, showing fragments and ill-defined forms. Subcultures to fresh medium at this

stage will again yield the typical rodshaped morphology. Bacilli may occur singly, in

pairs, or as short chains (Schalm and Beach, 1936).

2.7. Growth Requirements

The reduced form of NAD (NADH; 1.56-25 µg/mL medium) (Page, 1962; Rimler et

al., 1977) or its oxidized form (20-100 µg/mL) (Sato and Shifrine, 1965) is necessary

for the in vitro growth of most isolates of H. paragallinarum. The exceptions are the

isolates described in South Africa, which are NAD independent (Horner et al., 1992;

Mouahid et al., 1992; Bragg et al., 1993). Sodium chloride (NaCl) (1.0-1.5%) (Rimler

et al., 1977) is essential for growth. Chicken serum (1%) is required by some strains

(Hinz, 1973), whereas others merely show improved growth with this supplement

(Blackall and Reid, 1982). Brain heart infusion, tryptose agar, and chicken-meat

infusion are some basal media to which supplements are added (Hinz, 1973; Kume et

al., 1980; Sato and Shifrine, 1965). More complex media are used to obtain dense

growth of organisms for aracterization studies (Rimler, 1979; Blackall, 1983; Reid

11

and Blackall, 1987). The pH of various media varies from 6.9-7.6. A number of

bacterial species excrete V-factor that will support growth of H. aragallinarum (Page,

1962). The determination of the growth factor requirements of the avian haemophili is

not an easy process. Commercial growth factor disks used for this purpose may yield a

high percentage of cultures that falsely appear to be both X- and V-factor dependent

(Blackall and Farrah, 1985). The brand of disks and the medium to be used should be

checked are fully for their suitability. For well-equipped laboratories, the porphyrin

test (Kilian, 1974) is recommended for X factor testing. For classical X- and V-factor

testing, the use of purified hemin and NAD as supplements to otherwise complete

media may also be considered.

The organism is commonly grown in an atmosphere of 5% carbon dioxide; however,

carbon dioxide is not an essential requirement, because the organism is able to grow

under reduced oxygen tension or anaerobically (Eliot and Lewis, 1934; Page, 1962).

The minimal and maximal temperatures of growth are 25 and 45°C, respectively, the

optimal range being 34- 42°C. The organism is commonly grown at 37-38°C.

2.8. Colony Morphology

Tiny dewdrop, nonhemolytic colonies up to 0.3 mm in diameter develop on suitable

media. In obliquely transmitted light, mucoid (smooth) iridescent, rough

noniridescent, and other intermediate colony forms have been observed (Hinz, 1976;

Rimler, 1979; Sawata et al., 1979; Sawata and Kume, 1983).

2.9. Biochemical Properties

The ability to reduce nitrate to nitrite and ferment glucose without the formation of

gas is common to all the avian haemophili. Oxidase activity, the presence of the

enzyme alkaline phosphatase, and a failure to produce indole or hydrolyse urea or

gelatin are also uniform characteristics (Blackall, 1989). Considerable confusion

surrounds the carbohydrate fermentation patterns of the avian haemophili. Much of

the variability recorded in the literature may be due to the use of different basal media.

12

False-negative results are associated mainly with poor growth and can be a significant

problem (Blackall, 1983). In general, recent studies have used a medium consisting of

a phenol red broth containing 1% (w/v) NaCl, 25 µg/mL NADH, 1% (v/v) chicken

serum, and 1% (w/v) carbohydrate. For routine identification, the use of the phenol

red broth just described and a dense inoculum is a most suitable approach for

determining carbohydrate fermentation patterns. Alternatively, agarbased methods

(Blackall, 1983; Terzolo et al., 1993) may be used. A range of organisms that

superficially resemble H. paragallinarum can be found in chickens. In particular,

organisms once known as Haemophilus avium are common in chickens and are

regarded as nonpathogenic (Hinz and Kunjara, 1977).

Based on DNA hybridization studies, isolates of H. avium were found to be comprised

of at least three DNA homology groups (Mutters et al., 1985). They have been named

Pasteurella avium, P. volantium, and Pasteurella species A. Not all isolates of H.

avium, however, can be assigned to these three new taxa solely on the basis of

phenotypic properties (Blackall, 1988). Table 1. represents those properties that allow

a full identification of the avian haemophili. The failure of H. paragallinarum to

ferment either galactose or trehalose and its lack of catalase clearly separates this

organism from the other avian haemophili. The properties shown in the table for H.

paragallinarum have been found to be typical of isolates from Argentina, Australia,

Brazil, China, Germany, Indonesia, Japan, Kenya, Malaysia, and the United States

(Blackall et al., 1982; Blackall et al., 1994; Chen et al., 1993; Hinz and Kunjara,

1977; Kesler, 1997; Kume et al., 1978; Narita et al., 1978; Poernomo et al., 2000;

Rimler, 1979; Terzolo et al., 1993, Zaini et al., 1991). The main characteristics that

differentiate the NAD-independent from the NADdependent H. paragallinarum are

that the former does not have ß-galactosidase activity and does not ferment maltose

(Mouahid et al., 1992).

2.10. Susceptibility to Chemical and Physical Agents

H. paragallinarum is a delicate organism that is inactivated rather rapidly outside the

host. Infectious exudate suspended in tap water is inactivated in 4 hours at ambient

13

temperature; when suspended in saline, the exudate is infectious for at least 24 hours

at 22°C. Exudate or tissue remains infectious when held at 37°C for 24 hours and, on

occasion, up to 48 hours; at 4°C, exudate remains infectious for several days. At

temperatures of 45-55°C, hemophili are killed within 2-10 minutes. Infectious

embryonic fluids treated with 0.25% formalin are inactivated within 24 hours at 6°C,

but the organism survives for several days under similar conditions when treated with

thimerosal, 1:10,000 (Yamamoto, 1978). The organism may be maintained on blood

agar plates by weekly passages.

Table 1. Differential tests for the avian haemophili

Property Hemophilus

paragallinarium

H. avium Pasteurella

avium

P.

volantium

Pasteurella

species A

Pigment _ Yellow V _ Yellow U _

Catalase _ + + + +

Growth in

air

_ + + + +

ONPG + V _ + V

Acid from

Arabinose

_ V _ _ +

Galactose _ + + + +

Maltose + V _ + V

Mannitol + V _ + V

Sorbital V V _ V _

Sucrose V + + + +

Trehalose _ + + + +

Susceptibility to Chemical and Physical Agents

U = usually; V = variable; + = positive; _ = negative.

14

Young cultures maintained in a “candle jar” will remain viable for 2 weeks at 4°C.

Chicken embryos 6-7 days old may be inoculated with single colonies or broth

cultures via the yolk sac; yolk from embryos dead in 12-48 hours will contain a large

number of organisms that may be frozen and stored at -20 to -70°C or lyophilized

(Yamamoto, 1972.). A good suspension medium for lyophilization of H.

paragallinarum from agar cultures is used at the Animal Research Institute and

contains 6% sodium glutamate and 6% bacteriological peptone (filter sterilized). After

any storage, whether frozen or lyophilized, revival should include inoculation of a

suitable liquid growth medium (egg inoculation is even better) as well as an agar

medium.

2.11. Strain Classification

Antigenicity

Page (1962) classified his organisms of H. paragallinarum with the plate

agglutination test using whole cells and chicken antisera into serovars A, B, and C.

Although Page’s serovar A strain 0083 and B strain 0222 are available today, all the

serovar C strains were lost during the mid-1960s. Matsumoto and Yamamoto (1975)

isolated strain Modesto, which was later classified as a strain of serovar C by Rimler

et al. (1977). It is also possible to use a hemagglutination inhibition (HI) test to

serotype isolates by the Page scheme (Blackall et al., 1990). This HI test uses fixed

chicken erythrocytes and results in fewer nontypable isolates than the original

agglutination technology (Blackall et al., 1990) and is now the recommended

technique when performing serotyping by the Page scheme. The distribution of Page

serovars differs from country to country. Page serovar A has been reported in China

(Chen et al., 1993) and Malaysia (Zaini and Iritani, 1992); serovar C in Taiwan (Lin et

al., 1996); serovars A and B in Germany (Hinz, 1973); serovars A and C in Australia

(Blackall et al., 1988); and serovars A, B and C in Argentina (Terzolo et al., 1993),

Brazil (Blackall et al., 1994), Indonesia (Poernomo et al., 2000; Takagi et al., 1991),

Mexico (Fernández et al., 2000), the Philippines (Nagaoka et al., 1994), South Africa

(Bragg et al., 1996), Spain (Pages Mante and Costa Quintana, 1986), and the United

States (Page, 1962; Page et al., 1963).

15

Another method of assigning isolates of H. paragallinarum to a Page serovar is based

on the use of a panel of monoclonal antibodies developed by workers in Japan

(Blackall et al., 1991), but the technique is available only in a few laboratories due to

the limited availability of the monoclonal antibodies. Other sets of MABs have been

described but either lack serovar-specificity (Bragg et al., 1997; Zhang et al., 2000) or

detect only Page serovar A (Takagi et al., 1991). There have been suggestions that

Page serovar B is not a true serovar, but rather consists of variants of serovar A or C

that have lost their type-specific antigen (Kume et al., 1980; Sawata et al., 1980).

Recent studies, however, have shown conclusively that Page serovar B is a true

serovar (Yamaguchi et al., 1990). Kume et al. (1983) proposed an alternative

serologic classification based on an HI test using potassium thiocyanate- treated and -

sonicated cells, rabbit hyperimmune serums, and glutaraldehyde-fixed chicken

erythrocytes. In the original study, Kume et al. (1983) recognized three serogroups

and seven serovars. The terminology of the Kume scheme has been altered so that the

Kume serogroups match the Page serovars of A, B, and C (Blackall et al., 1990).

Thus, the nine currently recognized Kume serovars are A-1, A-2, A-3, A-4, B-1, C-1,

C-2, C-3, and C-4 (Blackall et al., 1990). Some Kume serovars seem to be unique in

terms of geographic origin—serovar A-3 has been found only in Brazil, serovar C-3

only in South Africa, and serovars A-4 and C-4 only in Australia (Blackall et al.,

1990, Eaves et al., 1989; Kume et al., 1983). Many isolates that were nontypable in

the Page scheme by agglutination tests were typed easily using the Kume scheme

(Eaves et al., 1989). Fernández et al. (2000) have reported the presence of Kume

serovars A-1, A-2, B-1, and C-2 in isolates of H. paragallinarum from Mexican

chickens. The Kume scheme has not been widely applied, as it is technically

demanding to perform. Hence, only a few laboratories are able to perform the

serotyping on a routine basis.

Other serological tests described in the literature include an agar-gel precipitin (AGP)

test (Hinz, 1980) and a serum bactericidal test (Sawata et al., 1984). Neither of these

tests has been widely used.

16

2.12. Immunogenicity or Protective Characteristics

Infectious coryza is relatively unique among common bacterial infections in that a

bacterin (inactivated whole cell vaccine) is protective against the disease when the

bacterin is adequately prepared. From the early days of bacterin production, it was

obvious that protection was limited (Matsumoto and Yamamoto, 1975). Later studies

confirmed a correlation between Page serovars and immunotype specificity (Kume et

al., 1980; Rimler et al., 1977). Chickens vaccinated with a bacterin prepared from one

serovar were protected against homologous challenge only. Evidence suggests that the

cross-protection within Page serovar B is only partial (Yamaguchi et al., 1991). Only

incomplete results are available on immunospecificity within the serogroups

recognized by the Kume scheme. Significant cross-protection has been shown

between Kume serovars C-1 and C-2 as well as between C-2 and C-4 (Blackall and

Reid, 1987; Kume et al., 1980). Only one serovar, B-1, exists within serogroup B of

the Kume scheme. However, reports have been made of undefined heterogeneity

within the B serogroup. Bivalent vaccines containing Page serovars A and C provide

protection against Page serovar B strain Spross but not against two South African

isolates of Page serovar B (Yamaguchi et al., 1991). Furthermore, only partial cross-

protection exists within various strains of Page serovar B (Yamaguchi et al., 1991).

Poor vaccine protection against IC due to serovar B strains in Argentina have been

explained by antigenic differences between field isolates and the “standard” serovar B

strains in commercial vaccines from North America or Europe (Terzolo et al., 1997).

One report supports the genetic uniqueness of serovar B strains isolated in Argentina

(Bowles et al., 1993). Vaccination/challenge exposure studies are needed to study the

antigenicity and immunospecificity of recent serovar B isolates. In both Argentina and

Brazil, isolates of Page serovar A are not recognized by a monoclonal antibody

specific for this serovar (Blackall et al., 1994; Terzolo et al., 1993). It has been

speculated that these “variant” Page serovar A isolates may be sufficiently different

from typical serovar A vaccine strains to cause vaccine failure (Terzolo et al., 1993).

South African workers have suggested that Kume serovar C-3 as well as other

serovars of NAD-independent H. paragallinarum are so antigenically different that

they are causing vaccine failure (Bragg et al., 1996; Bragg et al., 1997; Horner et al.,

17

1995). However, it has been shown that a commercial vaccine, specified as containing

serovars A, B, and C without details of the actual strains, provided acceptable levels

of protection against NAD-independent isolates of Page serovar A and Kume serovar

C-3 (Jacobs et al., 2000).

Overall, these recent results and field observations clearly indicate the need for further

vaccination/challenge studies. At this stage of our knowledge, no clear-cut definitive

publications negate the existence of cross-protection within Page serovars and Kume

serogroups. Indeed, the only publication to date, while not providing full details of the

vaccine seed strains, suggests that serological variation within a Page serovar is not a

cause of vaccine failure (Jacobs et al., 2000). There is no doubt that, on an ongoing

basis, debate will continue on the topic of whether commercially available trivalent

vaccines, containing serovars A, B, and C, give adequate protection if there are

significant antigenic differences between vaccine and field strains.

2.13. Molecular Techniques

DNA fingerprinting by restriction endonuclease analysis has been shown to be a

suitable typing technique with patterns being stable in vitro and in vivo (Blackall et

al., 1990; Blackall et al., 1991). Restriction endonuclease analysis has proven useful

in epidemiologic studies (Blackall et al., 1990). Ribotyping is another molecular

technique that has proven useful— being used to confirm that the recent NAD-

independent H. paragallinarum isolates from South Africa are clonal in nature (Miflin

et al., 1995) as well as examining the epidemiologic relationships among Chinese

isolates of H. paragallinarum (Miflin et al., 1997). ERIC-PCR, a DNA fingerprinting

method that uses the polymerase chain reaction technique, has been shown to be

capable of strain typing (Khan et al., 1998). The technique of multilocus enzyme

electrophoresis has been used to examine the genetic diversity of H. paragallinarum

isolates (Bowles et al., 1993). These nucleic acid techniques (including the

speciesspecific PCR discussed later in this chapter) are advancing to the stage where

they offer a rapid and convenient method for identification and typing. These

18

techniques are likely to replace time-consuming and cumbersome cultural,

biochemical, and serological means of identification and typing in the near future.

2.14. Diagnosis

2.14.1. Isolation and Identification of Causative Agent

Although H. paragallinarum is considered to be a fastidious organism, it is not

difficult to isolate, requiring simple media and procedures. Specimens should be taken

from two or three chickens in the acute stage of the disease (1—7 days’ incubation).

The skin under the eyes is seared with a hot iron spatula, and an incision is made into

the sinus cavity with sterile scissors. A sterile cotton swab is inserted deep into the

sinus cavity where the organism is most often found in pure form. Tracheal and air sac

exudates also may be taken on sterile swabs. The swab is streaked on a blood agar

plate, which is then cross-streaked with a Staphylococcus culture and incubated at

37°C in a large screw-cap jar in which a candle is allowed to burn out. Staphylococcus

epidermidis (Page, 1962) or S. hyicus (Blackall et al., 1982), which are commonly

used as “feeders,” should be pretested because not all strains actively produce the V

factor. Terzolo et al. (Terzolo et al., 1993) have reported the successful use of an

isolation medium that contains selective agents which inhibit the growth of gram-

positive bacteria. This medium has the advantage of not using either a “feeder”

organism or additives such as NADH.

At the simplest level, IC may be diagnosed on the basis of a history of a rapidly

spreading disease in which coryza is the main manifestation, combined with the

isolation of a catalase-negative bacterium showing satellitic growth. At this level, the

sinus exudate or culture should be inoculated into two or three normal chickens by the

intrasinus route. The production of a coryza in 24—48 hours is diagnostic; however,

the incubation period may be delayed up to 1 week if only a few organisms are present

in the inoculum, such as in long-standing cases. Better equipped laboratories should

attempt more complete biochemical identification as described earlier. Additional

studies of this nature are essential when isolates of NAD-independent H.

paragallinarum are suspected. To perform this biochemical testing, the suspect

19

isolates are best grown in pure culture on medium that does not require the addition of

a nurse colony. Many different media have been developed to support the growth of

H. paragallinarum (Kume et al., 1980; Otsuki, and Iritani. 1974; Rimler, 1979;

Terzolo et al., 1993). The medium described by Terzolo et al. (Terzolo et al., 1993) is

particularly suited for those laboratories that find the cost of such ingredients as

NADH and albumin expensive. The carbohydrate fermentation tests shown in Table

20.1 can be done in either a phenol red broth base (Rimler, 1979) or in an agar plate

format (4). The agar plate method can be performed in conventional petri dishes (9

cm), allowing multiple isolates to be tested at once, or in small petri dishes (2 cm),

allowing one to three isolates to be economically characterized. The agar plate method

(4) has also been modified to be performed as a tube method (Terzolo et al., 1993). A

PCR test specific for H. paragallinarum has been developed (Chen et al., 1996). This

test is rapid (results available within 6 hours compared with days for conventional

techniques) and has been shown to recognize all H. paragallinarum isolates tested,

including the NAD-independent H. paragallinarum from South Africa and the variant

Page serovar A isolates and the unusual Page serovar B isolates from Argentina (Chen

et al., 1996). The PCR, termed the HP-2 PCR, has been validated for use on colonies

on agar or on mucus obtained from squeezing the sinus of live birds (Chen et al.,

1996). When used directly on sinus swabs obtained from artificially infected chickens

in pen trials performed in Australia, the HP-2 PCR has been shown to be the

equivalent of culture—but much more rapid (Chen et al., 1996). When used in China,

direct PCR examination of sinus swabs outperformed traditional culture when used on

routine diagnostic submissions (Chen et al., 1998). The problems of poor samples,

delayed transport, and poor quality (but expensive) media mean that culture will have

a higher failure rate in developing countries than in developed countries—making the

PCR an attractive diagnostic option.

The HP-2 PCR is a robust test; sinus swabs stored for up to 180 days at 4°C or _20°C

were positive in the PCR (Chen et al., 1998). In contrast, culture of known positive

swabs failed to detect H. paragallinarum after 3 days of storage at 4°C or _20°C

(Chen et al., 1998). The HP-2 PCR has proven very useful in South Africa where the

20

diagnosis of infectious coryza is complicated by the presence of NAD-independent H.

paragallinarum, Ornithobacterium rhinotracheale, as well as the traditional form of

NAD-dependent H. paragallinarum (Miflin et al., 1999).

2.14.2. Serology

No totally suitable serological test exists for the diagnosis of infectious coryza.

However, despite this absence of a “perfect” test, serological results are often useful

for retrospective/epidemiological studies in the local area. A review of the techniques

that have been used in the past is presented by Blackall et al. (Blackall et al., 1997).

At this time, the best available test methodology is the HI test. Although a range of HI

tests have been described, three main forms of HI tests have been recognized—these

being termed simple, extracted, and treated HI tests (Blackall and Yamamoto, 1998).

Full details of how to perform these tests are available elsewhere (Blackall and

Yamamoto, 1998). In the following text, the advantages and disadvantages of the

three HI tests are briefly and critically discussed. The simple HI is based on whole

bacterial cells of Page serovar A H. paragallinarum and fresh chicken erythrocytes

(Iritani et al., 1977). Although simple to perform, this HI test can detect antibodies

only to serovar A. The test has been widely used to both detect infected as well as

vaccinated chickens (Blackall et al., 1997). The extracted HI test is based on KSCN-

extracted and sonicated cells of H. aragallinarum and glutaraldehydefixed chicken

erythrocytes (Sawata et al., 1982). This extracted HI test has been validated mainly for

the detection of antibodies to Page serovar C rganisms. The test has been shown to be

capable of detecting a serovar-specific antibody response in Page serovar C

vaccinated chickens (Sawata et al., 1982). A major weakness with this assay is that, in

chickens infected with serovar C, the majority of the birds remain seronegative

(Yamaguchi et al., 1988).

The treated HI test is based on hyaluronidase-treated whole bacterial cells of H.

paragallinarum and formaldehyde-fixed chicken erythrocytes (Yamaguchi et al.,

1989). The treated HI has not been widely used or evaluated. It has been used to detect

antibodies to Page serovars A, B, and C in vaccinated chickens with only serovar A

21

and C vaccinated chickens yielding high titers (Yamaguchi et al., 1991). The test has

been used to screen chicken sera in Indonesia for antibodies arising from infection

with serovars A and C (Takagi et al., 1991). Vaccinated chickens with titers of 1:5 or

greater in the simple or extracted HI tests have been found to be protected against

subsequent challenge (Sawata et al., 1982). Enough knowledge or experience is not

yet available to draw any sound conclusions on whether there is a correlation between

titer and protection for the treated HI test. An alternative serological test is a

monoclonal antibody-based blocking ELISA, the B-ELISA (Zhang et al., 1999).

While having shown very good specificity and acceptable levels of sensitivity, this

test has several drawbacks. As there are only monoclonal antibodies for Page serovar

A and C, the assay can detect only antibodies to these two serovars. The monoclonal

antibodies that form the heart of the assays are not commercially available, limiting

access to the assays. Finally, some isolates of H. paragallinarum do not react with the

monoclonal antibodies and, thus, infections associated with these isolates cannot be

detected with these ELISAs (Zhang et al., 1999). This ELISA has not been widely

evaluated, and there is no knowledge about any correlation between ELISA titer and

protection. The reduced sensitivity of the ELISA for serovar C infections indicates

that the test would have to be used as a flock test only (Zhang et al., 1999). A B-

ELISA kit based on the preceding B-ELISA has been developed (Miao et al., 2000).

Based on pen trial data, the serovar A B-ELISA kit had a sensitivity of 95% and a

specificity of 100%. The serovar C B-ELISA kit had a sensitivity of 73% and a

specificity of 100% (Miao et al., 2000).

Overall, the serological test of choice remains either the simple HI test (Iritani et al.,

1977) for either infections or vaccinations associated with serovar A, the extracted or

treated HI tests (Sawata et al., 1982; Yamaguchi et al., 1989) for vaccinations

associated with serovar C, and the treated HI test (Yamaguchi et al., 1989) for

infections associated with serovar C. There has been so little work performed on

serological assays for infections or vaccinations associated with serovar B that it is not

possible to recommend any test.

22

CHAPTER III

MATERIALS AND METHODS

The present research work was undertaken in The Department of Pathology,

Bangladesh Agricultural University, Mymensingh during January to May 2012 to

study the experimental pathogenesis in chicks by local isolate of Avibacterium

paragallinarum.

3.1. Preparation of experimental house

The experimental poultry house was properly cleaned, washed and then dried up. The

room was fumigated with formaldehyde and with ammonia before introduction of chicks.

The feeder, waterer and cages were cleaned with water and then fumigated with

ammonia.

3.2. Experimental Chicks

Twenty four, Cobb 500 day old broiler chicks were included in this study. The chicks

were collected from Kazi Hatchery, Gazipur. Vaccination of chicks with BCRDV was

performed on day 2.

3.3. Feed

The broiler chicks were provided with commercial broiler starter and grower feed (Champion

Starter and Champion Grower, Quality Feeds Ltd.) according to the age. The starter feed was

given from day 1 to day 10 and the grower feed was given from day 11 up to the end of

the experiment. The feed was stored in a dry place.

3.4. Experimental Pathogenesis Study

3.4.1. Groupings

Chicks (n = 24) at the age of day 14 were divided into 2 groups (group A and B) each

consisting of 12 birds. Chicks were reared in separate cages. The group A was maintained

as a control group without induction of infection. Birds of group B was maintained for

bacterial inoculation.

23

3.4.2. Inoculation of Bacteria

Chicks of group A were inoculated with 1 ml of 2 days old nutrient broth per bird at 14

days of age. Chicks of group B were inoculated at the age of 14 days with 2 days old

culture broth of Avibacterium paragallinarum at the dose rate of 1 ml/bird (Islam, 2010)

through intranasal route (Figure 1). The bacterial inoculum of A. paragallinarum was

taken from Akter (2012) who isolated and identified the bacteria from field samples.

Figure 1. Intranasal inoculation of A. paragallinarum on day 14 of age.

3.4.3. Post-mortem of chicks and sample collection

On day 3rd, 5th and 7th day of inoculation, four birds from each group were sacrificed for

post-mortem examination and samples collection (Table 4.)Nasal swab was collected

prior to post-mortem examination for isolation and identification head (nasal passage),

trachea, lungs, intestine and liver were examined for any changes and kept in10% neutral

buffered formalin for histopathological studies.

The gross lesions of different organs were graded as:

almost no lesions ( ±), mild lesions (+) and moderate lesions (++).

24

Table 2. No. of chicks slaughtered for sample collection on different days of post-

inoculation

Days of post-inoculation Group A Group B

Day 3 4 4

Day 5 4 4

Day 7 4 4

3.4.4. Histopathology

Nasal passage tissue, trachea, heart, lungs, and liver of different experimental

inoculated chicks were selected for histopathological study. The formalin fixed tissues

were trimmed, processed, sectioned and stained following standard procedure (Luna,

1968). Specific samples containing lesions from each group were used in

histopathological study. The histopathological lesions of different organs of chicks were

graded as:

almost no lesions (±), mild lesions (+), moderate lesions (++), severe lesions (+++).

3.4.4.1. Processing of tracheal tissue 1. The tissue samples were trimmed properly and fixed for 72 hrs with three

changes of fixative. 2. To remove the fixative, the tissues were kept in running tap water for

overnight after being fixed properly. 3. The tissues were dehydrated in grades of alcohol starting from 50%, 70%,

80%, 95% and in absolute alcohol, the tissues were changed at every 1hour interval.

4. The tissues were cleared by two changes in chloroform, one and half an hour for each.

5. The tissues were embedded with molten wax at 56oC: 2 changes, one and half an hour for each.

6. Paraffin blocks containing tissue pieces were made using templates.

25

7. The tissues were sectioned with a rotary microtome at 5µm thickness. Then the

sections were allowed to spread on hot water bath (450C) and taken on oil and

grease free glass slide. A small amount of gelatin was added to the water bath

for better adhesion of the sections to the slide. The slides containing sections

were air dried and kept in cool place until staining.

3.4.4.2. Processing of nasal passage tissue

1. The samples were placed in 10% buffered formalin in a volume 20 times that

of the specimens for 7 days. The solutions were changed three times during this

period.

2. After removing the fixative, the tissues were kept in running tap water for

overnight after being fixed properly.

3. Samples were fixing in decalcifying solution until decalcification completed.

The solution was changes daily for the first three weeks followed by changes

every other day for the remaining period.

4. End point of decalcification was determined by specimen flexibility method.

5. The tissues were dehydrated in grades of alcohol starting from 50%,

70%,80%,95%, and 100%, each were 12 hours interval in two changes.

6. The tissues were cleared by three changes in chloroform, 4 hours for each.

7. The tissues were embedded with molten wax at 560C: 4 changes, 8 hours for

each.

8. Paraffin blocks containing tissue pieces were made using templates.

9. The tissues were sectioned with a rotary microtome at 5µm thickness. Then the

sections were allowed to spread on warn water bath (450C) and taken on oil-

and grease-free glass slide. A small amount of gelatin was added to the water

bath for better adhesion of the sections to the slide. The slides containing

sections were air dried and kept in cool place until staining.

26

3.4.4.3. Preparation of decalcifying solution:

Formic Acid-sodium Citrate method:

Solution A Solution B

Ingredients Amount Ingredients Amount

Sodium citrate 50gm Formic acid (90%) l25ml

Distilled water 250m1 Distilled water 125ml Solution A and Solution B were mixed in equal amount for use. 3.4.4.4. Preparation of stains 3.4.4.4.1. Preparation of Harris Hematoxylin solution

Ingredients Amount

Hematoxylia crystals 5.0gm

Alcohol, 100% 50.0ml

Ammonium or potassium alum 100.0gm

Distilled water 1000.0ml

Mercuric oxide (red) 2.5gm

The Hematoxylin crystals were dissolved in the absolute alcohol and alum was added

and dissolved in water and heated. The two solutions were removed from heat and

thoroughly mixed and boiled as rapidly as possible. After removal from heat, mercuric

oxide was added slowly. It was reheated until it became dark purple and removed

from heat immediately and placed into a basin of cold water until cool. Just before

using, 2-4ml of glacial acetic acid was added per 100 ml of solution to increase the

precision of the nuclear stain. Before using the prepared solution was filtered.

3.4.4.4.2. Preparation of eosin solution

a) 1% Stock alcoholic eosin

Ingredients Amount

Eosin Y, water soluble 1.0gm

Distilled water 20.0ml

Dissolved and add alcohol, 95% 80.0ml

27

b) Working eosin solution

Ingredients Amount

Eosin stock solution 1 part

Alcohol, 80% 3 parts

Just before use 0.5 ml of glacial acetic acid was added to each 100 ml of stain and

stirred.

3.4.4.5. Routine Hematoxyrlin eosin staining procedure

1. The sectioned tissues were deparaffinized in 3 changes of xylene (3 minutes in

each)

2. Then the tissue sections were rehydrated through descending grades of alcohol.

(3 changes in absolute alcohol, 3 minutes in each; 95% alcohol for 2 minutes;

80% alcohol for 2 minutes; 70% alcohol for 2 minutes) followed by tap water

for 5 minutes.

3. The tissues (trachea) were stained with Harris Hematoxylin for 15 minutes and

the tissues (nasal sinus) were stained with Harris Hematoxylin for 1 hours.

4. Washed in running tap water for 15 minutes.

5. After washing the tissues were differentiated in acid alcohol: 2 to 4 dips (l part

HCI and99 parts 70% alcohol).

6. Then washed in tap water for 5 minutes followed by two to four dips in

ammonia water until sections were bright blue.

7. Stained with eosin I minute (tracheal tissue) and l0 seconds ( nasal sinuses)

tissue to visualize cytoplasmic componant.

8. Differentiated and dehydrated in alcohol: 95% alcohol: 3 changes, 3 dips each;

absolute alcohol: 3 changes 3 minutes for each.

9. Cleaned in xylene: 3 changes (5 minutes for each).

10. Finally the sections were mounted with coverslip using DPX.

28

3.4.4.6. Photomicrography

Photornicrography was taken at the Department of pathology using

photomicrographic camera (olympus pM-c 35 Model) onto fitted with Olympus

microscope (Olympus, Japan).

3.4.5. Reisolation of Avibacterium paragallinarum in Bacteriological Media

3.4.5.1. Preparation of various bacteriological culture media and different liquid solution

Different bacteriological media and reagents were prepared according to the procedures suggested by the manufacturer.

3.4.5.1.1. Nutrient broth

Nutrient broth was prepared by dissolving 13 grams of dehydrated nutrient broth

(HiMedia, India) into 1000 ml of distilled water and was sterilized by autoclaving at

121°C under 15 lb pressure per square inch for 15 minutes. Then the broth was

dispensed into tubes (10 ml/tube) and was incubated at 37°C for over night to cheek

their sterility and stored at 4°C in the refrigerator until used.

Ingredients (g/l)

Peptone 5.0

Sodium chloride 5.0

Beef extract 1.5

Yeast extract 1.5

Final Ph (at 250 C) 7.4±0.2

3.4.5.1.2. Nutrient agar

2.3 gms of Bacto-NA (Difco) was suspended in 100 ml cold distilled water taken in a

conical flask and heated to boiling to dissolved the medium completely. After

sterilization by autoclaving, the medium was poured in 10 ml quantities in sterile glass

petridishes (medium sized) and in 15 ml quantities in sterile glass petridishes (large

29

sized) to form a thick layer therein. To accomplish the surface be quite dry, the

medium was allowed to solidify for about 2 hours with the covers of the petridishes

partially removed. The sterility of the medium was judged by incubating overnight at

37°C and used for cultural characterization or stored at 4°C in refrigerator future use

(Carter, 1979).

Ingredients (g/l)

Peptic digest of animal tissue 5.0

Sodium chloride 5.0

Beef extract 1.5

Yeast extract 1.5

Agar 15.0

Final Ph (at 250 C) 7.4±0.2

3.4.5.1.3. Blood agar

Forty grams of blood agar base (HiMedia, India) was suspended in 1000 ml of

distilled water and heated for boiling to dissolve completely. The base was then

autoclaved and cooled at 50°C using water bath. Then sheep blood collected

aseptically was added at the rate of 5-7% of base. The medium was then poured in 20

ml quantities in to 15 X 100 mm petridishes and allowed to solidify. After

solidification of the medium in the plates, the plates were allowed for incubation at

37°C for over night to cheek their sterility.

Ingredients (g/l)

Aager 15.0

Peptone 10.0

Sodium chloride 5.0

Beef extract 10.0

Final Ph (at 250 C) 7.3±0.2

30

3.4.5.2. Isolation and identification of organisms

3.4.5.2.1. Isolation and identification of Staphylococcus aureus

3.4.5.2.1.1. Primary culture of Staphylococcus aureus

Primary growth of all kinds of bacteria was performed in nutrient broth. 24 nasal

swabs samples from live birds were collected with sterile cotton bud by gentle touch,

and then inoculated into the nutrient broth, incubated over night at 37°C to obtain the

primary culture.

3.4.5.2.1.2. Isolation of Staphylococcus aureus in pure culture

After primary culture of the organism, a small amount of inoculum from nutrient broth

was streaked onto mannitol salt agar showing characteristics morphology of

Staphylococcus aureus were selected for subculture on nutrient agar, blood agar.

3.4.5.2.2. Isolation and identification of Avibacterium paragallinarum

3.4.5.2.2.1. Primary culture of A. paragallinarum

Twenty four nasal swabs samples from live birds were collected with sterile cotton

bud by gentle touch, and then inoculated into the nutrient broth, incubated over night

at 37°C to obtain the primary culture.

3.4.5.2.2.2. Isolation of A. paragallinarum in pure culture

After primary culture of the organism, a small amount of inoculum from nutrient broth

was streaked onto blood agar showing characteristics morphology of A.

paragallinarum were selected for subculture. Tiny dewdrop colonies developed on

blood agar media was considered positive for A. paragallinarum.

3.4.5.3. Study of colony morphology for identification

The colony morphology of the isolates was studied as mentioned by Merchant and

Packer (1967). Morphological characteristics (shape, size, surface texture, edge,

elevation, color, opacity etc.) developed after 24hours of incubation were carefully

studied and recorded.

31

3.4.6. Staining

3.4.6.1. Preparation of Gram staining solution

Crystal violet solution

Stock crystal violet solution

Ingredients Amount

Crystal violet 10gm

Ethyl alcohol 1000ml

Stock oxalate solution

Ingredients Amount

Ammonium oxalate 1gm

Distilled water 1000ml

Working crystal violet solution

Ingredients Amount

Stock crystal violet solution 20ml

Stock oxalate solution 80ml

It was mixed and prepared when required.

Lugol’s iodine solution

Ingredients Amount

Iodine crystal 1gm

Potassiun iodide 2gm

These two were dissolved completely in 10 ml of distilled water and then distilled

water was added to make 300 ml and store in amber bottle.

Acetone alcohol

Ingredients Amount

Ethyl alcohol 250ml

Acetone 250ml

32

Safranin (counterstain) solution

Safranin stock solution

Ingredients Amount

Safranin 2.5ml

Ethyl alcohol (95%) 100ml

Safranin working solution

The stock safranin was diluted 1:4 with distilled water.

3.4.6.2. Microscopic study of the suspected colonies

Gram’s staining was performed to determine the shape, arrangement and Gram

reaction of the isolates as described by Merchant and Packer (1967). The procedure

was as follows:

1. A small colony was picked up with a bacteriological loop, a drop of distilled

water added then mixed and smeared on a glass slide and fixed by gentle

heating.

2. Ammonium oxalate crystal violet was added on to the smear and allowed to

react for ½ min.

3. Washed with running water.

4. Lugol’s iodine was then added to act as mordant for one minute and then again

washed with running water.

5. Acetone alcohol was then added, which act as a decolourizer, for 3- 5 seconds.

6. Washed thoroughly in water.

7. After washing with water, safranine was added as counter stain and allowed to

stain for two minutes.

8. The slide was then washed with water, blotted and dried in air and then

examined under microscope with high power objectives (100X) using

immersion oil.

3.4.7. Biochemical studies for the identification of organisms

Several biochemical tests were performed for confirmation of the isolates.

33

3.4.7.1. Reagents for biochemical test

i. Methyl Red and Voges-Proskauer broth (MR-VP broth) (Difco, USA)

ii. Peptone water

iii. Phosphate buffer solution

3.4.7.2. Sugars

i. Dextrose (LOBA Chemic Pvt. Ltd., India)

ii. Sucrose (Wako, Japan)

iii. Lactose (Merc, England)

iv. Maltose (Techno Pharma., India)

v. Manitol (Beximco Pharma., Germany)

vi. Galactose (LOBA Chemic Pvt. Ltd., India)

3.4.7.3. Carbohydrate fermentation test

Preparation of Carbohydrate fermentation test reagents

Bacteriological peptone

Ingredients Amount

Bacteriological peptone 10gm

Sodium chloride 5gm


Phenol red (0.2%)

Ingredients Amount

Phenol red 2gm


Sugar (10%)

Five basic sugar as dextrose, sucrose, maltose and mannitol were used for suger

fermentation test.

Ingredients Amount

Specific sugar 1gm


34

Preparation of sugar media and carbohydrate fermentation tests

The medium consists of peptone water to which fermentable sugar was added to the

proportion of 1 percent. Peptone water was prepared by adding one gram of Bacto

peptone (Difco, USA) and 0.5 grams of sodium chloride in 100 m1 distilled water.

The medium was boiled for 5 minutes, adjusted to pH 7.0, cooled and then filtered

through filter paper: Phenol red, an indicator at the strength of 0.2 percent solution

was added to peptone water and then dispensed in 5 m1 amount into cotton plugged

test tubes containing a Durham's fermentation tubes, placed inversely. These were

then sterilized in the autoclave machine. The sugars used for fermentation were

prepared separately as 10 percent solutions in distilled water (10 grams sugar was

dissolved in 100 ml of distilled water). A little heat was necessary to dissolve the

sugar completely.

The sugar solutions were sterilized in Arnold steam sterilizer at 100°C for 30 minutes

for three successive days. An amount of 0.5 ml of sterile sugar solution was added

aseptically in each culture tubes containing sterile peptone water and indicator. Before

use, the sterility of the sugar media was examined by incubating it for 24 hours at

37°C.

The carbohydrate fermentation test was performed by inoculating a loop full of

nutrient broth culture of the organisms into the tubes containing five basic sugars e.g.,

galactose, maltose, sucrose, and mannitol, glucose and incubated for 24 hours at 37°C.

Acid production was indicated by the color change reddish to yellow in the medium

and presence of no of gas bubbles in the inverted Durham's tubes indicate no gas

production.

35

3.4.7.4. Indole test

Two ml of peptone water was inoculated with 5 ml of bacterial culture and incubated

for 48 hours. 0.5 ml of Kovac’s reagent was added, shaked well and examined after 1

minute, no development of red color. In positive case there is a red color in the reagent

layer indicate indole.

3.4.7.5. Methyl-Red & Voges-Proskauer (MR-VP) test

Composition of MR-VP medium (DIFCO Laboratories, USA)

Ingredients Amount

Buffered peptone 7.0gm

Dextrose 5.0gm

Dipotassium phosphate 5.0gm

A quantity of 3.4 gm of Bacto MR-VP medium was dissolved in 250 m1 of distilled

water dispensed in 2 ml amount in each test tube and then the tubes were autoclaved.

After autoclaving, the tubes containing medium were incubated at 37°C for overnight

to check their sterility and then stored in a refrigerator for future use.

Two milliliters of sterile glucose phosphate peptone water were inoculated with the 5

ml of test organisms. It was inculated at 370C for 48 hours. A very small amount

(knife point) of creatine was added and mixed. Three milliliters of sodium hydroxide

were added and shaked well. The bottle cap was removed and left for an hour at room

temperature. It was observed closely for no development of pink color. In positive

cases there was the slow development of a pink color.

Methyl Red solution

Ingredients Amount

Methyl red 0.05gm

Ethanol (absolute) 28ml


The indicator phenyl red solution was prepared by dissolving 0.1 gm of Bacto methyl-

red in 300 ml of 95 percent alcohol and diluting to 500 ml with the addition of 200 ml

of distilled water.

36

The test was performed by inoculating a colony of the test organism in 0.5 ml sterile

glucose phosphate broth (as used in the VP test). After overnight incubation at 37°C, a

drop of methyl red solution was added. A negative methyl red test was shown by a

yellow or orange color. A positive test shown by the appearance of bright red color

indicated the acidity.

3.4.8. Enzyme activity test

3.4.8.1. Catalase test

The organism was grown on a slope of nutrient agar or other suitable medium. One ml

3% H2O2 was run down the slope and examined immediately and after 5 min for

evaluation of gas.

37

CHAPTER IV

RESULTS

The present investigation was taken to study the pathogenesis of Avibacterium

paragallinarum, the causal agent of IC in chicks. The isolation and identification of

the causal agent from layer chickens was performed by Akter (2012). However,

reisolation and identification of Avibacterium paragallinarum in experimental chicks

was included in investigation.

4.1. Clinical signs of chickens

Chicks of group A (inoculated with pure nutrient broth) did not show any remarkable

clinical signs up to the end of the experimental period. However, chicks of group B

(inoculated with A. paragallinarum) showed mild nasal discharge, conjunctivitis,

depression and inability to move.

4.2. Gross study

The results of gross study have been presented in Table 5 and Figures 2-5. Chicks of

group A did not reveal any lesion related to the IC on day 3, 5 and 7. On the other

hand, chicks of group B reveal lesions in different organs following bacterial

inoculation which were progressively massive (Table 5).

38

Table 3. Results of gross study

Groups Day after inoculation

Gross lesions related to IC

Group A (inoculated with

pure nutrient broth)

3 ±

5 ±

7 ±

Group B (inoculated with

A. paragallinarum)

3 Mucus in nasal passage (+)

5 Mucus in nasal passage (+)

Mild tracheal hemorrhage (+) (Fig. 5)

7 Mucus in nasal passage(+)

Conjunctivitis (+) (Fig. 3)

Swelling of sinuses and face(+)

Congested lungs(++) (Fig. 4)

± = almost no lesions, + = mild lesions, ++ = moderate lesions.

4.3. Histopathological study

The microscopic lesions have been presented in Tables 6 and 7 and illustrated in

Figure 10-21.

Table 4. Results of histopathological studies of group A (inoculated with nutrient

broth)

Day of inoculation

Nasal passage Lung Liver Heart

Day 3 ± ± ± ±

Day 5 ± + ± ±

Day 7 ± + ± ±

± = almost no lesions, + = mild lesions

39

Table 5. Results of histopathological studies of group B (Inoculated with A.

paragallinarum)

Day of inoculation

Nasal passage Lung Liver Heart

Day 3 Acanthosis of nasal epithelium,congested blood vessel, hyperplasia of mucous gland (+)

Mild pneumonic lesions (+)

Lymphocytic infiltration(+)

Fatty change and lipid nodule in macrophage (+)

Day 5 Acanthosis of nasal epithelium,congested blood vessel, hyperplasia of mucous gland (+)

Moderate pneumonic lesions (++)

Lymphocytic infiltration (+)

Fatty change and lipid nodule in macrophage (+)

Day 7 Acanthosis of nasal stratified epithelium (Fig. 9), congested blood vessel (Fig. 9), hyperplasia of serous gland and necrotic tissue debris found in serous gland, presence of inflammatory cells (heterophils and lymphocytes) (++)

Severe pneumonia(+++) (Fig. 15)

Focal hepatitis(++) (Fig. 20)

Fatty change and lipid nodule in macrophage (++)

+ = mild lesions, ++ = moderate lesions, +++ = severe lesions.

4.4. Reisolation of Avibacterium paragallinarum on day 7

Reisolation was performed only in tissues showing pathological lesions i.e. found on

day 7 of post inoculation (PI). Only 4 samples were processed for reisolation.

Reisolation procedures of Avibacterium paragallinarum have been illustrated in

Figures 6-9.

4.4.1. Results of Gram's stain

Tentatively diagnosed all samples from blood agar media were stained with Gram’s

stain. All of the suspected samples showed Gram- negative, red color, rod shaped

bacilli arranged as single or paired (Fig. 8).

40

4.4.2. Results of biochemical tests

Biochemical tests were performed from tentatively diagnosed 4 samples from blood

agar media, a series of biochemical tests especially selective for A. paragallinarum

were performed with the positive culture and Gram-negative rod shaped bacteria. The

results are furnished below:

4.4.2.1. Results of sugar fermentation test

Four isolates fermented four basic sugars (maltose, sucrose, and mannitol, glucose)

and produced acid and did not ferment galactose. Acid production was indicated by

the color change from reddish to yellow (Table 8, Fig. 7).

4.4.2.2. Results of other biochemical tests

Four isolates were then subjected to different biochemical tests such as methyl-red

test, VP test and Indole test. All the isolates were methyl-red negative; VP test

negative and Indole test negative (Table 8). Isolates revealed the following pattern of

biochemical reactions were regarded as A. paragallinarum.

Table 6. Results of biochemical characteristics of A. paragallinarum

Different Biochemical test Sample size Result Identification of

bacteria

Fermentation reaction

with five basic sugars

a. Glucose

4

+ A. paragallinarum

b. Sucrose + A. paragallinarum

c. Galactose _ A. paragallinarum

d. Maltose + A. paragallinarum

e. Mannitol + A. paragallinarum

Other biochemical test

Indole

4

_ A. paragallinarum

MR _ A. paragallinarum

VP _ A. paragallinarum

+ = Positive; - = Negative; MR = Methyl red; VP = Voges proskauer

41

4.5. Enzymatic activity test

4.5.1 Catalase activity test

Twelve isolates from blood agar media were subjected to catalase test. All the isolates

showed negative test (i.e. production of no buble) indicating A. paragallinarum .

In summary, the probable experimental pathogenesis might be started with inoculation

of A. paragallinarum through nasal passage and rhinitis was produced following

reached to the various visceral organs via blood and finally showed lesions. The

lesions that found in this experiment (rhinitis in association with focal hepatitis,

progressive pneumonic lesions, fatty change in heart with lipid granuloma) are not

normally present in adult and young chicks. Lesion was not found in control group but

time dependently intensity of lesions was found in different organs in experimental

inoculated group (inoculated with A. paragallinarum). This may be a latest finding of

this disease. However, further investigation is needed.

42

Conjunctivitis

Mild facial edema

Figure 2. Depression of chicks of group B (Inoculated with A. paragallinarum) on day 7 of post inoculation.

Figure 3. A chick of group B (Inoculated

with A. paragallinarum) with conjunctivitis and mild facial edema on day 7 of post inoculation.

Figure 4. Severely congested lung of a chicken of group B (Inoculated with A. paragallinarum) with on day 7 of post inoculation.

Figure 5. Mild tracheal haemorrhage of a chicken of group B (Inoculated with A. paragallinarum) on day 3 of post inoculation.

43

Figure 6. Staphylococcus aureus produces golden yellow color colony on mannitol salt agar media.

Glac Glu Su Ml Mn Cont.

Figure 7. Fermentation of glucose, sucrose, mannitol, maltose with production of only acid and no galactose by A. paragallinarum.

Figure 8. A. paragallinarum showing gram negative rod shaped bacilli ( Gram's staining. x830)

Figure 9. A. paragallinarum produce smooth iridescent colonies with no hemolysis on blood agar media.

44

Figure 10. Nasal passage of group A (inoculated with nutrient broth) on day 7 of post inoculation showing no lesion.

Figure 11. Nasal passage of group B (Inoculated with A. paragallinarum) on day 7 of post inoculation showing acanthosis (arrow).

Figure 12. Nasal passage of group B (Inoculated with A. paragallinarum) on day 5 of post inoculation showing presence of reactive leukocytes (arrow).

Figure 14. Section of lung of a chicken of group A showing almost no lesion

Figure 15. Lung of group B (Inoculated with A. paragallinarum) on day 3 of post inoculation showing mild pneumonic lesion. (+).

Figure 14. Section of lung of a chicken of group A showing almost no lesion (±).

Figure 13. Nasal passage of group B (Inoculated with A. paragallinarum) on day 7 of post inoculation showing congestion of blood vessels (arrow) and acanthosis.

45

Figure 17. Lung of group B on day 7 of post inoculation showing severe

Figure 19. Fatty change, lipid nodules in macrophages and micronodules in heart on day 7 of post inoculation.

Figure 18. Fatty change, lipid nodules in macrophages and micronodules in heart on day 5 of post inoculation.

Figure 20. Liver of group A (inoculated with nutrient broth) on day 7 of post inoculation.

Figure 21. Liver of group B (Inoculated with A. paragallinarum) on day 7 of post inoculation showing focal hepatitis.

Figure 16. Lung of group B (Inoculated with A. paragallinarum) on day 5 of post inoculation showing moderate pneumonic lesion (++).

Figure 17. Lung of group B (Inoculated with A. paragallinarum) on day 7 of post inoculation showing severe pneumonic lesion (+++).

46

CHAPTER V

DISCUSSION

Infectious coryza in chicken is a problem in poultry industries and the clinical signs

associated with this disease included nasal discharge, conjunctivitis with swelling of

the sinuses, face and wattles, diarrhoea, decreased feed and water consumption,

retarded growth in younger chickens and reduced egg production. In this study, 24

chicks of 14 days of age were grouped into two (A and B) each group containing 12

birds. Chicks of group A were inoculated with 1ml of 2 days old nutrient broth and

were kept as control group while group B were inoculated with 1 ml of 2 days old

culture broth of Avibacterium paragallinarum. To study the pathology, 4 birds from

each group were sacrificed on day 3, 5 and 7 of post inoculation. Sacrificed birds of

group A did not reveal any significant clinical sign and lesion. Chicks of group B

showed different clinical signs resemble infectious coryza which were mild nasal

discharge, conjunctivitis, depression and inability to move. The gross lesions of the

chicks of group B included mucous in nasal passage, conjunctivitis, swelling of

sinuses and face and congested lungs. The microscopic lesions of the chicks of this

group were acanthosis and congested blood vessels of nasal passage and pneumonic

lesion of lung which were progressively prominent on day 7 of bacterial inoculation.

All of these studies have very close agreements with studies of Fujiwara and Konno

(1965), Sawata et al. (1985) and Droual et al. (1990) who studied the microscopic

changes due to infectious coryza.

Avibacterium paragallinarum was reisolated from day 7 of post inoculation (PI) from

nasal passage of chicks in which lesions were prominent. Identification was carried

out according to the cultural properties in different media, staining characters and

biochemical tests. These reisolation procedures have the similarities with the study of

different researchers. The media used in this study were selected considering the

experience of the past researcher who worked elsewhere on various fields similar to

the present study (Page 1962; Blackall and Farrah, 1985; Sameera et al., 2001; lnzana

et al., 1987; Garcia et al., 2004). The colony characters on different media exhibited

characteristic reaction (Page 1963). The colony characteristics of A. paragallinarum

47

observed on Blood agar (BA) was similar to the findings of other authors (Page 1963;

Blackall 1989; Sameera et al., 2001). The differences in colony morphology may be

manifested by the isolates may be due to loosing or acquiring some properties by the

transfer of host or choice of host tissue observed by Dubreuil et al., (1991).

In Gram's staining the morphology of the isolated bacteria exhibited pink or red

(Gram’s stain color) small rod shaped Gram negative coccobacilli. These findings

were in aggrement with several authors such as Sameera et al. (2001), Yamamoto

(1991), Sawata et al. (1980), Jaswinder et al. (2004). Nasal swab isolates revealed a

complete fermentation of four basic sugar as stated by Blackall (1989), Hinz and

Kunjara (1977). A. paragallinarum isolates were able to ferment four basic sugars

with the by production of acid. Al1the isolates fermented glucose, sucrose maltose,

mannitol but failed to ferment galactose within 21h-48h of incubation.

It could be summarized that in the A. paragallinarum inoculated group (group B) the

clinical sings were nasal discharge, conjunctivitis, depression and inability to move

and the gross lesions were mucus in nasal passage, conjunctivitis, swelling of sinuses

and face and congested lungs. The microscopic lesions of the chicks of this group

were acanthosis and congested blood vessels of nasal passage and pneumonic lesion

of lung. These were progressively prominent on day 7 of inoculation. Avibacterium

paragallinarum was reisolated from day 7 of post inoculation (PI) from nasal passage

of chicks in which lesions were prominent.

In brief, the proposed experimental pathogenesis might be started with inoculation of

A. paragallinarum through nasal passage and produced rhinitis following reached to

the different organs via blood and finally revealed lesions. The lesions that found in

this experiment (rhinitis in association with focal hepatitis, fatty change in heart with

lipid granuloma, progressive pneumonic lesions) are not normally present in adult and

young birds. There was no lesion in control group but time dependently severity of

lesions was found in different organs in experimental inoculated group (inoculated

with A. paragallinarum). This may be a new finding of this disease. However, further

investigation is needed on this issue.

48

CHAPTER VI

SUMMARY AND CONCLUSION

The present study was conducted for experimental pathogenesis study of Infectious

Coryza caused by Avibacterium paragallibarum. One isolate of Avibacterium

paragallinarum was used to study the experimental pathogenesis. For this purpose, 24

chicks of 14 days of age were grouped into two (A and B) each group containing 12

birds. Chicks of group A were inoculated with 1ml of 2 days old nutrient broth and

were kept as control group while group B were inoculated with 1 ml of 2 days old

culture broth of Avibacterium paragallinarum. To study the pathology, 4 birds from

each group were sacrificed on day 3, 5 and 7 of post inoculation. Sacrificed birds of

group A did not reveal any significant clinical sign and lesion. Chicks of group B

showed different clinical signs resemble infectious coryza which were mild nasal

discharge, conjunctivitis, depression and inability to move. The gross lesions of the

chicks of group B included mucous in nasal passage, conjunctivitis, swelling of

sinuses and face and congested lungs. The microscopic lesions of the chicks of this

group were acanthosis and congested blood vessels of nasal passage, pneumonic

lesion of lung, focal hepatitis of liver and fatty change and lipid nodules in

macrophages of heart which were progressively prominent on day 7 of bacterial

inoculation.

Avibacterium paragallinarum was reisolated from day 7 of post inoculation (pi) from

nasal passages of chicks as lesions were prominent at this time point of experimental

study. The inoculated A. paragallinarum was confirmed by the cultural properties in

different media, staining characters and biochemical tests.

Therefore, in brief, the proposed experimental pathogenesis was as follows:

Inoculation of A. paragallinarum through nasal passage it produced rhinitis, then it

went to the different organs via blood and finally revealed lesions.

49

The lesions that we discussed here (rhinitis in association with focal hepatitis, fatty

change in heart with lipid granuloma, progressive pneumonic lesions) are not

normally present in adult and young birds. In this study there was no lesion in control

group (inoculated without A. paragallinarum). But in comparison with control group

time dependently severity of lesions was found in different organs in experimental

inoculated group (inoculated with A. paragallinarum). This may be a new finding of

this disease. However, it needs further investigation.

Further study in connection with this research work might be:

1. Serotyping and molecular characterization of A. paragallinarum isolated from

suspected clinical sample.

2. Vaccine development.

50

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