ISOLATION and IDENTIFICATION of Avibacterium Paragallinarum from LAYER CHICKENS

ISOLATION AND IDENTIFICATION OF Avibacterium paragallinarum FROM LAYER CHICKENS

A THESIS

Submitted to Bangladesh Agricultural University, Mymensingh In partial Fulfillment of the Requirements for the Degree of

Master of Science in Pathology

SALEHA AKTER

Roll No.: 11Vet Path JJ 14 M Registration No.: 38019, Session: 20011-12

Department of Pathology Faculty of Veterinary Science Bangladesh Agricultural University Mymensingh

May 2012

ISOLATION AND IDENTIFICATION OF Avibacterium paragallinarum FROM LAYER CHICKENS

A THESIS

Submitted to Bangladesh Agricultural University, Mymensingh In partial Fulfillment of the Requirements for the Degree of

Master of Science in Pathology

SALEHA AKTER

Approved as to style and content by

Prof. Dr. Priya Mohan Das Prof. Dr. Md. Mokbul Hossain Co- Supervisor Supervisor

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

May, 2012

ACKNOWLEDGEMENTS

The author is grateful and indebted to the almighty Allah without whose grace she would have ever been able to pursue her higher studies in this field of science and to complete her thesis for the degree of Master of Science (MS) in Pathology.

The author expresses her ever indebtedness, deepest sense of gratitude, sincere appreciation and profound regards to her reverend and beloved teacher and research Supervisor, Professor Dr. Md.Mokbul Hossian, Department of Pathology Bangladesh Bangladesh Agricultural University, (BAU) Mymensingh for his scholastic guidance, uncompromising principles, sympathetic supervision, valuable advice, constant inspiration, affectionate feeling, radical investigation and constructive criticism in all phases of this study and preparing the manuscript.

The author finds it a great pleasure in expressing her heartfelt gratitude and immense indebtedness to her honourable and respected Research Co –Supervisor Dr. Priya Mohan Das, Professor and Head, Department of Pathology, BAU, Mymensingh for his sympathy, kind co-operation, inspiration, scholastic supervision and suggestions, constructive criticism and valuable advice for the completion of the research work and preparation of the thesis.

The author wishes to express cordial respect and profound gratitude to her respected teachers Prof. Dr.Md. Iqbal Hossain, Prof. Dr. Md. Abdul Baki, Prof. Dr. Md. Rafiqul Islam, Prof. Dr. Md. Habibur Rahman, Prof. Dr. Emdadul Haque Chowdhury, Prof. Dr. Md. Abu Hadi Noor Ali Khan, Dr. Mrs. Jahanara Begum, Dr. Mrs. Munmun Parvin, and Dr. Md. Nuruzzaman lecturers Department of Pathology, BAU, Mymensingh for their valuable advice and constant inspiration throughout the entire period of study.

The author explicit gratefulness and cordial thanks to Dr. Abu Sufiun, and all other Ph.

iv D. students of Department of Pathology, BAU, Mymensingh for their valuable suggestions, practical help and co-operation in every step of the research work and in writing the thesis.

The author is also thankful to all office staffs, Department of Pathology, BAU, Mymensingh for their technical assistance during the research work.

The author expresses her thanks to Ali, Solaiman, Mamun, Sankar, Arif, Sharif, Shuvonkar, Mehedi, Poly, Luna and Sumi for their continuous help in conducting the research work.

Dictions not enough to express the author’s immense gratitude and endless love to her beloved parents for their heartiest blessing and encouragement for her higher education and inspiration throughout her academic life.

Last but not the least the author expresses her deeper sense of gratitude and sincere thanks to all her well wishers kith and kin for their constant inspiration and blessing throughout the entire period of her academic life.

May, 2012 The Author

v ABSTRACT The present research work was conducted for the isolation and identification of Avibacterium paragallinarum, the etiological agent of infectious coryza (IC) along with pathological study of the affected organs from layer chickens. A total of 30 nasal swab samples collected aseptically from the dead chickens from SK Veterinary Diagnostic Center, Mymensingh and 4 live samples: 2 from Gazipur, and 2 from Tangail. The used methods were culture of organisms in different media, staining, sugar fermentation test, biochemical tests of the isolated causal agent and histopathology of the affected tissues. A total of 30 swabs from nasal passage of dead birds died of other diseases, no A. paragallinarum was isolated and identified. Two out of 4 suspected clinical cases of IC confirmed by isolation of A. paragallinarum. The organism A. paragallinarum was canfirmed on the basis of colony morphology, staining characters, sugar fermentation test, MR test, VP test, indole test and catalase test. The clinical signs of all 4 cases were nasal discharge; conjunctivitis with swelling of the sinuses, face and wattles; decreased feed and water consumption and reduced egg production. At necropsy, the gross lesions noted were mucous in nasal passage and tracheal hemorrhage. Histopathology of the nose showed acanthosis, congestion, mucous glandular cell hyperplasia, hyperplasia of nasal sinus and parakeratosis. From Bangladesh, this is the first time A. paragallinarum was isolated and identified from clinical cases. Further study is essential involving the other areas of serological and molecular identification of the etiological agent of infectious coryza since the disease has great economic importance.

CONTENTS

CHAPTER LIST OF CONTENTS PAGE

NO. ACKNOWLEDGEMENTS iv ABSTRACT vi LIST OF FIGURES xii LIST OF TABLES xiv LIST OF ABBREVIATION AND SYMBOLS xv 1 INTRODUCTION 01 2 REVIEW OF LITERATURE 04 2.1 Avibacterium paragallinarum 04 2.1.1 History of A. paragallinarum 04 2.1.2 Etiology of infectious coryza 05 2.1.3 Host of infectious coryza 05 2.1.4 Clinical signs of the disease 06 2.1.5 Cultural properties of A. paragallinaru 08 2.1.6 Morphology and staining characteristics of 13 A. paragallinarum 2.1.7 Isolation and identification of A. paragallinarum 14 2.1.8 Biochemical properties of A. paragallinarum 18 2.1.9 Antibiotic sensitivity of A. paragallinarum 19 2.1.10 Pathogenicity of A. paragallinarum 20 2.1.11 Prevalence of infectious coryza 22 2.2. Staphylococcus aureus 26 2.2.1 Colonies 26 vii

CONTENTS(COND.)

CHAPTER LIST OF CONTENTS PAGE NO. 2.2.2 Staining characters 26 2.2.3 Biochemical tests of S. aureus 27 2.2.4 Pathology and pathogenesis of S. aureus 27 3 MATERIALS AND METHODS 28 3.1 Materials 28 3.1.1. Study area 28 3.1.2 Cotton swab 28 3.1.3 Bacteriological media for culture 29 3.1.3.1 Solid media 29 3.1.3.2 Liquid media (broth) 29 3.1.3.3 Chemicals, reagents and solutions 29 3.1.3.4 Sugars 29 3.1.3.5 Reagents for biochemical test 29 3.1.4 Glassware and other necessary instruments 30 3.1.5 Hexisol hand rub 30 3.1.6 20% sterile buffered glycerin 30 3.1.7 Preparation of various bacteriological culture 30 media and different liquid solution 3.1.7.1 Nutrient broth 30 3.1.7.2 Nutrient agar 31 3.1.7.3 Blood agar 31 3.1.7.4 Bacteriological peptone 32 3.1.7.5 MR-VP medium 32 3.1.7.6 Phosphate buffer solution 33 viii

CONTENTS(COND.)

CHAPTER LIST OF CONTENTS PAGE NO. 3.1.7.6 Phosphate buffer solution 33 3.1.7.7 10% buffer formalin 33 3.2 METHODS 33 3.2.1 Cleaning and sterilization of glassware 33 and plastic ware 3.2.2 Brief description of the experimental design 34 3.2.3 Collection and transportation of samples 37 3.2.4 Isolation and identification of organisms 37 3.2.4.1 Isolation and identification of S. aureus 37 3.2.4.1. Primary culture of S. aureus 37 3.2.4.1.2 Isolation of S. aureus in pure culture 37 3.2.4.2 Isolation and identification of pure culture 37 3.2.4.2.1 Primary culture of A. paragallinarum 37 3.2.4.2.2 Isolation of A. paragallinarum in pure culture 37 3.2.4.3 Identification of the isolates 38 3.2.4.3.1 Study of colony morphology for identification 38 3.2.4.3.2 Morphological characterization by Gram’s 38 staining method 3.2.4.3.2.1 Preparation of Gram staining solution 38 3.2.4.3.2 Microscopic study of the suspected colonies 39 3.2.4.4 Biochemical studies for the identification 40 of organism 3.2.4.4.1 Carbohydrate fermentation test 40 3.2.4.4.2 Indole test 42 ix

CONTENTS(COND.)

CHAPTER LIST OF CONTENTS PAGE NO. 3 .2.4.4.3 Methyl-Red & Voges-Proskauer (MR-VR) test 42 3.2.4.4.4 Alpha- napthanol solution 43 3.2.4.4.5 Potassium hydroxide solution 43 3.2.4. Enzyme activity test 43 3.2.4.5.1 Catalase test 43 3.2.5 Maintenance of the stock culture 44 3.2.5.1 Phosphate buffered saline solution 44 3.2.5.2 20% sterile buffered glycerin method 44 3.2.6 Pathological studies 44 3.2.6.1 Gross pathology 44 3.2.6.2 Histopathology 44 3.2.6.2.1 Processing of nasal passage tissue 45 3.2.6.2.2 Preparation of decalcifying solution 45 3.2.6.2.3 Preparation of stains 46 3.2.6..2.4 Routine hematoxyrlin eosin staining procedure 47 3.2.7 Photomicrography 47 4 RESULTS 48 4.1 Results of isolation and identification of S. aureus 48 4.1.1 Results of cultural examination 48 4.1.1.1 Culture on nutrient broth 48 4.1.1.2 Culture on mannitol salt agar 48 4.1.1.3 Culture on nutrient agar 48 4.1.1.4 Culture on blood agar 49 4.1.2 Results of Gram's stain 49 x

CONTENTS(COND.)

CHAPTER LIST OF CONTENTS PAGE NO. 4.1.3 Enzymatic activity test 49 4.1.3.1 Catalase activity test 49 4.2 Results of isolation and identification of 49 etiological agent, A. paragallinarum 4.2.1 Results of cultural examination 49 4.2.1.1 Culture on nutrient broth 50 4.2.1.2 Culture on blood agar media containing S. aureus 50 4.2.2 Results of Gram's stain 50 4.2.3 Results of biochemical tests 50 4.2.3.1 Results of sugar fermentation test 50 4.2.3.2 Results of other biochemical tests 51 4.2.4 Enzymatic activity test 51 4.2.4.1 Catalase activity test 51 4.2.5 Bulk culture and storage of bacteria 52 4.2.5 Gross study 52 4.2.6 Histopathological study 52 5 DISCUSSION 58 6 SUMMARY AND CONCLUSION 61 7 REFERENCES 63

LIST OF FIGURES

FIGURE NO. TOPIC PAGE NO. Figure 1 Isolation and identificotion design of 35 Staphylococcus aureus

Figure 2 Isolation and Identification design of A. 36 paragallinarum Figure 3 S. aureus produces turbidity in nutrient broth 53 Figure 4 S.aureus produces golden yellow color colony 53 on nutrient agar media. Figure 5 S. aureus produces golden yellow color colony 53 on mannitol salt agar media. Figure 6 S. aureus produces yellowish colony on blood 53 agar media. Figure 7 S. aureus showing gram positive, cocci and arranged 53 in grape like clusters (Modified Gram's staining. x830). Figure 8 Production of bubbles which indicate catalase 53 positive for S. aureus. Figure 9 Frothy oculo-nasal discharge (A and B) in 54 infectious coryza infected chicken. Figure 10 Mucoid exudates in the nasal passage (A) and 54 congested trachea (B) in infectious coryza infected chicken. Figure 11 A. paragallinarum produces turbidity in nutrient 54 Broth. Figure 12 A. paragallinarum produce smooth iridescent 54 colonies with no hemolysis on blood agar media. xii

LIST OF FIGURES (COND.)

FIGURE NO. TOPIC PAGE NO. Figure 13 A. paragallinarum showing gram negative rod 55 shaped bacilli (Modified Gram's staining. x830) Figure 14 Fermentation of glucose, sucrose, mannitol, 55 maltose with production of only acid and no galactose by A. paragallinarum Figure 15 A. paragallinarum produces yellow color in 55 VP test, indicate negative reaction. Figure 16 A. paragallinarum produces yellow color in 55 MR-VP test, indicate negative reaction. Figure 17 Color change was not seen in indole test which 55 indicate negative reaction by infectious coryza Figure 18 Production of no bubbles which indicate 55 catalase nagative for A. paragallinarum Figure 19 Bulk culture of A. paragallinarum in nutrient broth 56

Figure 20 Storage of A. paragallinarum 56

Figure 21 Congestion and mucous glandular cells 57 hyperplasia of A. paragallinarum affected nasal passage of chickens (H&E stain, X 333). Figure 22 Acanthosis, parakeratosis and inflammatory 57 cells of A. paragallinarum affected nasal passage of chickens (H&E stain, X 333).

xiii

LIST OF TABLES

TABLE NO. TOPIC PAGE NO.

Table 1 Number of nasal swab samples collected from 28 different areas. Table 2 Results of Biochemical characteristics of 51 A. paragallinarum.

xiv

LIST OF ABBREVIATIONS AND SYMBOLS BA = Blood Agar

BAU = Bangladesh Agricultural University CON = Control ELISA = Enzyme linked immunosorbent assay H. paragallinarum = Haemophilus paragallinarum A. paragallinarum = Avibacterium paragallinarum Et al. = Associated Etc. = Etcetra FAT = Flurescent antibody test Fig. = Figure Glu = Glucose Hrs = Hours i.e. = That is e.g. = Example Ltd. = Limited L = Lactose mg = Milligram ml = Millilitre Min. = Minute MN = Mannitol ML = Maltose mm = Millimetre MR = Methyl Red MS = Master of Science NA = Nutrient agar xv NB = Nutrient broth No. = Number NaCl = Sodium chloride OIE = Office International Des Epizooties P = Penicillin PBS = Phosphate buffered solution PCR = Polymerase chain reaction Prof. = Professor R = Resistant S = Sensitive SAT = Serum agglutination test Sl. = Serial Sp = Species Su = Sucrose USA = United States of America VP = Voges-Proskauer OC = Degree Celcius - = Negative + = Positive % = Percentage ≤ = Less than or equal ≥ = More than or equal µg = Mcrogram µl = Microlitre / = Per

xvi CHAPTER I INTRODUCTION

Since early 1995 poultry farming is being considered as a well established business in Bangladesh although commercial poultry production was started around 1980 (Islam and Ali, 2009). A large number of private companies started investing huge amount of money to improve poultry farming in Bangladesh. Many of the unemployed young male and female of the rural and urban areas of the country are trying to improve their socio-economic condition through poultry farming. There are about 0.15 million poultry farmers in the country and 6 million people directly dependent on poultry industry for their livelihoods (Hossain and Ali, 2009). Approximately, 90% of the rural households rear native chickens (indigenous type) while 0.15 million farms are involved in the production of the hi-breed layer and broiler chickens in country (Raha, 2009). The entire population of poultry species in Bangladesh is about 222 million (Bhuyian, 2007) in which chicken population is around 90% followed by duck (8%) and small number of pigeon, goose and quail (2%) (Das et al., 2007). A total of 135 hatcheries of Bangladesh are producing 6 million day-old-chicks (DOC) per week (Khaleduzzaman and Khandaker, 2009). Poultry production is an important business for the persons of low income-food deficit countries, which is considered as an appropriate system to supply with high quality animal protein to the fast growing population. Poultry meat and eggs production accounts for more than 30% of all animal protein worldwide and the share is up to 40% of all animal protein by the year 2015 (Ferrell, 2000).

Bangladesh is a developing country where poultry industry is a growing sector. There are several constraints for the expansion of poultry industries in Bangladesh. Development of poultry sector in Bangladesh is being hampered by a number of factors of which infectious diseases are considered as the major factor causing 30% mortality of chickens per year (Das et al., 2005). Among the bacterial diseases, infectious coryza (IC) is one of the major threats to the poultry industry and the causal agent is a bacterium, Avibacterium paragallinarum new nomenclature of

Haemophillus paragallinarum (Mendoza et al., 2009) belonging to genus Haemophilus and Pasteurellaceae family.

A. paragallinarum causes an acute respiratory disease in chickens known as infectious coryza, a disease first recognized as a distinct entity in the late 1920's (De Blieck, 1932) and described as roup, cold, contagious or infectious catarrh and uncomplicated coryza (Yamamoto, 1991). Chicken (Gallus gallus) is the natural host for A. paragallinarum and birds of all ages are susceptible. The disease is usually transmitted through drinking water contaminated with infective nasal exudates (Page, 1962). Infection may also occur by contact and by air-borne infected dust or droplet.

Infectious coryza is regarded as a disease limited to the upper respiratory tract (Roberts et al., 1964; Reid and Blackall, 1984) and infection in the lower respiratory tract (Adler and Page, 1962) may be due to synergism between A. paragallinarum and other respiratory tract pathogens (Reid and Blackall, 1984) like fowl cholera, pox, CRD, and Pseudomonas aeruginosa infection (Giurov, 1984). Due to the phenomenon that the disease proved to be infectious only in the nasal passages the name "Infectious Coryza" was adopted (Beach and Schalm, 1936).

The clinical signs associated with this disease include a nasal discharge, conjunctivitis with swelling of the sinuses, face and wattles, diarrhoea, decreased feed and water consumption, retarded growth in younger chickens, increased number of culls and reduced egg production (10-40%) (Eaves et al., 1989; Calnek et al., 1991). Lesions associated with the disease reflect an acute catarrhal inflammation of the upper respiratory tract.

A. paragallinarum is a Gram negative, polar staining, non-motile bacterium. In 24- 48hrs cultures, it appears as short rods or coccobacilli 1-3µm in length and 0.4- 0.8µm in width, with a tendency for filament formulation. The organism undergoes degeneration within 48-60 hours, showing fragments and indefinite forms (Yamamoto, 1991).

In Bangladesh, the information on IC is very scanty (Talha et al., 2001). Infectious coryza has been diagnosed on the basis of postmortem examination of the dead birds but no attempt was taken to isolate the causal agent of infectious coryza, A. paragallinarum.

Therefore our present investigation is undertaken with the following objectives: 1. To isolate and identify the etiological agent of infectious coryza A. paragallinarum from layer chickens. 2. To determine the pathological lesions in the affected organs.

CHAPTER 2 REVIEW OF LITERATURE

The purpose of this chapter is to provide a selective review of the works related to the present study entitled “Isolation and identification of the A. paragallinarum from layer chickens". The works done in the recent past are reviewed below:

2.1 A. paragallinarum 2.1.1 History of A. paragallinarum Mendoza et al. (2009) represented the first serotyping study of 24 isolates of A. paragallinarum obtained from different regions of Peru during 1998-2008. All isolates were characterized as beta-nicotinamide adenine dinucleotide dependent.

Garcia et al. (2004) mentioned that it was the first report of NAD independent H. paragallinarum outside South Africa and was the first time that NAD independent H. paragallinarum of serovar B was reported.

Zhang et al. (2003) reported the first isolation of H. paragallinarum serovar B from an outbreak of infectious coryza in China. They found that layer flock suffered from H. paragallinarum outbreak in which 50% of the 100000 birds showed the typical clinical signs and mortality associated with the outbreak was estimated to be between 2 and 5%. They also added that outbreak caused an average of 10% drop in egg production.

Poernomo et al. (2000) characterized 18 isolates of H. paragallinarum isolated from chickens in Indonesia. They confirmed the presence of all 3 Page serovars (A, B and C) for the first time in Indonesian chickens.

Yamamoto (1984) mentioned that hemophilic organisms were first isolated from birds in the 1930s when De Blieck (1932) isolated an organism, he termed `Bacillus haemoglobinophilus coryzae gallinarum' from chickens suffering an upper respiratory tract disease now known as infectious coryza.

Rashid et al. (1984) reported an outbreak of infectious coryza (IC) in a Badash, Iraq, and poultry farm in 1982. It appears to be the first published report of IC in Iraq, although claims of previous occurrence of the disease have been made.

Beach et al. (1936) stated that due to the phenomenon that the disease proved to be infectious only in the nasal passages the name "Infectious Coryza" was adopted.

De Blieck (1932) repored that H. paragallinarum causes an acute respiratory disease in chickens known as infectious coryza, a disease first recognized as a distinct entity in the late 1920's.

2.1.2 Etiology of infectious coryza Soriano et al. (2004) Studied on the etiological agent of infectious coryza and found that the bacterium H. paragallinarum is the etiological agent of infectious coryza, an upper respiratory disease of poultry. The bacteriological characteristic of the etiological agent are described in a manner that allocates it within the Pasteurellaceae family, and also shows its relationship to other potentially pathogenic agents in poultry.

Blackall (1984) mentioned that infectious coryza is a well-recognized and commonly encountered upper respiratory tract disease of chickens that is caused by the bacterium H. paragallinarum.

Giurov (1984) stated that with regard to differential diagnosis coryza caused by H. paragallinarum should not be confused with fowl cholera, pox, CRD, and Pseudomonas aeruginosa infection which may run their course in a mixed infection.

2.1.3 Host of infectious coryza Haunshi et al. (2006) reported an outbreak of infectious coryza in 10 weeks old Vanaraja chickens from Meghalaya.

Horner et al. (1995) reported that the disease affected all strains of chickens in an overall age range of 14 days to 64 weeks. The organism was responsible for upper respiratory disease of broilers and layers and implicated in lower respiratory disease of broilers. It was commonly isolated from diseased adult birds previously vaccinated against typical H. paragallinarum. Broilers were most commonly infected from 3 weeks of age and layers within the placement to peak production period.

Yamamoto (1991) established that chronically ill or healthy carrier birds serve as the main reservoir of infection, with infectious coryza occurring mostly during winter, although such seasonal patterns may be coincidental to management practices, an example which is the introduction of susceptible pullets onto farms where infectious coryza is present.

Delaplane et al. (1934) stated that the natural host for H. paragallinarum is the chicken but infectious coryza has been reported in pheasants.

2.1.4. Clinical signs of the disease Welchman et al. ( 2010) mentioned that the age range of the laying strain birds were from 5 weeks to 72 weeks with 50% of the recorded age being between 20 and 30 weeks. Commercial broilers ranged in age from 19 days to 9 weeks and broiler breeders from 27 to 40 weeks. The clinical signs observed in all types of chickens were those of an acute upper respiratory disease manifesting as facial swelling, nasal discharge and lacrimation. The condition was usually unilateral but occasionally bilateral. Internal pathology revealed a translucent mucoid sinusitis and excess mucus in the choanal cleft. Sometimes a mucoid tracheitis was also present.

Miao et al. (2007) diagnosed avian infectious coryza in 50 layers which were characterized by sneezing, coughing and head swelling in Henan province and Shandong province, China.

Haunshi et al. ( 2006) reported an outbreak of infectious coryza in 10 weeks old Vanaraja chickens from Meghalaya. The clinical signs they recorded were nasal discharge, swelling of sinuses, severe foamy lacrimation, conjunctivitis and reduction in feed intake. Necropsy showed that gross lesions were restricted only to the upper respiratory tract, mainly in the nasal cavity.

Jaswinder et al. (2005) mentioned that pathological studies on naturally occurring infectious coryza in chickens revealed swelling of the face, nasal discharge and conjunctivitis as the clinical signs. The authors stated pathological changes were mainly seen in the upper respiratory tract, which included hyperplasia of sinus and tracheal epithelium, along with an increase in goblet cells, their exfoliation and infiltration of heterophils, lympho-mononuclear cells and plasma cells in the sub- epithelium and in some cases, pathological changes were also seen in the lungs.

Soriano et al. (2004) found the epizootiology of infectious coryza, an upper respiratory tract disease of poultry which was characterized by sneezing, nasal discharge and facial swelling. However, very virulent strains had also been described as causing lesions of pneumonia, air sacculitis and arthritis.

Ibrahim et al. (2004) conducted clinical, bacteriological and postmortem examinations in 205 broilers and 162 layer chickens, of various ages and breeds and from different farms in Beni-Suif, Elmenia, Assiut and Sohag Governorates in Upper Egypt, suffering from infectious coryza. They found that the infected broiler chickens showed severe respiratory signs; similar clinical signs were observed in layer chickens, in addition to decreased egg production (by 3-40%) and the morbidity and mortality rates were 10-30% and 0.5-2% and 30-60% and 1-10% in broiler and layer chickens, respectively. The course of the disease was longer in layers (3-6 weeks) compared to broilers (2-4 weeks). However, postmortem examination revealed conjunctivitis, infra-orbital sinusitis, tracheitis, air sacculitis, fibrinous pericarditis, peritonitis and enteritis. In layers, salpingitis and oophoritis were observed. Chickens infected with H. paragallinarum and M. gallisepticum showed severe clinical signs like swollen wattles; dyspnoea and rales; decreased

7 body weight and decrease in egg production by 40-50%. Postmortem lesions were sinusitis, tracheitis, air sacculitis, fibrinous pericarditis, peritonitis, salpingitis, presence of caseous materials in the oviduct and oophoritis. In addition the group infected with H. paragallinarum and E. coli. H. paragallinarum only kept under unhygienic conditions showed similar clinical and postmortem findings.

Sameera et al. (2001) reported infectious coryza from a commercial layer flock in Arifwala (total population 20,000 birds; Breed white leghorn). The age of the flock were 35 weeks and there were more than 50% flock showing respiratory signs. The history disclosed that the sick birds were dull and depressed, nasal/lacrimal discharge, facial swelling and open mouth breathing. Also added that there was delay in feed consumption and production was dropped from 85 to 60% over three days of sickness.

Kurkure et al. (2001) reported an outbreak of infectious coryza in a commercial layer farm around Nagpur, India. The authors found that a commercial layer farm with a capacity of 22500 layers and 7500 growers, were affected with infectious coryza and the clinical symptoms observed were serous discharges from nostrils, facial edema, conjunctivitis, and inappetance, drop in egg production and mortality percentage varied with the age of birds and grower birds did not show any mortality.

Eaves et al. (1989) observed the clinical signs associated with this disease include a nasal discharge, conjunctivitis with swelling of the sinuses, face and wattles, diarrhea, decreased feed and water consumption, retarded growth in younger chickens and reduced egg production.

2.1.5 Cultural properties of A. paragallinarum Jaswinder et al. (2004) isolated H. paragallinarum from 19 of the 65 samples taken from 135 outbreaks. The isolates showed characteristic tiny dew drop colonies.

Garcia et al. (2004) carried out a study on the presence of nicotinamide adenine dinucleotide-independent H. paragallinarum in Mexico and obtained two isolates of H. paragallinarum from layers (n=80000) in Mexico. The isolates were nicotinamideadenine dinucleotide (NAD) independent and growing on blood agar without the need of a nurse colony as well as on a complex medium that lacked both NAD and chicken serum.

Bragg et al. (2004) mentioned that H. paragallinarum, the causative agent of infectious coryza in poultry, is an extremely fastidious organism requiring specific growth conditions for isolation and described need of modification and testing of transport media, which ensured the survival of the causative agents in suspected infectious coryza cases for transport to a laboratory where the bacterium could be isolated and serotyped. They established that the bacterium remained viable for up to 18 days in Amies-Transport Medium containing all the supplements when stored at 40C or 370C. At room temperature or 250C, there was no difference in the survival of H. paragallinarum in commercial Amies Transport Medium (without charcoal) and Amies Transport Medium with supplements.

Sobti et al. (2001) found field isolates of avian haemophili and other related organisms from cases of infectious coryza in Jabalpur. The growth characteristics and growth requirements of the isolates were studied using differential media. 22 isolates had the typical phenomenon of characteristics satellite growth. Six isolates of H. paragallinarum were NAD-dependent, and one strain was found to be chromogenic.

Sameera et al. (2001) reported a disease in a commercial flock in Arifwala, Pakistan (total population of 20000, white leghorns). Samples from the sick (n=5) and dead birds (n=5) were collected and bacterial growth was observed only in the nasal swabs on blood agar, chocolate agar and tryptose agar. The colonies appeared as small (1 mm) dew drops. Isolated organism revealed that the causal agent was H. paragallinarum.

Blackall (1999) mentioned that when the Haemophilus sp. were first isolated it was suggested that the organism required them (X factor) for growth, it was also noted that the organisms only grew close to Staphylococcus sp. colonies and thus also required NAD (V factor) for growth. The requirement for haem in the growth media was reported by a number of other workers who all suggested that blood or blood products where needed in the medium.

Horner et al. (1995) reported an unusual bacterium causing respiratory disease in chickens emerged in South Africa in February 1989. The disease resembled infectious coryza but the organism differed from typical H. paragallinarum especially in that it did not require V-factor for growth. It has been termed an NAD-independent H. pagallinarum. They further mentioned that a study of avian haemophili isolated from diseased chickens in Kwazulu-Natal over the past five years revealed the presence of typical H. paragallinarum, NAD-independent H. paragallinarum and H. avium (now transferred to the genus Pasteurella).

Yamamoto (1991) reported that Brain heart infusion (BHI), tryptose agar, and chicken meat infusion are some basal media to which supplements are added. The pH of these media varies from 6.9-7.6. Other sources of NAD include yolk from chicken embryos, fresh yeast extract and chicken or sheep serum. Chicken serum (I%) is required by some strains.

Blackall (1989) mentioned that colonies of H. paragallinarum are typically tiny (0.3mm after 24hrs of growth) with a dew drop shape.

Blackall et al. (1989) examined the growth factor requirements and other properties of two strains of avian haemophili, labelled as ‘H. gallinarum’ (an X- and V factor-dependent organism) and stored since the 1940s and 1950s, they found that both strains were X-factor independent and V-factor dependent and possessed the typical biochemical, serological and pathological properties of H. paragallinarum. In experiments repeating the tests used in the 1930s that reported the existence of X- and V-factor dependent avian haemophili, they found that the

10 methodology used resulted in reference strains of H. paragallinarum (X-factor independent and V-factor dependent) appearing to be X- and V-factor dependent. They concluded that the early descriptions of the etiological agent of infectious coryza as an X- and V-factor dependent organism.

Inzana et al. (1987) mentioned that a broth system was developed for rapid identification of the requirement for X factor (hemin), or V factor (NAD), or both for growth of Hemophilic species. This system was compared to growth around paper discs/strips impregnated with factors X and/or V. The broth system consisted of three tubes, each containing brain-heart infusion broth supplemented with V factor, X factor, or both. Each tube was inoculated with a saline suspension of a Hemophilic isolate, and broths were shaken for aeration at 370C. They noted that under these dictions’ turbidity or clumping was usually evident after 4-5 hr only in the broth(s) containing the required supplement(s). A few strains requiring only V factor required overnight incubation. One hundred fifty-six Hemophilic isolates were tested for growth around supplemented discs/strips or in supplemented broths: 129 were H. influenzae/aegypticus, 25 were of various species that required only V factor, and 2 were H. hamophilus.

Blackall et al. (1985) evaluated the ability of 2 brands of growth factor discs (Oxoid and Mast) to identify correctly the growth factor requirements of 41 isolates of H. paragallinarum and 17 isolates of H. avium. The percentage of isolates correctly identified as requiring V factor varied with both the brand of the disc and the medium used. On basic nutrient media both brands gave low percentages of correct results: on Isosensitest Agar (Oxoid) the Oxoid discs gave 26% and the Mast discs 24% while on Heart Infusion Agar (Gibco) the Oxoid discs gave 54% and the Mast discs 28%. However, on more complex medium (TM/S) the percentage of correct results was considerably higher with the Oxoid discs 100% and the Mast discs 52% accurate.

Yamamoto (1984) mentioned that colonies of H. paragallinarum are typically tiny (up to 0.3 mm after 24 hrs of growth) and dew drop in shape.

Rimler (1979) shown the colony morphology to range from mucoid (smooth) iridescent and rough non-iridescent to other intermediate colony forms when inspected under obliquely transmitted light. Examined isolate from 7 different countries, including South Africa, and he could not find any isolates that required haem for growth.

Rimler et al. (1977) mentioned that the reduced form of NAD (NADH) (1.56- 25mglml media) or its oxidized form and sodium chloride (NaCI) (1-1.5%) are essential for growth of H. paragallinarum.

Rimler et al. (1976) found that the organism can grow under reduced oxygen tension or anaerobically and the normal temperature for growth varies between 34- 42°C.

Hinz (1976) investigated bacteria of 2 phenotypic forms of one strain of H. paragallinarum morphologically, culturally, biochemically and serologically. Found that on transparent solid media bacteria of the M variant (mucoid) were capsulated and showed iridescence in oblique transmitted light throughout the first 8-14 hrs of incubation. Iridescence disappeared completely after 36hrs incubation. Bacteria of this variant formed stable homogeneous suspensions in 0.15 M NaCI and an homogeneous cloudiness after growth in liquid media. Bacteria of the R variant (rough) derived by dissociation of iridescent colonies were not capsulated and showed no iridescence throughout 8-14 hrs incubation (minus variant).

Rimler et al. (1975) mentioned that a serum-free modified Casman broth medium was developed to grow H. paragallinarum to titers of 10-18 organisms per ml.

Page (1962) explained that the organism may be maintained on blood agar plates by weekly passages and young cultures maintained in a candle jar would remain viable for 2 weeks at 4°C. A number of bacterial species excrete NAD and have been used as "feeder" cultures to support growth of H. paragallinarum. The author

12 also added that currently media used for isolation, growth and maintenance of the bacteria require NADH (1.56-25 mg/ml media), the reduced form of NAD.

Page et al. (1963) mentioned that the minimum and maximum 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. Tiny dew drop colonies up to 0.3mm in diameter develop on suitable media. In obliquely transmitted light, mucoid (smooth) iridescent and rough non iridescent and other intermediate colony forms have been observed.

Elliot et al. (1934) stated that the organism is commonly grown in an atmosphere of carbon dioxide; however, it is not an essential requirement, since the organism is able to grow under reduced oxygen tension or anaerobically.

McGaughey (1932) reported that his organism only required the presence of V factor for growth, but his work was largely overlooked until the 1960's when various workers reported that the isolates made from chickens were not dependant on haem.

2.1.6 Morphology and staining characteristics of A. paragallinarum Jaswinder et al. (2004) observed while studying the epidemiology of infectious coryza H. paragallinarum on Gram’s staining revealed gram-negative, pleomorphic, coccobacilli or rods.

Sameera et al. (2001) mentioned that morphologically the organism revealed that it was coccobacilli, non-spore farming, non motile (hanging drop method). Capsule was undetectable. The staining results showed that the organism was Gram's negative (Gram's staining).

Yamamoto (1991) stated that H. paragallinarum is a gram negative, polar staining, non motile bacterium. In 24-48 hrs cultures, it appears as short rods, or coccobacilli 1-3mm in length and 0.4-0.8mm in width, with a tendency for filament

13 formulation. The organism undergoes degeneration within 48-60 hrs, showing fragments and indefinite forms

Hinz (1973) observed that Some H. paragallinarum strains possess a capsule.

Sawata et al. (1980) mentioned H. parogallinarum as a Gram-negative, polar staining, non-motile bacterium appearing as short rods or coccobacilli in 24hrs old cultures. Filamentous forms of the bacteria also occur with the older cultures showing pleomorphism.

Delaplane et al. (1934) reported that the bacilli may occur singly, in pairs, or as short chains.

2.1.7 Isolation and identification of A. paragallinarum Mendoza et al. (2009) studied serotyping of 24 isolates of A. paragallinarum obtained from different regions of Peru during 1998-2008. All isolates were characterized as beta -nicotinamide adenine dinucleotide dependent. According to the Page scheme, modified by Blackall, they found that eight isolates were classified as serogroup A, seven isolates as serogroup B, and five isolates as serogroup C, while four isolates could not be serotyped.

Gayatri et al. (2009) made an study to isolate H. paragallinarum from the nasal swabs, 4 swabs (from live birds) and caseous infra orbital sinus and tracheal exudates swabs from dead birds from commercial poultry farms Anand, Kheda and Mahua region of Saurashtra area of Gujrat state. The authors obtained 6 H. paragallinarum isolates from 109 samples suspected of infectious coryza infection.

Sun et al. (2007) conducted a study on ability of blocking ELISA and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either A. (H.) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection and the results found were the positive rate of serovar A

14 specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post- infection. The highest rate of serovar C antibodies was 20% at the fifth and sixth weeks post-infection. The authors further added that the results had provided further evidence of the suitability of the serovar A and C B-ELISAs for the isolation of infectious coryza.

Miao et al. (2007) isolated pathogenic bacteria during 2003-2006 from 50 diseased laying hens characterized by sneezing, coughing and head swelling in Henan province and Shandong province, China. Based on the results of gram staining microscopic examination test, biochemistry test, agglutination test and animal regression test, they found that the diseases infecting the 50 layers were diagnosed as avian infectious coryza, which was mainly caused by type A and type C of H. avium.

Haunshi et al. (2006) reported an outbreak of infectious coryza in 10 weeks old Vanaraja chickens from Meghalaya. They isolated H. paragallinarum from the inter-orbital and nasal secretions of both infected and dead birds.

Ibrahim et al. (2004) mentioned that a total of 22 morphologically selected isolates were subjected to biochemical, serological and biological investigations. Fifteen of the 22 isolates had haemagglutination activity against formaldehyde-fixed chicken erythrocytes. Serological testing revealed the presence of H. paragallinarum serotypes A, B and C. M. gallisepticum was also isolated from chronic or complicated cases of coryza (25%).

Garcia et al. (2004) obtained two isolates of H. paragallinarum from layers (n=80000) in Mexico. The isolates were confirmed as H. paragallinarum by polymerase chain reaction and conventional biochemical identification. One isolate was Page serovar B/Kume serovar B-1 and the other isolate was Page serovar C/Kume serovar C-2.

Bragg (2002) collected various isolates of H. paragallinarum from a severe outbreak of infectious coryza in poultry from Zimbabwe, serotyped and found the isolates to belong to serovar C-3.

Soriano et al. (2001) conducted a study to determine the occurrence of Kume serovars of H. paragallinarum in Mexico. A total of 42 isolates of H. paragallinarum from 6 Mexican states were serotyped by the Kume haemagglutinin scheme. Serovars A-l, A-2, B-1, and C-2 were recognized among 11 (26.2%), 7 (16.6%), 4 (9.5%), and 14 (33.3%) isolates, respectively. However, six isolates (14.3%) showed haemagglutinating activity but could not be classified into any serovar. Commercial vaccines containing Kume serovars A-1, A-2, B-1, and C-2 may provide better protection than those bi or trivalent infectious coryza vaccines currently used in Mexico.

Sobti et al. (2001) isolated avian hemophilic and other related organisms from cases of infectious coryza in Jabalpur characterized on the basis of their morphological, cultural and biochemical properties. Among 253 samples of infectious coryza were 28 avian hemophilic, 17 Pasteurella, 10 Streptococcus, and 11 Corynebacterium isolates. The growth characteristics and growth requirements of the isolates were studied using differential media. Catalase activity, carbohydrate fermentation test, indole production, midrate ate reduction and urease activiiy of bacterial isolates were determined. 22 isolates had the typical biochemical properties of H. paragallinarum with characteristic satellite growth phenomenon. Six isolates of H. paragallinarum were NAD-dependent, and one strain was found to be chromogenic.

Sameera et al. (2001) reported a disease in a commercial flock in Arifwala, Pakistan (total population of 20000, white leghorns). Samples from the sick (n=5) and dead birds (n=5) were collected. History, clinical signs and cultural properties of the isolated organism revealed that the causal agent was H. paragallinarum.

Kurkure et al. (2001) reported outbreak of infectious coryza in a commercial layer farm around Nagpur, India. Isolates were confirmed as H. paragallinarum based on morphological, cultural, and biochemical characters.

Fernandez et al. (2000) studied the carbohydrate fermentation, antimicrobial drug resistance and serological properties of 40 isolates of H. paragallinarum from outbreaks of infectious coryza in Mexic. Four biochemical biovariants and five antimicrobial drug resistance patterns were recognized. All isolates were serotyped by the Page scheme, foun 21 isolates were serogroup A, five isolates were serogroup B and 14 isolates were serogroup C.

Poernomo et al. (2000) characterized 18 isolates of H. paragallinarum isolated from chickens in Indonesia. The isolates were identified to species level by traditional phenotypic methods. The authors stated that six of the isolates were also identified by a species-specific PCR, 14 were examined for resistance to a panel of 7 antimicrobial agents using a disc diffusion method. All 18 isolates were serotyped according to the Page scheme using reference antiserum in a haemagglutination inhibition test. Seven isolates were Page serovar A, 4 were Page serovar B and 7 were Page serovar C.

Blackall et al. (1994) described the biochemical and serological properties of 29 isolates of avian hemophili obtained from chickens in Brazil. Twenty-seven of the isolates had the typical biochemical properties of H. paragallinarum. The two remaining isolates had the typical properties of Pasteurella avium, formerly known as H. avium.

Terzolo et al. (1993) described the biochemical and serological properties of H. paragallinarum isolates recovered from 11 recent outbreaks of infectious coryza in

17 layer hens and one case of swollen-head syndrome in broilers in Argentina. Twenty-four isolates had the typical biochemical properties of H. paragallinarum.

Chen et al. (1993) studied biochemical and serological properties of 11 isolates of H. paragallinarum from outbreaks of infectious coryza in the People's Republic of China are described. All 11 isolates had the typical biochemical properties of H. paragallinarum, and all belonged to Page serovar A.

Blackall et al. (1982) examined a total of 60 isolates of Haemophilus spp. from chickens, including four reference strains of and one of H. avium, for their physiological and biochemical properties. The authors added that H. avium can be differentiated from H. paragallinarum by its possession of the enzymes catalase and alpha-glycosidase, capacity to grow in air, production of acid from galactose, and by the fact that its growth is not improved by the addition of chicken serum. In addition, the majority of H. avium isolates, unlike H. paragallinarum, possesses a yellow pigment and produce acid from trehalose.

2.1.8 Biochemical properties of A. paragallinarum Jaswinder et al. (2004) isolated H. paragallinarum from 19 of the 65 samples taken from 35 outbreaks. Majority of the isolates were catalase negative, showed oxidase activity, failed to produce indole, reduced nitrate and did not ferment galactose or trehalose.

Sameera et al. (2001) reported a disease in a commercial flock showing respiratory signs in Arifwala, Pakistan. The sample was cultured on different media. Purified growth was studied for its morphological and biochemical characters. The organism produced acid after fermentation of glucose, sucrose, lactose, manitol and negative to galactose, sorbitol. The authors also stated that the isolate was negative to indol, vogas proskauer test, methyl red test was negative and H2S was not produced, this revealed that the causal agent was H. paragallinarum.

Blackall (1989) studied on diagnosis of inrectious coryza and found that hydrogen sulfide and indole are not produced, gelatin is not liquefied, and litmus and methylene blue milk are not changed.

Blackall et al. (1988) determined the biochemical properties of 39 strains of Hemophilic avium from chickens. All the strains produced acid from fructose, galactose, glucose and mannose but not from lactose. Variable reactions were found for arabinose, maltose, mannitol, sorbitol, trehalose and xylose. No strains showed urease activity or produced indole, while beta-galactosidase and/or ornithine decarboxylase activity was present in some strains.

Piechulla et al. (1985) observed that the ability to reduce nitrate to nitrite and ferment glucose without the formation of gas is common to all of the avian haemophili. Oxidase activity, the presence of the enzyme alkaline phosphatase, and a failure to produce indole or hydrolyze urea or gelatin are also uniform characteristics.

Hinz et al. (1977) described that biochemical properties of H. paragallinarum include the ability to produce acid when grown in fructose, glucose and mannose and an inability to ferment galactose and trehalose.

2.9 Antibiotic sensitivity of A. paragallinarum Haunshi et al. (2006) reported an outbreak of infectious coryza in 10 weeks old Vanaraja chickens from Meghalaya. They isolated H. paragallinarum were sensitive to enrofloxacin, ciprofloxacin, gentamicin and chloramphenicol but resistant to ampicillin, cloxacillin and penicillin.

Sameera et al. (2001) reported a disease in a commercial flock showing respiratory signs and their production was dropped from 85 to 60% in three days in Arifwala, Pakistan (total population of 20000, white leghorns). The isolate was also subjected to antibiotic sensitivity test and found that the organism was sensitive to

19 gentamicin, amoxicillin, enrofloxacillin, chloramphenicol and resistant to tylosin, norfloxacin, tribriseen and revealed that the causal agent was H. paragallinarum.

Kurkure et al. (2001) reported an outbreak of infectious coryza in a commercial layer farm around Nagpur, India. A commercial layer farm with a capacity of 22500 layers and 7500 growers, reported respiratory diseases. Isolates were confirmed as H. paragallinarum based on morphological, cultural and biochemical characters. The isolates were sensitive to ciprofloxacin and gentamicin, and moderately sensitive pefloxacin, and resistant to norfloxacin and cephalexin.

Poernomo et al. (2000) characterized 18 isolates of H. paragallinarum isolated from chickens in Indonesia. The isolates were identified to species level by traditional phenotypic methods. The authors stated that 14 were examined for resistance to a panel of 7 using a disc diffusion method. During the test with antimicrobial agents the authors found that 11 isolates were resistant to erythromycin and streptomycin, 10 to neomycin, 8 to oxytetracycline, 5 to doxycycline, 3 to sulphamethoxazol-trimethoprim, and 1 to ampicillin.

Sobti et al. (2000) tested isolates from cases of infectious coryza from Jabalpur, India, like H. paragallinarum (28 isolates), NAD independent H. paragallinarum (6), Pasteurella spp. (17), Streptococcus spp. (10) and Corynebacterium spp. (11) for their sensitivity to cloxacillin, cotrimoxazole, enrofloxacin, gentamicin, pefloxacin, ampicillin, cefalexin and oxytetracycline.The authors demonstrated that H. paragallinarum was highly sensitive to gentamicin (50%) and enrofloxacin (40.91%). NAD independent H. paragallinarum was highly sensitive to gentamicin (66.67%).

2.1.10 Pathogenicity of A. paragallinarum Zhao et al. (2010) performed a experiment for evaluation of two experimental infection models for A. paragallinarum. The clinical symptoms of infectious coryza are multiple and include nasal discharge, facial swelling, lacrimation, and anorexia. In general, the disease is not fatal to chicken; so, in experiments where

20 birds are infected with A. paragallinarum, there have been debates about conducting the challenge model and evaluating the clinical signs. The author sin their experiment, 150 chickens of aged 30 days, randomly divided into different groups. Some groups were infected with the 'in-contact' challenge model and others with the artificial intra-sinus-injection-route model with three field isolates of different serogroups of A. paragallinarum, including Hpg-8 (Page serovar A), CCM6075 (Page serovar B) and Hpgb68 (Page serovar C). The final results observed that the 'in-contact' challenge model of the three isolates showed a more reliable representation of the natural infection under field conditions than the artificial intra-sinus-injection-route model. Thus, on carrying out birds experiments, the effect of 'in-contact' challenge model is more accurate than the artificial intra- sinus-injection-route model.

Awad et al. (2005) prepared twelve inactivated oil adjuvant vaccines (4 for egg drop syndrome (EDS) virus, 4 for H. paragallinarum and 4 for bivalent vaccines) by using different ratios of Tween 80, span 80. Hydrophil-lipophil Balance (HLB) was determined for all vaccines. It was shown that the combined vaccines protected chickens against infectious coryza with no interference in the immune responses. The vaccines that had a HLB of 4.98 resulted in higher antibody titers than those having 5.4, 6.01 and 6.58 HLB, respectively.

Soriano et al. (2004) investigated the virulence of the reference strains of the nine currently recognized Kume serovars of H. paragallinarum. The capacity of the H. paragallinarum strains to cause the typical clinical signs of upper respiratory tract disease associated with infectious coryza in unvaccinated, nasal-challenged chickens was assessed. Differences in virulence were assessed by means of a standardized scoring system for clinical signs. Results found by the authors were all nine strains were pathogenic to chickens, producing typical clinical signs of infectious coryza. The highest clinical signs score was obtained for serovar C-1 (1.72), while the lowest clinical signs score was obtained for serovar C-4 (0.32). They concluded that the results indicate virulence differences exist among the serovars of H. paragallinarum.

Ibrahim et al. (2004) carried out pathogenicity testing of identified isolates in embryonated chicken eggs and layer chickens. Experimental infection using H. paragallinarum resulted to signs of coryza and decreased egg production within 24-72 hours, while the course of the disease lasted for 7-10 days. Chickens infected with H. paragallinarum showed severe clinical signs like swollen wattles, dyspnea, rales and decreased body weight after 24 hours and decrease in egg production by 40-50%. Postmortem lesions were sinusitis, tracheitis, air sacculitis, fibrinous pericarditis, peritonitis, salpingitis, presence of caseated materials in the oviduct and oophoritis.

Garcia et al. (2004) obtained two isolates of H. paragallinarum from layers (n=80000) in Mexico. Both isolates were pathogenic, causing the typical clinical signs of infectious coryza in susceptible chickens.

Bragg (2002) investigated the virulence of four South African field isolates of NAD dependent H. paragallinarum, representing the four serovars known to occur in the country. The challenge model consisted of the direct challenge, via intra- sinus injection of one chicken in a row of interconnected layer cages, containing 10 chickens, which are subsequently infected by natural routes. A scoring system of the clinical signs was established in which a score is given to the ability of the isolate to produce clinical signs in the challenge birds. It had been demonstrated using this scoring system that the South African serogroup C isolates appear to be more virulent than the South African serogroup A or B isolates. It was further established that the serovar C-3 isolate appeared to be the most virulent.

2.1.11 Prevalence of infectious coryza Gayatri et al. (2009) stated that among infectious diseases, infectious coryza is one of the major problems affecting commercial poultry industry in the developing countries like India.

Haunshi et al. (2006) reported an outbreak of infectious coryza in 10 weeks old Vanaraja chickens from Meghalaya. Out of the 501 birds, 205 belonging to the

Institute farm and farmers' fields were affected by infectious coryza. The authors found that Out of the 205 birds affected, 2 from the Institute farm and 5 from the farmers' fields died due to infectious coryza.

Rahman et al. (2004) conducted bacterio-pathological studies on moribund and dead chickens presented at the Bangladesh Rural Advancement Committee Poultry Disease Diagnostic Centre in Nagapara, Gazipur from January to December 2002. Bacteriological specimens were collected aseptically during necropsy were detected cases of colibacillosis (n=147), salmoneliosis (n =385), and fowl cholera (n=114), to occur at a higher rate compared with staphylococcosis (n=6), gangrenous dermatitis (n=17), necrotic enteritis (n=24) and infectious coryza (n=4).

Jaswinder et al. (2004) studied the epidemiology of infectious coryza by analysing poultry postmortem records of the poultry disease diagnostic laboratory, Department of Veterinary Pathology, Punjab Agricultural University, Ludhiana, India, obtained from 1997 to 2001 and reported that H. paragallinarum was isolated from 19 of the 65 samples taken from 35 outbreaks.

Hossain et al. (2004) conducted a study to determine the prevalence of poultry diseases in Rajshahi, Bangladesh, during the period from January 2001 to February 2002. A total of 327 cases were studied, of which some were sick and the others were dead. Diagnosis of different diseases was made based on the history, clinical findings, pathological findings, age, isolation and identification of causative agents, serology and response to treatment. The incidence rates of infectious coryza (1.22%) among various infectious diseases.

Sarbiiand et al. (2003) undertook a study by collecting data from 83 broiler farms in the district of Chakwal, Punjab, Pakistan between 1998 and 1999 to investigate the disease incidence, mortality and reduction in farm income due to mortality and reported the incidence of infectious coryza was >11% among various infectious diseases.

Islam et al. (2003) performed pathological investigation on the occurrence of poultry diseases in Sylhet region of Bangladesh during November 2001 to October 2002. A total of 1352 samples of either dead or sick birds were brought from different upazillas in the Sylhet region. Diagnosis of different disease conditions was made on the basis of the history, age of birds, clinical signs, gross, and microscopic lesions. The diagnosed diseases included infectious bursal disease (IBD; 24.26%), Newcastle disease (ND; 6.73%), infectious bronchitis (0.29%), omphalitis (2.81%), fowl cholera (0.44%), salmonellosis (6.73%), colibacillosis (5.17%), necrotic enteritis (0.44%), aspergillosis (17.53%), infectious coryza (0.37%), chronic respiratory disease (CRD)/mycoplasmosis (5.32%), coccidiosis (9.46%), and deficiency disorders stress condition (1.03%).

Ashenafi et al. (2003) conducted a study to identify the major infections of local chickens (n=190) raised under traditional management system in central Ethiopia (Jeldu, Sebeta and Awash-Melka-Kontire) from October 2000 to April 2001. The results revealed the existence of various infections of local chickens in the three study sites. 83 (43.7%) cases of Newcastle disease were confirmed using indirect ELISA test. A sero prevalence study of S. pullorum and/or S. gallinarum using the rapid serum agglutination test indicated that 122 (64.2%) chickens were seropositive. Other observed infections and/or syndromes included lymphoid leukosis, 1.9%; marek's disease, 1.1%; infectious coryza, 2.1% and colibacillosis, 1.9%.

Hasan et al. (2002) found a high mortality among broiler flocks in various areas of Pakistan the bacteriological examination of the respiratory organs of clinically sick birds yielded Hemophilic and pathogenic E. coli. Further added, infectious coryza and colibacillosis played precipitating role in the problem.

Farooq et al. (2002) carried out a study during the years 2000-2001 in 109 flocks to investigate prevalent diseases and mortality in egg type layers in Chakwal district. Mortality was defined as the sum of culled and dead birds during a 52 week period prior to disposal of the flock. About 7% of loss was due to disease

24 outbreaks including E. coli, infectious coryza and chronic respiratory disease (CRD).

Talha et al. (2001) undertook a pathological investigation on the poultry diseases occurring in Mymensingh district, Bangladesh, during the period from July 1998 to October 1999. A total of 381 samples of either dead or sick birds were collected from different upazillas of Mymensingh district. The diagnosed diseases included infectious bursal disease (IBD; 19.16%), Newcastle disease (ND; 10.24%), lymphoid leukosis (1.57%), infectious laryngotracheitis (0.26%), fowl cholera (3.15%), salmonellosis (13.12%), colibacillosis (5.51%), necrotic enteritis (0.52%), ulcerative enteritis (0.26%), non-specific enteritis (1.05%), pneumonia (5.77%), infectious coryza (0.52%), chronic respiratory disease (CRD)/mycoplasmosis (11.55%), aspergillosis (4.20%), aflatoxicosis (0.52%), coccidiosis (5.51%), visceral gout (0.79%), helminthiasis (1.05%), deficiency disorders (8.14%) and miscellaneous disease conditions (7.09%).

Sobti et al. (2001) mentioned that field isolates of avian hemophilic and other related organisms from cases of infectious coryza in Jabalpur were characterized based on their morphological, cultural and biochemical properties. The result was that isolated from 253 samples of infectious coryza were 28 avian hemophilic, 17 Pasteurella, 10 Streptococcus, and 11 Corynebacterium isolates.

Miao et al. (2001) conducted laboratory test to measure the antibodies in sera from chickens vaccinated with bivalent vaccine of infectious coryza (H. paragallinarum) by B-ELISA, HI [haemagglutination inhibition test] and SPA [serum plate agglutination test].The B-ELISA antibody to serovar A and HI antibodies remained a high level post vaccination; the C serovar B-ELISA antibody and SPA antibodies reached their vertex at 5-10 days and 50-60 days, respectively, and after that the level of the antibodies decreased slowly. Further added, all the antibodies could be detected more than 390 days post-vaccination. These results also demonstrated the good sensitivity of the B-ELISA and SPA, and gave a reference for the further

25 application of the bivalent vaccine, all the diagnostic methods and correlative products.

Kurkure et al. (2001) reported outbreak of infectious coryza in a commercial layer farm around Nagpur, India. A commercial layer farm with a capacity of 22500 layers and 7500 growers, reported respiratory diseases. Clinical symptoms were serous discharges from nostrils, facial edema, conjunctivitis, and inappetance. There was a drop in egg production and mortality percentage varied with the age of birds.

Sobti et al. (2000) conducted a retrospective study of infectious coryza cases diagnosed at the Phoenix Disease Diagnostic Laboratory, Jabalpur, India, from 1996 to 1999. A total of 9591 cases were recorded, which constituted 23.02% of the respiratory diseases recorded for that span of time. The disease was more commonly observed in broilers (75.63%) and birds below 14 weeks of age. The highest incidence was observed during the months of January to April.

2.2. S. aureus 2.2.1 Colonies Freeman (1985) reported that S. aureus produce non hemolytic colonies but S. hemolyticus produce hemolytic colonies were surrounded by a zone of hemolysis on blood agar.

Merchant et al. (1967) described the colonies of Staphylococcus sp. on nutrient agar plate. It was reported that these colonies were round, smooth, glistening, opaque, low, convex, amorphous, edge and entire colonies were golden yellow or white in color.

2.2.2 Staining characters Brooks et al. (2002) reported that Gram’s stained S. aureus appears Gram-positive cocci in pair, tetrads and clusters.

Carter (1979) showed the cluster of Gram-positive cocci of S. aureus using Gram’s staining.

2.2.3 Biochemical tests of S. aureus Brooks et al. (2002) described that S. aureus produced coagulase, an enzyme like protein that caused clots of oxalated or citrated plasma in the presence of a factor contained in sera. Coagulase might deposit fibrin on the surface of Staphylococcus sp., perhaps altering their ingestion by phagocytic cells or their destruction within such cells.

Cheebrough (1985) reported that Staphylococcus sp. produced bubbles of oxygen and water from hydrogen peroxide and water in catalase test.

Carter (1979) demonstrated the catalase positive test of S. aureus, which was also coagulase positive and fermented mannitol.

2.2.4 Pathology and pathogenesis of S. aureus White et al. (2003) observed S. aureus as an important opportunist that can cause superficial to life-threatening illness in a variety of animal species. In poultry, this organism has been implicated in osteomyelities, synovitis and cellitis.

Chauhan et al. (1996) described various form of Staphylococcusis. There were haemorrhage or gangrenous dermatitis, bumble foot, septicemia, arthritis and sterna bursitis.

Nicoll et al. (1987) studied that S. epidermidis strain 115 was avirulent when administered to 3-days old chicks oral, aerosol, or intravenous route. Strain 115 adhere specifically to tracheal, lung, air-sac, and liver cells in vitro and interfered with subsequent colonization by virulent S. aureua.

CHAPTER 3 MATERIALS AND METHODS

The present research work was divided in three phases. First phase included isolation and identification of S. aureus, the second phase included isolation and identification of A. paragallinarum and the third phase included pathological study of affected organs. The study was conducted during June 2011 to May 2012.

3.1 Materials 3.1.1. Study area For the isolation of S. aureus, 10 nasal swab samples from nasal passages of 10 alive chickens were collected aseptically from BAU poultry farm. For the isolation and identification of A. paragallinarum, 30 nasal swab samples and heads from 30 dead birds died due to different diseases were collected from SK Vet. Diagnostic Center, Mymensingh and 4 clinical samples: 2 from Gazipur and 2 from Tangail. The field samples comprising nasal swabs were aseptically collected into nutrient broth (NB) (Table-1) and carried to the laboratory for the isolation and identification of the etiological agent.

Table 1. Number of nasal swab samples collected from different areas No. Sources and Location Total no. of nasal swab Type of birds samples collected 1 BAU poultry farm 10 Alive 1 SK Vet. Diagnostic Center 30 Dead 2 Gazipur 2 Alive 3 Tangail 2 Alive Total 44

3.1.2 Cotton swab Packed sterilized cotton tripped swabs and tubes were used for the collection of swabs from nasal passage.

3.1.3 Bacteriological media for culture 3.1.3.1 Solid media The following solid media were used for this experiment- i. Blood agar (Techno Pharma., India) ii. Nutrient agar (Techno Pharma., India) iii. Mannitol salt agar (Techno Pharma., India)

3.1.3.2 Liquid media (broth) The following liquid medum was used for this experiment- i. Nutrient broth (HiMedia Lab. Pvt., India)

3.1.3.3 Chemicals, reagents and solutions Crystal violate (LOBA Chemic Pvt. Ltd., India), acetone alcohol, 70% alcohol, Gram's iodine, methylene blue (Merck, Darmstad), basic funchsion(NEN Tech.

Ltd., UK), MR-VP solution¸ phenol red GR (LOBA Chemic Pvt. Ltd., India), methyl red (Techno Pharma.Chem., India), 10% KOH, creatinine (Merck, Germany), hydrogen peroxide (A.B. Chemical, Bangladesh), potassium iodide (Fisher Scientific Company, UK), sodium hydroxide (BDH, USA), sodium chloride (BDH, USA), potassium permanganate (BDH, USA), copper sulfate (BDH, USA), phosphate buffered saline (PBS), kovac's reagent, peptone water, safranine solution(Techno Pharma. Chem., India).

3.1.3.4 Sugars I. Dextrose (LOBA Chemic Pvt. Ltd., India) II. Sucrose (Wako, Japan) III. Lactose (Merc, England) IV. Maltose (Techno Pharma., India) V. Mannitol (Beximco Pharma., Germany) VI. Galactose (LOBA Chemic Pvt. Ltd., India)

3.1.3.5 Reagents for biochemical test i. Methyl Red and Voges-Proskauer broth (MR-VP broth) (Difco, USA)

ii. Peptone water iii. Phosphate buffer solution

3.1.4 Glassware and other necessary instruments In this research work following glassware and instruments were used: test tubes (with and without Durham's fermentation tube), petridishes, conical flasks, pipette, slides, compound light microscope, sterilized cotton, immersion oil, bacteriological incubator, jar, ice boxes, balance, hand gloves, spirit lamps, match lighter, bacteriological loop, glass spreader, forceps, screw capped vials, test tube rack, autoclave machine, centrifuge tube and machine, refrigerator, distilled water, cover slip, toothpick, marker pen, hanging drop slide and vaseline etc.

3.1.5 Hexisol hand rub Hexisol hand rub (100 ml bottle) was used for hand disinfection prior to collection of nasal swab samples from chickens.

3.1.6 20% sterile buffered glycerin 20% sterile buffered glycerin was used for the a preservation of A. paragallinarum.

3.1.7 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.

3.1.7.1 Nutrient broth Nutrient broth was prepared by dissolving 13g 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 overnight at 37°C to cheek their sterility and stored at 4°C 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

3.1.7.2 Nutrient agar 2.3g 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 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 for 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

3.1.7.3 Blood agar Forty g of blood agar base (HiMedia, India) was suspended in 1000ml 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 20ml quantities

31 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 overnight to cheek their sterility.

Ingredients (g/l) Agar 15.0 Peptone 10.0 Sodium chloride 5.0 Beef extract 10.0 Final pH (at 250 C) 7.3±0.2

3.1.7.4 Bacteriological peptone: Ten bacteriological peptone was dissolved in 1000ml of distilled water and 5g of sodium chloride was added to the solution. Then 50ml of 0.2% phenol red and 10% sugar were mixed for the sugar fermentation test. The mixture was sterilized in the autoclave for 15lb pressure at 1210C for 15 minutes. It was used as an ingredient of media for the investigation of numbers organism through various biochemical tests.

Ingredients (g/l) Total nitrogen 14 Amino nitrogen 2.6 Sodium chloride 1.6 Final pH (at 250 C) 7.2±0.2

3.1.7.5 MR-VP medium: The medium was rehydrated by dissolving 17g in 1000ml fresh distilled water and sterilized in the autoclave for 15lb pressure at 121oC for 15 minutes. Cultures for the methyl red test were incubated at 300C for five days and those for the Voges-Proskauer reaction for twenty four to forty eight hours. Ingredients (g/l) Buffered Peptone 7.0

Di-Potassium phosphate 7.0 Bacto- dextrose 5.0 Final pH (at 250 C) 7.4±0.2

3.1.7.6 Phosphate buffer solution: PBS was prepared by dissolving the following ingredients:

Ingredients Amount Sodium chloride 8.0g Potassium chloride 0.2g

Disodium hydrogen phosphate (Na2HPO4) 2.89g

Potassium di-hydrogen phosphate (KH2PO4) 0.2g Distilled water 1000ml

The pH of the solution was adjusted to 7.0-7.2. The solution was then sterilized by autoclaving at 1210C under 15lb pressure for 15minutes and stored at 4-80C in refrigerator until used.

3.1.7.7 10% buffer formalin: 10% buffer formalin was prepared by dissolving the following ingredients:

Ingredients Amount Absolute formalin 10ml Phosphate buffer solution (pH 7.4) 90ml

3.2 METHODS 3.2.1 Cleaning and sterilization of glassware and plasticware New and previously used glassware and plastic ware were immersed in 2% sodium hypochloride solution and left there until cleaned. After having been soaked in a household dishwashing detergent solution (‘Trix,’ Reckitt and Colman Bangladesh Ltd), the glass ware were cleaned with brush and washed thoroughly in running tap

33 water and rinsed in distilled water. The cleaned glassware were then dried in a drier at 600C. The graduated pipettes were taken in a large cylinder filled with water, washed thoroughly by continuous filling and removal of water from the cylinder and finally washed in the same manner using distilled water. The pipettes were dried in a drier at 600C. Before being sterilized the graduated cylinders were sealed in aluminum foil cover. The petridishes were wrapped in brown paper and sterilized by dry heat at 1600C for one and half hour in a drier. However, the bottles with plastic caps or rubber lined aluminum caps were sterilized by autoclaving for 15minutes at 1210C under 15lb pressure per sq. inch. During autoclaving, the caps were loosely fitted at the bottles. After autoclaving the glassware were dried in a drier at 600C and the caps of the bottles were tightened after cooling. All sterile glassware and plastic ware were kept in a dust free place.

3.2.2 Brief description of the experimental design The experimental design is schematically presented in Figure 1 and 2. The entire study was divided into three steps. The first step included isolation of S. aureus, second step is the isolation and identification of A. paragallinarum and third step included pathological study of affected organs.

EXPERIMENTAL DESIGN

Collection of nasal swab from layer chicken

Inoculation into nutrient broth

Culture on Mannitol salt agar

Cultural properties and colony morphology

Sub culture bacteriological agar media (Nutrient agar and blood agar)

Gram’s stain

Gram positive Gram negative

Discarded Biochemical test

Catalase test positive

S. aureus

Fig. 1: Isolation and identification design of S. aureus

EXPERIMENTAL DESIGN

Collection of nasal swab from layer chicken

Sample preservation and transport to the laboratory

Inoculation into nutrient broth

Initially streaking on to bacteriological agar media ( Blood agar with S. aureus nurse colony)

Cultural properties and colony morphology Gram’s staining

Gram positive Gram negative

Discarded Sub culture on to Blood agar with S. aureus nurse colony and picked up the single colony

Biochemical test

Sugar fermentation test Indole test MR VP Catalase test

Confirmation of the organism (A. paragallinarum)

Fig-2:Isolation and Identification design of A. paragallinarum

3.2.3 Collection and transportation of samples Nasal swab samples from 10 live birds were used for isolation of S. aureus. Thirty dead birds and 4 live birds for the isolation and identification of A. paragallinarum were used with the help of sterile cotton bud and transferred the bud immediately into nutrient broth in sterile screw capped test tubes. At the time of collection, precaution was taken to prevent cross-contamination of samples. Each sample was labeled with an identification mark. The samples were carried to the laboratory in an ice box at 4°C temperature. After collection, the samples were transported to the laboratory as soon as possible for the isolation and identification of organisms.

3.2.4 Isolation and identification of organisms 3.2.4.1 Isolation and identification of S. aureus 3.2.4.1.1 Primary culture of S. aureus Primary growth of all kinds of bacteria was performed in nutrient broth. Ten nasal swab samples from live birds were collected with sterile cotton bud by gentle touch, and then inoculated into the nutrient broth , incubated overnight at 37°C to obtain the primary culture.

3.2.4.1.2 Isolation of S. aureus in pure culture After primary culture of the organism, a small amount of inoculums from nutrient broth was streaked onto mannitol salt agar showing characteristic morphology of S. aureus were selected for sub cultured on nutrient agar and blood agar.

3.2.4.2 Isolation and identification of A. paragallinarum 3.2.4.2.1 Primary culture of A. paragallinarum Thirty nasal swab samples from dead birds and 4 nasal swab samples from live birds were collected with sterile cotton bud by gentle touch, and then inoculated into the nutrient broth, incubated overnight at 37°C to obtain the primary culture.

3.2.4.2.2 Isolation of A. paragallinarum in pure culture

After primary culture of the organism, a small amount of inoculums from nutrient broth was streaked onto blood agar showing characteristic morphology of A. paragallinarum, were selected for subculture. Tiny dew drop colonies developed on blood agar media was considered positive for A. paragallinarum.

3.2.4.3 Identification of the isolates 3.2.4.3.1 Study of colony morphology for identification The colony morphology of the isolates were studied as mentioned by Merchant and Packer (1967). Morphological characteristics (shape, size, surface texture, edge, elevation, color, opacity etc.) developed after 24hrs of incubation were carefully studied and recorded.

3.2.4.3.2 Morphological characterization by Gram’s staining method 3.2.4.3.2.1 Preparation of Gram stain Crystal violet solution Stock crystal violet solution Ingredients Amount Crystal violet 10g Ethyl alcohol 1000ml

Stock oxalate solution Ingredients Amount Ammonium oxalate 1g Distilled water 1000ml

Working crystal violet solution Ingredients Amount Stock crystal violet solution 20ml Stock oxalate solution 80ml It mixed and prepared when required.

Lugol’s iodine solution Ingredients Amount Iodine crystal 1g Potassium iodide 2g

These two were dissolve 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

Safranin (counter stain) 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.

3.2.4.3.2 Microscopic analysis 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 was added then mixed and smeared on a glass slide and fixed by gentle heat.

2. Ammonium oxalate crystal violet was added on to the smear and allowed to react for 30 sec. 3. Washed in running tape water. 4. Lugol’s iodine was then added to act as mordant for 60sec and then again washed with running water. 5. Acetone alcohol was then added and decolorize the staining, for 3- 5 seconds. 6. Washed thoroughly in tape water. 7. After washing with water, safranin 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.

3.2.4.4 Biochemical studies for the identification of organisms Several biochemical tests were performed for confirmation of the isolates.

3.2.4.4.1 Carbohydrate fermentation test Preparation of Carbohydrate fermentation test reagents Bacteriological peptone Ingredients Amount Bacteriological peptone 10g Sodium chloride 5g Distilled water 1000ml

Phenol red (0.2%) Ingredients Amount Phenol red 2g Distilled water 1000ml

Sugar (10%) Five basic sugars such as dextrose, sucrose, galactose, maltose and mannitol were used for sugar fermentation test. Ingredients Amount Specific sugar 1g Distilled water 10ml

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 1g of Bacto peptone (Difco, USA) and 0.5g of sodium chloride in 100m1 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 5m1 amount into cotton plugged test 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 (10g sugar was dissolved in 100ml 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 0.5ml 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 (only for two samples that were already isolated) by inoculating a loop full of nutrient broth culture of the organisms into the tubes containing five basic sugars e.g., galactose, maltose, sucrose, mannitol and glucose and incubated for 24 hours at 37°C. Acid production was indicated by change of color from reddish to yellow in the medium.

3.2.4.4.2 Indole test Two ml of peptone water was inoculated with 5ml of bacterial culture and incubated for 48 hours. 0.5ml of Kovac's reagent was added, shaked well and examined after 1 minute. Development of red color indicate positive test.

3 .2.4.4.3 Methyl-Red & Voges-Proskauer (MR-VR) test Composition of MR-VP medium (DIFCO Laboratories, USA) Ingredients Amount Buffered peptone 7.0g Dextrose 5.0g Di-potassium phosphate 5.0g

A quantity of 3.4g of Bacto MR-VP medium was dissolved in 250m1 of distilled water dispensed in 2ml 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 ml of sterile glucose phosphate peptone water were inoculated 5ml of test organisms. It was inoculated at 370C for 48 hours. A very small amount (knife point) of creatine was added and mixed. Three ml 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 the development of pink color. Positive cases there was noted by the development of a pink color slowly.

Methyl Red solution Ingredients Amount Methyl red 0.05g Ethanol (absolute) 28ml Distilled water 22ml

The indicator phenyl red solution was prepared by dissolving 0.1g of Bacto methyl- red in 300ml of 95 percent alcohol and diluting to 500ml with the addition of 200ml of distilled water.

The test was performed by inoculating a colony of the test organism in 0.5ml 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 was shown by the appearance of bright red color was an indication of acid production.

3.2.4.4.4 Alpha- napthanol solution V-P reagent-1 Ingredients Amount Ethanol (95%) 10ml Α-nepthol 0.5g Alpha- napthanol solution was prepared by dissolving 5g of 1- apthanol in 100ml of 95% of ethyl alchohol.

3.2.4.4.5 Potassium hydroxide solution V-P reagent-2 Ingredients Amount Potassium hydroxide 40g Creatinine 0.3g Distilled water 100ml Cotton blue dye 0.05g Potassium hydroxide (KOH) solution was prepared by adding 40g of Potassium hydroxide crystals in 100ml of cold distilled water.

3.2.4.5 Enzyme activity test 3.2.4.5.1 Catalase test The organism was grown on a slope of nutrient agar for S. aureus and blood agar for A. paragallinarum. One ml 3% H2O2 was run down the slope and examined

43 immediately and after 5min for evaluation of gas. Positive test was indicated by the production of bubbles.

3.2.5 Maintenance of the stock culture 3.2.5.1 Phosphate buffered saline solution For preparation of PBS solution, 8g of sodium chloride, 2.89g of disodium phosphate, 0.2g of potassium chloride and 0.2g of potassium hydrogen phosphate were suspended in 1000ml of distilled water. The solution was heated to dissolve completely. The solution was then sterilized by autoclaving and stored at 4oC until use. The pH of the solution was adjusted to 7-7.2 and measured by a pH meter (Cheesbrough, 1985).

3.2.5.2 20% sterile buffered glycerin solution was prepard by Mixing of 20 parts pure glycerin and 80 parts of PBS. Then a loop full of thick bacterial culture of each isolate was mixed with 20% sterile buffered glycerin in small vial and preserved at -20°C. This method is more appropriate for preserving bacteria with no deviation of their original characters for long time even for several years.

3.2.6 Pathological studies 3.2.6.1 Gross pathology As sample was collected from live birds, so gross study was not done for 10 samples of S. aureus. Postmortem examination was carried out carefully on 30 dead chickens and 4 live chickens after killing . Gross tissue changes such as mucus filled sinuses were observed and recorded carefully, and representative samples containing lesions were fixed in 10% neutral buffered formalin for histopathological examination.

3.2.6.2 Histopathology Nasal passage of representative samples, from which isolation and identification of bacteria already done, were selected for routine histopathological study. The

44 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.

3.2.6.2.1 Processing of nasal passage tissue 1. The tissues were placed in 10% buffered neutral formalin in a volume 20 times more than the specimens for 7 days. The solutions were changed 24 hours after fixing. 2. The tissues were kept in running tap water for overnight after being fixed properly. 3. Tissues were putted in decalcifying solution until decalcification was 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 ascending 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, 4-5 hour for each. 7. The tissues were embedded with molten wax at 560C: 4 changes, 8-10 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.

3.2.6.2.2 Preparation of decalcifying solution : Formic acid-sodium citrate method:

Solution A Solution B

Ingredients Amount Ingredients Amount Sodium citrate 50g Formic acid (90%) l25ml Distilled water 250m1 Distilled water 125ml Solution A and Solution B were mixed in equal amount for use.

3.2.6.2.3 Preparation of stains 1. Preparation of Harris Hematoxylin solution Ingredients Amount Hematoxylin crystals 5.0g Alcohol, 100% 50.0ml Ammonium or potassium alum 100.0g Distilled water 1000.0ml Mercuric oxide (red) 2.5g

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. 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.

ii. Preparation of eosin solution a. 1% Stock alcoholic eosin Ingredients Amount Eosin, water soluble 1.0g Distilled water 20.0ml Dissolved and add alcohol, 95% 80.0ml

b) Working eosin solution Ingredients Amount Eosin stock solution 1 part Alcohol, 80% 3 parts Just before use 0.5ml of glacial acetic acid was added to each 100ml of stain and stirred.

3.2.6.2.4 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 tissue (nasal sinus) were stained with Harris Hematoxylin for 1 hours. 4. Section were washed in running tap water for 15 minutes. 5. After washing, the tissues were differentiated in acid alcohol: 2 to 3 quick dips (l part HCI and 99 parts 70% alcohol). 6. Then washed in tap water for 5 minutes followed by two to three quick dips in ammonia water until sections were bright blue. 7. Stained with eosin for l0 seconds ( nasal sinuses) to visualize cytoplasmic component. 8. Differentiated and dehydrated in alcohol: 95% alcohol: 3 changes, 2-4 dips each; absolute alcohol: 3 changes 2-3 minutes in each. 9. Cleaned in xylene: 3 changes (5 minutes in each). 10. Finally the sections were mounted with coverslip using Dpx.

3.2.7 Photomicrography Photomicrography was taken at the Department of Pathology using photomicrographic camera (olympus pM-c 35 Model) onto fitted with Olympus microscope (Olympus, Japan).

CHAPTER 4 RESULTS

The results presented below demonstrate the isolation and identification of the etiological agent of infectious coryza from suspected cases of layer chickens. The samples collected from field cases were subjected to various cultural and biochemical investigation for the isolation of causal agent, and pathological study of the affected tissue in the laboratory, Department of Pathology, BAU, Mymensingh.

4.1 Results of isolation and identification of S. aureus For the successful growth of A. paragallinarum the association of S. aureus bacterial colony is needed for supplying of additional nicotinamideadenine dinucleotide (NAD) for the growth of A. paragallinarum.

4.1.1 Results of cultural examination 4.1.1.1 Culture on nutrient broth Swab samples from the nasal passage of 10 alive chickens were inoculated separately in nutrient broth for 24 hours at 370C and presence of bacteria was indicated by the turbidity of broth. All 10 samples showed turbidity in Nutrient broth (Fig. 3)

4.1.1.2 Culture on mannitol salt agar Mannitol salt agar media is one of the special media for the growth of S. aureus. Ten swab samples inoculated separately in mannitol salt agar and incubated for 24 hours at 370C and the colony characteristics were round, smooth, shiny, opaque, golden yellow color resembling the colony characteristics of S. aureus (Fig. 5).

4.1.1.3 Culture on nutrient agar Tentatively diagnosed five S. aureus from mannitol salt agar media were streaked separately on nutrient agar and incubated for 24 hours at 37oC. The colony

48 characteristics of all five inoculated bacteria were round, smooth, shiny, opaque, golden yellow color which was indicating the colony characteristics of S. aureus (Fig. 4).

4.1.1.4 Culture on blood agar Tentatively diagnosed five S. aureus from mannitol salt agar media were streaked separately on blood agar and incubated for 24 hours at 37oC . All of the samples showed yellowish, smooth colonies with no hemolysis were observed indicating the colony characteristics of S. aureus (Fig. 6).

4.1.2 Results of Gram's stain Tentatively diagnosed five S. aureus isolates from blood agar media were stained with Gram’s stain. All five samples showed Gram-positive cocci and arranged in grape like clusters (Fig. 7).

4.1.3 Enzymatic activity test 4.1.3.1 Catalase activity test Tentatively diagnosed five S. aureus from blood agar media were subjected to catalase test. All five samples showed positive test (i. e. production of bubbles) indicating S. aureus (Fig. 8)

4.2 Results of isolation and identification of etiological agent, A. paragallinarum For the successful growth of A. paragallinarum additional nicotinamideadenine dinucleotide (NAD) is needed. However S. aureus grow in blood agar can produce additional NAD for the growth of A. paragallinarum.

4.2.1 Results of cultural examination For the isolation of A. paragallinarum nasal samples from 30 dead bird were collected and prepared following the adapted procedure described elsewhere. All the samples revealed no growth of A. paragallinarum. However, 4 suspected

49 clinical cases (nasal discharge, conjunctivitis with swelling of the sinuses, face and wattles) (Fig. 9) were subjected to isolation and identification of A. paragallinarum. A. paragallinarum was isolated and identified from 2 out of 4 clinical cases.

4.2.1.1 Culture on nutrient broth Four clinical samples from nasal passages were inoculated separately in nutrient broth and incubated for 24 hours at 370C. All of the 4 samples showed turbidity in nutrient broth (Fig. 11 ).

4.2.1.2 Culture on blood agar media containing S. aureus Four samples from nutrient broth were cultured on blood agar media containing S. aureus. Two out of 4 samples produced tiny dewdrops, mucoid, smooth iridescent colonies with no hemolysis in media resembling with the colony characteristics of A. paragallinarum (Fig. 12).

4.2.2 Results of Gram's stain Tentatively diagnosed 2 out of 4 samples from blood agar media were stained with Gram’s stain. All of the suspected 2 samples showed Gram- negative, red color, rod shaped bacilli arranged as single or paired (Fig. 13).

4.2.3 Results of biochemical tests Biochemical tests were performed from tentatively diagnosed 2 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 result were furnished below:

4.2.3.1 Results of sugar fermentation test All 2 isolates fermented four basic sugars (maltose, sucrose, mannitol and glucose) and produced acid and did not ferment galactose. Acid production was indicated by the color change from reddish to yellow (Table 2, Fig. 14).

4.2.3.2 Results of other biochemical tests Two 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 2, Fig. 15, 16, 17). Isolates revealed the following pattern of biochemical reactions regarded as A. paragallinarum.

Table 2. Results of Biochemical characteristics of A. paragallinarum Different Biochemical test Sample size Result Identification of bacteria Fermentation reaction with five basic sugars a. Glucose 2 + A. paragallinarum b. Sucrose 2 + A. paragallinarum c. Galactose 2 _ A. paragallinarum d. Maltose 2 + A. paragallinarum e. Mannitol 2 + A. paragallinarum Other biochemical test Indole 2 _ A. paragallinarum MR 2 _ A. paragallinarum MR-VP 2 _ A. paragallinarum + = Positive; - = Negative; MR = Methyl red; VP = Voges proskauer

4.2.4 Enzymatic activity test 4.2.4.1 Catalase activity test Two isolates from blood agar media were subjected to catalase test. All the isolates showed negative test (i. e. production of no bubble) indicating A. paragallinarum (Fig. 18).

4.2.5 Bulk culture and storage of A. paragallinarum Single colony of A. paragallinarum cultured in nutrient broth and store in ependrof tube (80µl broth with A. paragallinarum and 20µl 20% sterile buffer glycerin) (Fig. 19; Fig. 20).

4.2.6 Gross study Sample collected from 30 dead birds died due to other diseases, so produced lesions were not similar to infectious coryza. However 4 samples from alive birds showed clinical signs i. e. nasal discharge; conjunctivitis; swelling of the sinuses, face and wattles indicating cases of infectious coryza (Fig. 9). But only 2 samples out of 4 showed mucous in nasal passage and hemorrhage in trachea (Fig. 10).

4.2.7 Histopathological study Thirty nasal passages from 30 dead birds died due to other diseases were not processed for histopathology as A. paragallinarum was not isolated from this cases. Two samples diagnosed of infectious coryza were processed for histopathological study. Microscopically, the section of the nose showed acanthosis (Fig. 21), congestion, mucous glanduler cell hyperplasia (Fig. 20), hyperplasia of nasal sinus and parakeratosis and / mucous (Fig. 21).

Fig. 3. S. aureus produces turbidity in Fig. 4. S. aureus produces golden nutrient broth. yellow colored colony on nutrient agar media.

Fig. 5. S. aureus produces golden Fig. 6. S. aureus produces yellow colored colony on mannitol yellowish colony on blood agar media. salt agar media.

Fig. 8. Production of bubbles which indicate catalase positive for S. aureus FFig. 7. S. aureus showing gram positive, cocci and arranged in grape like clusters ( Gram's staining. x830).

A B Fig. 9. Frothy oculo-nasal discharge (A and B) in infectious coryza infected chickens.

A B Fig. 10. Mucoid exudates in the nasal passage (A) and congested trachea (B) in infectious coryza infected chickens.

Fig. 11. A. paragallinarum produces Fig. 12. A. paragallinarum produced turbidity in nutrient broth smooth iridescent colonies with no hemolysis on blood agar media.

Glac Glu Su Ml Mn Cont - ve + ve +ve +ve +ve

Fig. 13. A. paragallinarum showing Fig. 14. Fermentation of glucose, sucrose, gram negative rod shaped bacilli mannitol, maltose with production of only

(Gram's staining. x830) acid and no galactose by A. paragallinarum

VP, - ve MR-VP, -ve

Fig. 15. A. paragallinarum produces Fig. 16. A. paragallinarum produces yellow color in VP test, indicate negative yellow color in MR-VP test, indicate reaction. negative reaction.

Fig. 17. Color change was not seen in Fig. 18. Production of no indole test which indicate negative bubbles which indicate catalase reaction by infectious coryza nagative for A. paragallinarum

Control

Fig.19. Bulk culture of A. paragallinarum in nutrient broth

Fig. 20. Storage of A. paragallinarum

Fig. 21. Congestion and mucous glandular cell hyperplasia of the affected nasal passage of chickens (H&E stain, X 333) was seen.

Fig. 22. Acanthosis, parakeratosis and inflammatory cells of the affected nasal passage of chickens (H&E stain, X 333) was seen.

CHAPTER 5 DISCUSSION

The present investigation was performed for the isolation and identification of A. paragallinarum, the causal agent of infectious coryza, from layer chickens by cultural, morphological and biochemical examinations along with pathological investigation of the affected tissues.

For isolation and identification of A. paragallinarum additional NAD is required. However, the use of s. aureus bacterial colony in the same media influences the growth of A. paragallinarum as it is reported that s. aureus bacterial colony supply additional NAD for A. paragallinarum. For the isolation of A. paragallinarum an attempt was taken to isolate feeder bacteria s. aureus. In this study 10 nasal swab samples of live chickens were collected and all of them were found to be positive for S. aureus infection and were identified following cultural and morphological examination. These swabs were cultured in nutrient agar (NA), mannitol salt agar (MSA) and blood agar (BA) media. The colony characteristics of S. aureus observed on NA and MSA were smooth, shiny, opaque, golden yellow color and on BA showed yellowish, smooth colonies with no hemolysis was similar to the findings of Freeman, (1985); Merchant et al., (1967); Brooks et al., (2002). In Gram's staining the morphology of the isolated S. aureus appeared Gram-positive cocci and arranged in grape like clusters. These findings were in agreeable with several authors such as Brooks et al., (2002); Carter, (1979). The isolates also revealed positive reaction in catalase tests which was in agreement with Carter, (1979); Cheebrough, (1985).

In this study, a total of 34 nasal swab samples were collected (30 from dead birds and 4 from suspected clinical cases). Out of 34 nasal swab samples 2 were found to be positive for A. paragallinarum infection and were identified following cultural, morphological, biochemical examination and pathological study of the affected tissues. The colony characters on different media exhibited characteristic feature.

The colony characteristics of A. paragallinarum observed on blood agar (BA) media containing S. aureus tiny dewdrops, mucoid, smooth iridescent colonies with no hemolysis, similar to the findings of Sameera et al., (2001); Blackall (1989); Page (1963). The differences in colony morphology 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). However our isolates did not show any variation in colony morphology.

In Gram's staining the morphology of the isolated bacteria exhibited pink or small rod shaped Gram negative coccobacilli. These findings were agreeable with several authors such as Sameera et al., (2001), Yamamoto (1991), Sawata et al., (1980) and Jaswinder et al., (2004).

Our two isolates of A. paragallinarum was confirmed by sugar fermentation test. In this study 5 basic sugars were used as stated by Blackall, (1989) and Hinz et al., (1977). All the isolates fermented glucose, sucrose maltose, mannitol but failed to ferment galactose within 24-48h of incubation. The result of biochemical analysis suggested the species A. paragallinarum by Blackall (1989), Hinz et al., (1977), Sameera et a1., (2001) and Jaswinder et al., (2004).

To reconfirm the isolates as A. paragallinarum some other biochemical test was performed. The isolates also revealed negative reaction in MR test, Indole test, V- P test and catalase tests which was in agreement with the findings of Sameera et al., (2001) and Jaswinder et al., (2004).

Clinically suspected infectious coryza was characterized by nasal discharge, conjunctivitis with swelling of the sinuses, face and wattles, decreased feed and water consumption, and reduced egg production were seen. At necropsy examination excess mucous in nasal passage and hemorrhage in trachea seen, corresponding with the findings of Welchman et al., (2010), Miao et al., (2007), Haunshi et al., (2006), Ibrahim et al., (2004), Kurkure et al., (2001) and Evanes et

59 al., (1989). Histopathological study was done in two cases from where A. paragallinarum was isolated. Histopathology of the nose showed acanthosis, congestion, mucous glanduler cell hyperplasia, hyperplasia of nasal sinus and parakeratosis. Such findings were supportive of the presence of A. paragallinarum infection in layer chickens (Miao et al., 2007; Haunshi et al., 2006; Jaswinder et al., 2005; Ibrahim et al., 2004; Kurkure et al., 2001 and Sameera et al., 2001). Literature available indicated that this is the first study on A. paragallinarum in Bangladesh and a tight link between S. aureus and A. paragallinarum for growing in artificial culture media.

CHAPTER 6 SUMMARY AND CONCLUSION

The present study was conducted for the isolation and identification of A. Paragallinarum, the causal agent of infectious coryza, along with pathological study of the affected tissues in layer chickens. In this study, an isolates of S. aureus was used in culture as a feeder media for the growth of A. paragallinarum.

A total of 10 nasal swab samples were collected and all of them were found to be positive for S. aureus infection and were identified following cultural and morphological examination. Major findings of the morphological characterization revealed that the isolates were Gram-positive cocci and arranged in grape like clusters as revealed on Gram’s staining. On cultural examination, the isolates produced characteristic colonies on different media such as on nutrient agar, blood agar and mannitol salt agar the colony of bacteria showing round, smooth, shiny, opaque, golden yellow in color with no hemolysis. Catalase were positive all the test result supported the presence of S. aureus.

A total of 30 nasal swab samples from dead birds from SK Veterinary Diagnostic Center Mymensingh, two nasal swab samples of live birds from Gazipur and two nasal swab samples of live birds from Tangail, were subjected to various cultural and biochemical tests and investigations for the isolation and identification of A. paragallinarum, the etiological agent of infectious coryza. A. paragallinarum was failed to isolate and identify from nasal swabs of dead birds. This may be due to fact that the bacteria might have spoiled for delayed collection or died due to other diseases. However two isolates from four live birds were identified on the basis of colony characters, Gram's staining, sugar fermentation test, biochemical properties and histopathological study. Cinically IC affected birds showed nasal discharge, conjunctivitis with swelling of the sinuses, face and wattles, decreased feed and water consumption, and reduced egg production and by necropsy, all four cases revealed rhinitis and tracheitis.

As it is an important disease of poultry, often seen to suppress growth and productivity of chickens. It needs extensive investigation to know the percentage of chickens in our country carrying such infection. Result of which will enable us to design future prevention or control strategy about IC.

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