EXPERIMENTAL PATHOGENESIS STUDY OF INFECTIOUS CORYZA IN CHICKS BY LOCAL ISOLATE OF Avibacterium paragallinarum. A Thesis

Transcription

1 EXPERIMENTAL PATHOGENESIS STUDY OF INFECTIOUS CORYZA IN CHICKS BY LOCAL ISOLATE OF Avibacterium paragallinarum A Thesis Submitted to Bangladesh Agricultural University, Mymensingh In partial Fulfillment of the Requirements for the Degree of Master of Science in Pathology By MOHAMMAD ALI Roll No.: 11Vet Path JJ 04 M Registration No.: 31947, Session: Department of Pathology Bangladesh Agricultural University Mymensingh May, 2012 i

2 EXPERIMENTAL PATHOGENESIS STUDY OF INFECTIOUS CORYZA IN CHICKS BY LOCAL ISOLATE OF Avibacterium paragallinarum A Thesis Submitted to Bangladesh Agricultural University, Mymensingh In partial Fulfillment of the Requirements for the Degree of Master of Science in Pathology By MOHAMMAD ALI Approved as to Style and Contents by (Prof. Dr. Md. Abu Hadi Noor Ali Khan) Co-Supervisor (Prof. Dr. Md. Mokbul Hossain) Supervisor (Prof. Dr. Priya Mohan Das) Chairman, BOS & Head Department of Pathology May, 2012 ii

3 ACKNOWLEDGEMENTS All panegyrics are due to the Almighty Allah, the Supreme Authority of the Universe, Who has kindly enabled the author to conduct the research and thesis work successfully for the degree of Master of Science in Pathology. The author would like to express his heartfelt gratitude, indebtedness and profound respect to his honorable teacher and research supervisor Professor Dr. Md. Mokbul Hossain, Department of Pathology, BAU, Mymensingh for his generosity, scholastic guidance, invaluable advice, suggestions, constructive criticism, untiring help and constant inspiration throughout the course of this research work and immense help in preparing the thesis manuscript. The author wishes to convey his profound respect and sincere gratitude to his honorable teacher and research co-supervisor Professor Dr. Md. Abu Hadi Noor Ali Khan, Department of Pathology, BAU, Mymensingh, for his affectionate encouragement, constructive criticism, kind co-operation, necessary correction and instruction to complete this manuscript. It is a great opportunity for the author to express his gratefulness, sincere appreciation, high indebtedness and deep respect to Professor Dr. Priya Mohan Das, Head, Department of Pathology for his valuable suggestion, encouragement and help throughout the research period and preparation of the thesis. The author would like to express his immense indebtedness to Professor Dr. Md. Iqbal Hossain, Professor Dr. Md. Abdul Baki, Professor Dr. Md. Rafiqul Islam, Professor Dr. Md. Habibur Rahman, Professor Dr. A. S. Mahfuzul Bari, Professor Dr. Emdadul Haque Chowdhury, DR. Rokshana Parvin, DR. Jahan Ara Begum, DR. Mohammed Nooruzzaman and DR Munmun Pervin Department of Pathology, Bangladesh Agricultural University, Mymensingh, for giving encouragement, advice and facilitating the lab equipments and reagents to conduct the research work. iii

4 The author also wishes to express his gratefulness and sincere appreciation to all PhD and MS students of Department of Pathology, Bangladesh Agricultural University, Mymensingh and DR. Md. Zubaed Hossain, MS student, Department of Physiology, Bangladesh Agricultural University, Mymensingh for their inspirations and assistances during the course of the research. The author would like to express his cheerful acknowledgements to the sweet surroundings of well wisher specially DR. Sankar, DR. Mehedi, DR. Mamun, DR. Harun, DR. Tarek, DR. Shuvo, DR. Sujon, DR. Sulaiman and DR. Saleha Akter for their kind cooperation throughout the whole research period. The author expresses his thanks to all technicians and staff especially Md. Idris Ali and Md. Raihan, Department of Pathology for their assistance. The author gratefully acknowledges to his beloved elder sisters Most. Angumanara Begum and Most. Samina Nargis, younger brother Md. Morshedul Alam and brother-in-law Md. Samsul Alam for their marvelous sacrifices, inspiration and blessing throughout his life. Finally indebtedness is due to his beloved father Md. Balel Uddin Sarker and mother Most. Momotaz Begum for their sacrifices, inspiration, cooperation and blessing to get him to this position. The Author May, 2012 iv

5 ABSTRACT This research work was undertaken to study the experimental pathogenesis of infectious coryza by a local isolate of Avibacterium paragallinarum in broiler chicks in Bangladeah. For this purpose, 24 chicks of 14 days of age were grouped into two (A and B), each group containing 12 birds. Chicks of group A were inoculated with 1 ml of 2 days old nutrient broth and were kept as control group while group B were inoculated with 1 ml of 2 days old culture broth of Avibacterium paragallinarum. To study the pathology, 4 birds from each group were sacrificed on day 3, 5 and 7 of post inoculation. Sacrificed birds of group A did not reveal any significant clinical sign and lesion. Chicks of group B showed mild nasal discharge, conjunctivitis, depression and inability to move. The gross lesions of the chicks of group B included mucus in nasal passage, conjunctivitis, swelling of sinuses and face and congested lungs. The microscopic lesions in this group were acanthosis and congested blood vessels of nasal passage, pneumonic lesion of lung, focal hepatitis of liver and fatty change and lipid nodules in macrophages of heart which were progressively prominent on day 7 of bacterial inoculation. Avibacterium paragallinarum was reisolated from day 7 of post inoculation (PI) from nasal passage of chicks in which lesions were prominent. The proposed experimental pathogenesis might be inoculation of A. paragallinarum through nasal passage it produced rhinitis following reached to the different organs via blood and finally revealed lesions. The lesions that found in this experiment (rhinitis in association with focal hepatitis, fatty change in heart with lipid granuloma, progressive pneumonic lesions) are not normally present in adult and young birds. In this study there was no lesion in control group (inoculated without A. paragallinarum). But in comparison with control group time dependently severity of lesions was found in different organs in experimental inoculated group (inoculated with A. paragallinarum). This may be a new finding of this disease. However, it needs further investigation. v

6 CONTENTS CHAPTER TITLE PAGE NO. ACKNOWLEDGEMENTS iii-iv ABSTRACT v LIST OF CONTENTS vi-ix LIST OF TABLES x LIST OF FIGURE xi LIST OF ABBREVIATIONS AND SYMBOLS xii CHAPTER I INTRODUCTION 1-2 CHAPTER II REVIEW OF LITERATURE History Economic Significance Public Health Significance Etiology Pathobiology and Epizootiology Incidence and Distribution Natural and Experimental Hosts Age of Host Most Commonly Affected Transmission, Carriers, and Vectors Incubation Period Pathogenicity Virulence Factors Clinical Signs Morbidity and Mortality Pathology Immunity Morphology and Staining Growth Requirements Colony Morphology 11 vi

7 CONTENTS (Contd.) CHAPTER TITLE PAGE NO Biochemical Properties Susceptibility to Chemical and Physical Agents Strain Classification Immunogenicity or Protective Characteristics Molecular Techniques Diagnosis Isolation and Identification of Causative Agent Serology CHAPTER III MATERIALS AND METHODS Preparation of experimental house Chicks Feed Experimental Pathogenesis Study Groupings Inoculation of Bacteria Post-mortem of chicks and sample collection Histopathology Processing of tracheal tissue Processing of nasal passage tissue Preparation of decalcifying solution Preparation of stains Preparation of Harris Hematoxylin solution Preparation of eosin solution Routine Hematoxyrlin eosin staining procedure Photomicrography Reisolation of Avibacterium paragallinarum in Bacteriological Media vii

8 CONTENTS (Contd.) CHAPTER TITLE PAGE NO Preparation of various bacteriological culture media 28 and different liquid solution Nutrient broth Nutrient agar Blood agar Isolation and identification of organisms Isolation and identification of Staphylococcus 30 aureus Primary culture of Staphylococcus aureus Isolation of Staphylococcus aureus in pure culture Isolation and identification of Avibacterium 30 paragallinarum Primary culture of A. paragallinarum Isolation of A. paragallinarum in pure culture Study of colony morphology for identification Staining Preparation of Gram staining solution Microscopic study of the suspected colonies Biochemical studies for the identification of organisms Reagents for biochemical test Sugars Carbohydrate fermentation test Indole test Methyl-Red & Voges-Proskauer (MR-VP) test Enzyme activity test Catalase test 36 viii

9 CONTENTS (Contd.) CHAPTER TITLE PAGE NO. CHAPTER IV RESULTS Clinical findings of chickens Gross study Histopathological study Reisolation of Avibacterium paragallinarum on day Results of Gram's stain Results of biochemical tests Results of sugar fermentation test Results of other biochemical tests Enzymatic activity test Catalase activity test 41 CHAPTER V DISCUSSION CHAPTER VI SUMMARY AND CONCLUSION REFERENCES ix

10 LIST OF TABLES TABLE TITLE PAGE Table 1 Differential tests for the avian haemophili 13 Table 2 No. of slaughtered chicks for sample collection on different days 24 Table 3 Results of gross study 38 Table 4 Results of histopathological studies of group A (inoculated 38 with nutrient broth) Table 5 Results of histopathological studies of group B (inoculated 39 with A. paragallinarum) Table 6 Results of biochemical characteristics of A. paragallinarum 40 x

11 LIST OF FIGURES FIGURE TITLE PAGE Fig. 1 Intranasal inoculation of A. paragallinarum on day 14 of age 23 Fig. 2 Depression of chicks of group B( inoculation with A. paragallinarum ) on 42 day 7 of post inoculation Fig. 3 A chick of group B ( inoculation with A. paragallinarum ) with 42 conjunctivitis and mild facial edema on day 7 of post inoculation Fig. 4 Severely congested lung of a chicken of group B (inoculation with A. 42 paragallinarum ) with on day 7 of post inoculation. Fig. 5 Mild tracheal haemorrhage of a chicken of group B ( inoculation with A. paragallinarum ) on day 3 of post inoculation Fig. 6 Staphylococcus aureus produces golden yellow color colony on manitol 43 salt agar media. Fig. 7 Fermentation of glucose, sucrose, mannitol, maltose with production of 43 only acid and no galactose by A. paragallinarum. Fig. 8 A. paragallinarum showing gram negative rod shaped bacilli (Gram's 43 staining. x830) Fig. 9 A. paragallinarum produce smooth iridescent colonies with no hemolysis 43 on blood agar media Fig. 10 Nasal passage of group A on day 7 of post inoculation showing no lesion. 44 Fig. 11 Nasal passage of group B (inoculation with A. paragallinarum ) on day 7 of 44 post inoculation showing acanthosis. Fig. 12 Nasal passage of group B ( inoculation with A. paragallinarum ) on day 5 of 44 post inoculation showing presence of reactive leukocytes Fig. 13 Nasal passage of group B ( inoculation with A. paragallinarum ) on day 7 of 44 post inoculation showing congestion of blood vessels and acanthosis Fig. 14 Section of lung of a chicken of group A showing almost no lesion (±) 44 Fig. 15 Lung of group B (inoculation with A. paragallinarum ) on day 3 of post 44 inoculation showing mild pneumonic lesion. (+). Fig. 16 Lung of group B ( inoculation with A. paragallinarum ) on day 5 of post 45 inoculation showing moderate pneumonic lesion (++) Fig. 17 Lung of group B ( inoculation with A. paragallinarum ) on day 7 of post 45 inoculation showing severe pneumonic lesion (+++) Fig. 18 Fatty change, lipid nodules in macrophages and micronodules in heart on 45 day 5 of post inoculation Fig. 19 Fatty change, lipid nodules in macrophages and micronodules in heart on 45 day 7 of post inoculation Fig. 20 Liver of group A on day 7 of post inoculation 45 Fig. 21 Liver of group B ( inoculation with A. paragallinarum ) on day 7 of post inoculation showing focal hepatitis 45 xi

12 LIST OF ABBREVIATION AND SYMBOLS A. : Avibacterium Av. : Avibacterium BAU : Bangladesh Agricultural University BCRDV : Baby chick Ranikhet disease vaccine cm : Centimeter Co. : Company dl : Deciliter et al. : Associate etc. : Etcetera Fig. : Figure H. : Haemophilus IC : Infectious Coryza Kcal : Kilocalorie kg : Kilogram g : Gram Ltd. : Limited Max. : Maximum Min. : Minimum No. : Number PI : Post inoculation rpm : Rotation per minute µm : Micrometer ºC : Degree Celsius ± : Plus minus NAD : Nicotinamide adenine dinucleotide ELISA : Enzyme linked immunosorbent assy MR : Methyl red VP : Voges proskauer xii

13 CHAPTER I INTRODUCTION There is no denying fact that nowadays the poultry sub-sector is crucial in the context of agricultural growth and improvement of diet for the people in Bangladesh. This sub-sector is particularly important in the sense that it is a significant source for the supply of protein and nutrition in a household s nutritional intake. At present chicken contributes 51% of total meat production of the country (Raha, 2007). It is an attractive economic activity as well, especially to women and the poorer sections. Poultry farms in Bangladesh have witnessed a rapid growth in recent times. Poultry industry is an emerging agribusiness started practically during 1980s in Bangladesh (Huque, 2001). The poultry sector in Bangladesh is very important for the reduction of poverty and creation of employment opportunities. Many people are directly dependent on this industry for their livelihood. A total of 5 million people are engaged in this sector (Saleque, 2006). About 110,800 different size poultry farms have been established in the country (Anon, 2006). Per capita annual consumption of meat is 5.99 kg against the universal standard is 80 kg per head (Raha, 2007). Though there is satisfactory growth of poultry industry in Bangladesh it also faces some constraints. Among the constraints, emerging and reemerging diseases play a pivotal role for the development of this sector. Of all the emerging and reemerging diseases, there are lots of diseases that can affect the upper respiratory tact of chickens resulting low production of meat and egg. Among the respiratory diseases infectious coryza is an acute respiratory disease in chickens. The disease has worldwide economic recognition and causes infection in both broiler and layer flocks. The disease has a low mortality rate but leads to a drop in egg production of up to 40 % in layer hens and increased culling in broilers and thus poses significant financial liability to chicken farmers (Mouahid et al., 1989). The morbidity and mortality rate of broilers is 10-30% and 0.5 to 2%, respectively (Ibrahim et al., 2004). The disease is caused by gram negative bacteria Avibacterium 1

14 paragallinarum (previously called Haemophilus paragallinarum) which are classified into three serovars (A, B and C) using a slide agglutination test (Page, 1962). Clinically, the disease is characterized by rapid onset and high morbidity in flock, decreased of feed consumption, decreased egg production/growth, oculonasal conjunctivitis, edema of the face, respiratory noise, swollen infraorbital sinus, and exudates in the conjuncivital sac (Eaves et al., 1989). Respiratory sign of infectious coryza persists for a few weeks if complicated by Fowl pox, Mycoplasma gallisepticum, Newcastle Disease, Infectious Bronchitis, Pasteurellosis and Infectious laryngotracheitis (Yamamoto, 1972; Sandoval et al., 1994). So, certainly it has a huge negative impact in poultry industry. Clinically and grossly, the disease was diagnosed as infectious coryza in Bangladesh (Talha et al., 2001) but the causal agent was not identified. The confirmatory diagnosis of the disease in poultry of Bangladesh is inevitable. Pathological study (gross and microscopic) on the disease, isolation and identification of the causal agent; and study of pathogenesis by local isolate of this bacterium in experimental bird will enrich the knowledge to identify the disease rapidly and that will reflect the prevention and control measures of the disease. To my knowledge, experimental pathogenesis study by local isolate of the bacteria was not performed. Therefore, the present investigation was undertaken with the following objective: To study the pathogenesis of the disease in experimental chicks with local isolate of Avibacterium paragallinarum. 2

15 CHAPTER II REVIEW OF LITERATURE There is no doubt that infectious coryza (IC) caused by Avibacterium paragallinarum (formerly known as Haemophilus paragallinarum) has a great negative impact in poultry industry of Bangladesh. A few relevant published information on the experiment have been reviewed in the following paragraphs History As early as 1920, Beach believed that IC was a distinct clinical entity. The etiologic agent eluded identification for a number of years, because the disease was often masked in mixed infections and with fowl pox in particular. In 1932, De Blieck isolated the causative agent and named it Bacillus hemoglobinophilus coryzae gallinarum Economic Significance The greatest economic losses result from poor growth performance in growing birds and marked reduction (10-40%) in egg production. The disease can have significant impact in meat chickens. In California, two cases of infectious coryza, one complicated by the presence of Mycoplasma synoviae, caused increased condemnations, mainly due to airsacculitis, which varied from % (Droual et al., 1990). In Alabama, an infectious coryza outbreak in broilers, which was not complicated by any other disease agent, caused a condemnation rate of 69.8%, virtually all due to airsacculitis (Hoerr et al., 1994). When the disease occurs in chicken flocks in developing countries, the added presence of other pathogens and stress factors can result in disease outbreaks that are associated with greater economic losses than those reported in high health flocks in developed countries. In China, outbreaks of infectious coryza have been associated with morbidities of 20-50% and mortalities of 5-20% (Chen et al., 1993). In Morocco, outbreaks on 10 layer farms caused egg drops that ranged from 17-41% and mortalities of % (Mouahid et al., 1989). A study of village chickens in Thailand has shown that the most common 3

16 cause of death in chickens less than 2 months old and those more than six months old was infectious coryza (Thitisak et al., 1988). It was only in chickens that were between 2 and 6 months of age that other diseases, such as Newcastle disease and fowl cholera, killed more chickens than infectious coryza (Thitisak et al., 1988). Overall, considerable evidence shows that infectious coryza outbreaks can have a much greater impact in developing countries than in developed countries Public Health Significance The disease is limited primarily to chickens and has no public health significance Etiology Based on studies conducted during the 1930s, the causative agent of IC was classified as H. gallinarum because of its requirement for both X-(hemin) and V-(nicotinamide adenine dinucleotide NAD) factors for growth (Eliot et al., 1934; Schalm et al., 1936). Since 1962, however, Page, 1962 and others (Narita et al., 1978; Rimler, 1979; Hinz, 1980) have found that all isolates recovered from cases of IC required only the V-factor for growth. This led to the proposal and general acceptance of a new species, H. paragallinarum (Zinnemann, and Biberstein, 1974), for organisms requiring only the V-factor. H. gallinarum and H. paragallinarum are identical in all other growth characteristics and diseaseproducing potential (Rimler, 1979). These observations, in addition to the apparent abrupt change in the X-factor requirement of all isolates recovered worldwide since 1962, have led some workers to question the validity of tests used by earlier workers in classifying their isolates as H. gallinarum (Rimler, 1979). Indeed, it has been suggested that the early descriptions of the causative agent of IC as an X- and V-factor dependent organism were incorrect (Blackall, and Yamamoto, 1989). More recently, V-factor independent isolates of H. paragallinarum have been recovered from chickens with coryza in South Africa (Horner et al., 1992; Bragg et al., 1993). Thus, it is apparent that classification of hemophili based strictly on in vitro growth factor requirements may be misleading, as suggested by Kilian and Biberstein (Kilian et al., 1984). 4

17 2.5. Pathobiology and Epizootiology Incidence and Distribution Infectious coryza occurs whereever chickens are raised. The disease is a common problem in the intensive chicken industry; significant problems have been reported in California, southeastern United States, and most recently in the northeastern regions of the United States. The disease has also been reported in other, less intensive situations. As an example, infectious coryza has been a problem in kampung (village) chickens in Indonesia (Poernomo et al., 2000) Natural and Experimental Hosts The chicken is the natural host for H. paragallinarum. Several reports indicate that the village chickens of Asia are as susceptible to infectious coryza as normal commercial breeds (Zaini and Kanameda, 1991; Poernomo et al., 2000). Although there have been reports of IC due to H. paragallinarum in a number of bird species other than chickens, reviewed by Yamamoto (1991)), these reports need to be interpreted carefully. As a range of hemophilic organisms, none of which are H. paragallinarum, have been described in birds other than chickens (Grebe and Hinz. 1975; Piechulla et al., 1985; Devriese et al., 1988), only those studies that involve detailed bacteriology can be regarded as definitive proof of the presence of H. paragallinarum in birds other than chickens. The following species are refractory to experimental infection: turkey, pigeon, sparrow, duck, crow, rabbit, guinea pig, and mouse (Yamamoto, 1972; Yamamoto, 1978) Age of Host Most Commonly Affected All ages are susceptible (Yamamoto, 1991), but the disease is usually less severe in juvenile birds. The incubation period is shortened, and the course of the disease tends to be longer in mature birds, especially hens with active egg production Transmission, Carriers, and Vectors Chronic or healthy carrier birds have long been recognized as the main reservoir of infection. The application of molecular fingerprinting techniques has confirmed the 5

18 role of carrier birds in the spread of IC (Blackall et al., 1990). Infectious coryza seems to occur most frequently in fall and winter, although such seasonal patterns may be coincidental to management practices (e.g., introduction of susceptible replacement pullets onto farms where IC is present). On farms where multiple-age groups are brooded and raised, spread of the disease to successive age groups usually occurs within 1-6 weeks after such birds are moved from the brooder house to growing cages near older groups of infected birds (Clark and Godfrey, 1961). Infectious coryza is not an egg-transmitted disease. Whereas the sparrow could not be implicated as a vector, epidemiologic studies suggested that the organism may be introduced onto isolated ranches by the airborne route (Yamamoto, and Clark, 1966) Incubation Period The characteristic feature is a coryza of short incubation that develops within hours after inoculation of chickens with either culture or exudate. The latter will more consistently induce disease (Rimler, 1979). Susceptible birds exposed by contact to infected cases may show signs of the disease within hours. In the absence of a concurrent infection, IC usually runs its course within 2-3 weeks Pathogenicity As a general observation, the pathogenicity of H. paragallinarum can vary according to both the growth conditions and passage history of the isolate and the state of the host. Some specific evidence of variation in pathogenicity exists amongst H. paragallinarum isolates. Yamaguchi et al. (1990) found that one of four strains of H. paragallinarum serovar B failed to produce clinical signs. Horner et al. (1995) have suggested that the NAD-independent isolates may cause airsacculitis more commonly than the classic NAD-dependent H. paragallinarum isolates Virulence Factors A range of factors has been associated with the pathogenicity of H. paragallinarum. Considerable attention has been paid to HA antigens. In both Page serovar A and C, mutants lacking HA activity have been used to demonstrate that the HA antigen plays 6

19 a key role in colonization (Sawata and Kume, 1983; Yamaguchi et al., 1993). The capsule has also been associated with colonization and has been suggested to be the key factor in the lesions associated with IC (Sawata and Kume, 1983; Sawata et al., 1985). The capsule of H. paragallinarum has been shown to protect the organism against the bactericidal activity of normal chicken serum (Sawata et al., 1984). It has been suggested that a toxin released from capsular organisms during in vivo multiplication was responsible for the clinical disease (Kume et al., 1984). H. paragallinarum can acquire iron from chicken and turkey transferrin, suggesting that iron sequestration may not be an adequate host defense mechanism (Ogunnariwo et al., 1992). In contrast, two strains of H. avium were unable to acquire iron from these transferrins, despite apparently having the same receptor proteins (Ogunnariwo et al., 1992). Crude polysaccharide extracted from H. paragallinarum is toxic to chickens and may be responsible for the toxic signs that may follow the administration of bacterin (Iritani et al., 1981). The role, if any, of this component in the natural occurrence of the disease is unknown Clinical Signs The most prominent features are an acute inflammation of the upper respiratory tract including involvement of nasal passage and sinuses with a serous to mucoid nasal discharge, facial edema, and conjunctivitis facial edema. Swollen wattles may be evident, particularly in males. Rales may be heard in birds with infection of the lower respiratory tract. A swollen head-like syndrome associated with H. paragallinarum has been reported in broilers in the absence of avian pneumovirus, but in the presence or absence of other bacterial pathogens such as M. synoviae and M. gallisepticum (Droual et al., 1990; Sandoval et al., 1994). Arthritis and septicemia have been reported in broiler and layer flocks, respectively, in which the presence of other pathogens has contributed to the disease complex (Sandoval et al., 1994). Birds may have diarrhea, and feed and water consumption usually is decreased; in growing birds, this means an increased number of culls; and in laying flocks, this means a reduction in egg production (10 40%). A foul odor may be detected in flocks in which the disease has become chronic and complicated with other bacteria. 7

20 Morbidity and Mortality IC is usually characterized by low mortality and high morbidity. Variations in age and breed may influence the clinical picture (Blackall, 1983). Complicating factors such as poor housing, parasitism, and inadequate nutrition may add to severity and duration of the disease. When complicated with other diseases such as fowl pox, infectious bronchitis, laryngotracheitis, Mycoplasma gallisepticum infection, and pasteurellosis, IC is usually more severe and prolonged, with resulting increased mortality (Sandoval et al., 1994; Yamamoto, 1972) Pathology Gross H. paragallinarum produces an acute catarrhal inflammation of mucous membranes of nasal passages and sinuses. Frequently, a catarrhal conjunctivitis and subcutaneous edema of face and wattles occur. Typically, pneumonia and airsacculitis are rarely present; however, reports of outbreaks in broilers have indicated significant levels of condemnations (up to 69.8%) due to airsacculitis, even in the absence of any other recognized viral or bacterial pathogens (Droual et al., 1990; Hoerr et al., 1994). Microscopic Fujiwara and Konno (1965) studied the histopathologic response of chickens from 12 hours to 3 months after intranasal inoculation. Essential changes in the nasal cavity, infraorbital sinuses, and trachea consisted of sloughing, disintegration, hyperplasia of mucosal and glandular epithelia, and edema and hyperemia with heterophil infiltration in the tunica propria of the mucous membranes. Pathologic changes first observed at 20 hours reached maximum severity by 7-10 days, with subsequent repair occurring within days. In birds with involvement of the lower respiratory tract, acute catarrhal ronchopneumonia was observed, with heterophils and cell debris filling the lumen of secondary and tertiary bronchi; epithelial cells of air capillaries were swollen and showed hyperplasia. Catarrhal inflammation of air sacs was characterized by 8

21 swelling and hyperplasia of the cells, with abundant heterophil infiltration. In addition, a pronounced infiltration of mast cells was observed in the lamina propria of the mucous membrane of the nasal cavity (Sawata et al., 1985). The products of mast cells, heterophils, and macrophages may be responsible for the severe vascular changes and cell damage leading to coryza. A dissecting fibrinopurulent cellulitis similar to that seen in chronic fowl cholera has been reported in broiler and layer chickens (Droual et al., 1990) Immunity Chickens that have recovered from active infection possess varying degrees of immunity to reexposure. Pullets that have experienced IC during their growing period are generally protected against a later drop in egg production. Resistance to reexposure among individual birds may develop as early as 2 weeks after initial exposure by the intrasinus route (Sato and Shifrine, 1964). Experimentally infected chickens develop a crossserovar (Page scheme) immunity (Rimler and Davis, 1977). In contrast, as discussed earlier, bacterins provide only serovar-specific immunity (Blackall and Reid, 1987; Kume et al., 1980; Rimler et al., 1977). This suggests that cross-protective antigens are expressed in vivo that are either not expressed or expressed at very low levels in vitro. The protective antigens of H. paragallinarum have not been definitively identified. It has been suggested that the capsule of H. paragallinarum contains protective antigens (Sawata et al., 1984). Using both a Page serovar A and C strains, a crude polysaccharide extract was shown to provide serovar-specific protection (Iritani et al., 1981). Considerable attention has been paid to the role of HA antigens as protective antigens. It has been long noted that for Page serovar A organisms, a close correlation exists between HI titer and both protection (Otsuki and Iritani, 1974; Kume et al., 1980) and nasal clearance of the challenge organism (Kume et al., 1984) in vaccinated chickens. Purified HA antigen from a Page serovar A organism has been shown to be protective (Iritani et al., 1980). Takagi and colleagues have shown that a monoclonal antibody specific for the HA of Page serovar A provides passive tection and that the 9

22 HA antigen purified by use of this antibody is also protective (Takagi et al., 1991; Takagi et al., 1992). Based on studies conducted to date, considerable evidence shows that the protective antigens of H. paragallinarum are surface located. The antigens implicated have been the antigens detected during Page serotyping, HA antigens, and some component or components of the polysaccharide content of the cell. It seems probable that a number of different antigens (outer-membrane proteins, polysaccharides, lipopolysaccharides) are all likely to be involved Morphology and Staining H. paragallinarum is a gram-negative nonmotile bacterium. In 24-hour cultures, it ppears as short rods or coccobacilli 1-3 mm in length and mm in width, with a tendency for filament formation. A capsule may be demonstrated in virulent strains (Hinz, 1973; Sawata et al., 1980). The organism undergoes degeneration within hours, showing fragments and ill-defined forms. Subcultures to fresh medium at this stage will again yield the typical rodshaped morphology. Bacilli may occur singly, in pairs, or as short chains (Schalm and Beach, 1936) Growth Requirements The reduced form of NAD (NADH; µg/ml medium) (Page, 1962; Rimler et al., 1977) or its oxidized form ( µg/ml) (Sato and Shifrine, 1965) is necessary for the in vitro growth of most isolates of H. paragallinarum. The exceptions are the isolates described in South Africa, which are NAD independent (Horner et al., 1992; Mouahid et al., 1992; Bragg et al., 1993). Sodium chloride (NaCl) ( %) (Rimler et al., 1977) is essential for growth. Chicken serum (1%) is required by some strains (Hinz, 1973), whereas others merely show improved growth with this supplement (Blackall and Reid, 1982). Brain heart infusion, tryptose agar, and chicken-meat infusion are some basal media to which supplements are added (Hinz, 1973; Kume et al., 1980; Sato and Shifrine, 1965). More complex media are used to obtain dense growth of organisms for aracterization studies (Rimler, 1979; Blackall, 1983; Reid 10

23 and Blackall, 1987). The ph of various media varies from A number of bacterial species excrete V-factor that will support growth of H. aragallinarum (Page, 1962). The determination of the growth factor requirements of the avian haemophili is not an easy process. Commercial growth factor disks used for this purpose may yield a high percentage of cultures that falsely appear to be both X- and V-factor dependent (Blackall and Farrah, 1985). The brand of disks and the medium to be used should be checked are fully for their suitability. For well-equipped laboratories, the porphyrin test (Kilian, 1974) is recommended for X factor testing. For classical X- and V-factor testing, the use of purified hemin and NAD as supplements to otherwise complete media may also be considered. The organism is commonly grown in an atmosphere of 5% carbon dioxide; however, carbon dioxide is not an essential requirement, because the organism is able to grow under reduced oxygen tension or anaerobically (Eliot and Lewis, 1934; Page, 1962). The minimal and maximal temperatures of growth are 25 and 45 C, respectively, the optimal range being C. The organism is commonly grown at C Colony Morphology Tiny dewdrop, nonhemolytic colonies up to 0.3 mm in diameter develop on suitable media. In obliquely transmitted light, mucoid (smooth) iridescent, rough noniridescent, and other intermediate colony forms have been observed (Hinz, 1976; Rimler, 1979; Sawata et al., 1979; Sawata and Kume, 1983) Biochemical Properties The ability to reduce nitrate to nitrite and ferment glucose without the formation of gas is common to all the avian haemophili. Oxidase activity, the presence of the enzyme alkaline phosphatase, and a failure to produce indole or hydrolyse urea or gelatin are also uniform characteristics (Blackall, 1989). Considerable confusion surrounds the carbohydrate fermentation patterns of the avian haemophili. Much of the variability recorded in the literature may be due to the use of different basal media. 11

24 False-negative results are associated mainly with poor growth and can be a significant problem (Blackall, 1983). In general, recent studies have used a medium consisting of a phenol red broth containing 1% (w/v) NaCl, 25 µg/ml NADH, 1% (v/v) chicken serum, and 1% (w/v) carbohydrate. For routine identification, the use of the phenol red broth just described and a dense inoculum is a most suitable approach for determining carbohydrate fermentation patterns. Alternatively, agarbased methods (Blackall, 1983; Terzolo et al., 1993) may be used. A range of organisms that superficially resemble H. paragallinarum can be found in chickens. In particular, organisms once known as Haemophilus avium are common in chickens and are regarded as nonpathogenic (Hinz and Kunjara, 1977). Based on DNA hybridization studies, isolates of H. avium were found to be comprised of at least three DNA homology groups (Mutters et al., 1985). They have been named Pasteurella avium, P. volantium, and Pasteurella species A. Not all isolates of H. avium, however, can be assigned to these three new taxa solely on the basis of phenotypic properties (Blackall, 1988). Table 1. represents those properties that allow a full identification of the avian haemophili. The failure of H. paragallinarum to ferment either galactose or trehalose and its lack of catalase clearly separates this organism from the other avian haemophili. The properties shown in the table for H. paragallinarum have been found to be typical of isolates from Argentina, Australia, Brazil, China, Germany, Indonesia, Japan, Kenya, Malaysia, and the United States (Blackall et al., 1982; Blackall et al., 1994; Chen et al., 1993; Hinz and Kunjara, 1977; Kesler, 1997; Kume et al., 1978; Narita et al., 1978; Poernomo et al., 2000; Rimler, 1979; Terzolo et al., 1993, Zaini et al., 1991). The main characteristics that differentiate the NAD-independent from the NADdependent H. paragallinarum are that the former does not have ß-galactosidase activity and does not ferment maltose (Mouahid et al., 1992) Susceptibility to Chemical and Physical Agents H. paragallinarum is a delicate organism that is inactivated rather rapidly outside the host. Infectious exudate suspended in tap water is inactivated in 4 hours at ambient 12

25 temperature; when suspended in saline, the exudate is infectious for at least 24 hours at 22 C. Exudate or tissue remains infectious when held at 37 C for 24 hours and, on occasion, up to 48 hours; at 4 C, exudate remains infectious for several days. At temperatures of C, hemophili are killed within 2-10 minutes. Infectious embryonic fluids treated with 0.25% formalin are inactivated within 24 hours at 6 C, but the organism survives for several days under similar conditions when treated with thimerosal, 1:10,000 (Yamamoto, 1978). The organism may be maintained on blood agar plates by weekly passages. Table 1. Differential tests for the avian haemophili Property Hemophilus paragallinarium H. avium Pasteurella avium P. volantium Pasteurella species A Pigment _ Yellow V _ Yellow U _ Catalase _ Growth in _ air ONPG + V _ + V Acid from _ V + Arabinose Galactose _ Maltose + V _ + V Mannitol + V _ + V Sorbital V V _ V _ Sucrose V Trehalose _ Susceptibility to Chemical and Physical Agents U = usually; V = variable; + = positive; _ = negative. 13

26 Young cultures maintained in a candle jar will remain viable for 2 weeks at 4 C. Chicken embryos 6-7 days old may be inoculated with single colonies or broth cultures via the yolk sac; yolk from embryos dead in hours will contain a large number of organisms that may be frozen and stored at -20 to -70 C or lyophilized (Yamamoto, 1972.). A good suspension medium for lyophilization of H. paragallinarum from agar cultures is used at the Animal Research Institute and contains 6% sodium glutamate and 6% bacteriological peptone (filter sterilized). After any storage, whether frozen or lyophilized, revival should include inoculation of a suitable liquid growth medium (egg inoculation is even better) as well as an agar medium Strain Classification Antigenicity Page (1962) classified his organisms of H. paragallinarum with the plate agglutination test using whole cells and chicken antisera into serovars A, B, and C. Although Page s serovar A strain 0083 and B strain 0222 are available today, all the serovar C strains were lost during the mid-1960s. Matsumoto and Yamamoto (1975) isolated strain Modesto, which was later classified as a strain of serovar C by Rimler et al. (1977). It is also possible to use a hemagglutination inhibition (HI) test to serotype isolates by the Page scheme (Blackall et al., 1990). This HI test uses fixed chicken erythrocytes and results in fewer nontypable isolates than the original agglutination technology (Blackall et al., 1990) and is now the recommended technique when performing serotyping by the Page scheme. The distribution of Page serovars differs from country to country. Page serovar A has been reported in China (Chen et al., 1993) and Malaysia (Zaini and Iritani, 1992); serovar C in Taiwan (Lin et al., 1996); serovars A and B in Germany (Hinz, 1973); serovars A and C in Australia (Blackall et al., 1988); and serovars A, B and C in Argentina (Terzolo et al., 1993), Brazil (Blackall et al., 1994), Indonesia (Poernomo et al., 2000; Takagi et al., 1991), Mexico (Fernández et al., 2000), the Philippines (Nagaoka et al., 1994), South Africa (Bragg et al., 1996), Spain (Pages Mante and Costa Quintana, 1986), and the United States (Page, 1962; Page et al., 1963). 14

27 Another method of assigning isolates of H. paragallinarum to a Page serovar is based on the use of a panel of monoclonal antibodies developed by workers in Japan (Blackall et al., 1991), but the technique is available only in a few laboratories due to the limited availability of the monoclonal antibodies. Other sets of MABs have been described but either lack serovar-specificity (Bragg et al., 1997; Zhang et al., 2000) or detect only Page serovar A (Takagi et al., 1991). There have been suggestions that Page serovar B is not a true serovar, but rather consists of variants of serovar A or C that have lost their type-specific antigen (Kume et al., 1980; Sawata et al., 1980). Recent studies, however, have shown conclusively that Page serovar B is a true serovar (Yamaguchi et al., 1990). Kume et al. (1983) proposed an alternative serologic classification based on an HI test using potassium thiocyanate- treated and - sonicated cells, rabbit hyperimmune serums, and glutaraldehyde-fixed chicken erythrocytes. In the original study, Kume et al. (1983) recognized three serogroups and seven serovars. The terminology of the Kume scheme has been altered so that the Kume serogroups match the Page serovars of A, B, and C (Blackall et al., 1990). Thus, the nine currently recognized Kume serovars are A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4 (Blackall et al., 1990). Some Kume serovars seem to be unique in terms of geographic origin serovar A-3 has been found only in Brazil, serovar C-3 only in South Africa, and serovars A-4 and C-4 only in Australia (Blackall et al., 1990, Eaves et al., 1989; Kume et al., 1983). Many isolates that were nontypable in the Page scheme by agglutination tests were typed easily using the Kume scheme (Eaves et al., 1989). Fernández et al. (2000) have reported the presence of Kume serovars A-1, A-2, B-1, and C-2 in isolates of H. paragallinarum from Mexican chickens. The Kume scheme has not been widely applied, as it is technically demanding to perform. Hence, only a few laboratories are able to perform the serotyping on a routine basis. Other serological tests described in the literature include an agar-gel precipitin (AGP) test (Hinz, 1980) and a serum bactericidal test (Sawata et al., 1984). Neither of these tests has been widely used. 15

28 2.12. Immunogenicity or Protective Characteristics Infectious coryza is relatively unique among common bacterial infections in that a bacterin (inactivated whole cell vaccine) is protective against the disease when the bacterin is adequately prepared. From the early days of bacterin production, it was obvious that protection was limited (Matsumoto and Yamamoto, 1975). Later studies confirmed a correlation between Page serovars and immunotype specificity (Kume et al., 1980; Rimler et al., 1977). Chickens vaccinated with a bacterin prepared from one serovar were protected against homologous challenge only. Evidence suggests that the cross-protection within Page serovar B is only partial (Yamaguchi et al., 1991). Only incomplete results are available on immunospecificity within the serogroups recognized by the Kume scheme. Significant cross-protection has been shown between Kume serovars C-1 and C-2 as well as between C-2 and C-4 (Blackall and Reid, 1987; Kume et al., 1980). Only one serovar, B-1, exists within serogroup B of the Kume scheme. However, reports have been made of undefined heterogeneity within the B serogroup. Bivalent vaccines containing Page serovars A and C provide protection against Page serovar B strain Spross but not against two South African isolates of Page serovar B (Yamaguchi et al., 1991). Furthermore, only partial crossprotection exists within various strains of Page serovar B (Yamaguchi et al., 1991). Poor vaccine protection against IC due to serovar B strains in Argentina have been explained by antigenic differences between field isolates and the standard serovar B strains in commercial vaccines from North America or Europe (Terzolo et al., 1997). One report supports the genetic uniqueness of serovar B strains isolated in Argentina (Bowles et al., 1993). Vaccination/challenge exposure studies are needed to study the antigenicity and immunospecificity of recent serovar B isolates. In both Argentina and Brazil, isolates of Page serovar A are not recognized by a monoclonal antibody specific for this serovar (Blackall et al., 1994; Terzolo et al., 1993). It has been speculated that these variant Page serovar A isolates may be sufficiently different from typical serovar A vaccine strains to cause vaccine failure (Terzolo et al., 1993). South African workers have suggested that Kume serovar C-3 as well as other serovars of NAD-independent H. paragallinarum are so antigenically different that they are causing vaccine failure (Bragg et al., 1996; Bragg et al., 1997; Horner et al., 16

29 1995). However, it has been shown that a commercial vaccine, specified as containing serovars A, B, and C without details of the actual strains, provided acceptable levels of protection against NAD-independent isolates of Page serovar A and Kume serovar C-3 (Jacobs et al., 2000). Overall, these recent results and field observations clearly indicate the need for further vaccination/challenge studies. At this stage of our knowledge, no clear-cut definitive publications negate the existence of cross-protection within Page serovars and Kume serogroups. Indeed, the only publication to date, while not providing full details of the vaccine seed strains, suggests that serological variation within a Page serovar is not a cause of vaccine failure (Jacobs et al., 2000). There is no doubt that, on an ongoing basis, debate will continue on the topic of whether commercially available trivalent vaccines, containing serovars A, B, and C, give adequate protection if there are significant antigenic differences between vaccine and field strains Molecular Techniques DNA fingerprinting by restriction endonuclease analysis has been shown to be a suitable typing technique with patterns being stable in vitro and in vivo (Blackall et al., 1990; Blackall et al., 1991). Restriction endonuclease analysis has proven useful in epidemiologic studies (Blackall et al., 1990). Ribotyping is another molecular technique that has proven useful being used to confirm that the recent NADindependent H. paragallinarum isolates from South Africa are clonal in nature (Miflin et al., 1995) as well as examining the epidemiologic relationships among Chinese isolates of H. paragallinarum (Miflin et al., 1997). ERIC-PCR, a DNA fingerprinting method that uses the polymerase chain reaction technique, has been shown to be capable of strain typing (Khan et al., 1998). The technique of multilocus enzyme electrophoresis has been used to examine the genetic diversity of H. paragallinarum isolates (Bowles et al., 1993). These nucleic acid techniques (including the speciesspecific PCR discussed later in this chapter) are advancing to the stage where they offer a rapid and convenient method for identification and typing. These 17

30 techniques are likely to replace time-consuming and cumbersome cultural, biochemical, and serological means of identification and typing in the near future Diagnosis Isolation and Identification of Causative Agent Although H. paragallinarum is considered to be a fastidious organism, it is not difficult to isolate, requiring simple media and procedures. Specimens should be taken from two or three chickens in the acute stage of the disease (1 7 days incubation). The skin under the eyes is seared with a hot iron spatula, and an incision is made into the sinus cavity with sterile scissors. A sterile cotton swab is inserted deep into the sinus cavity where the organism is most often found in pure form. Tracheal and air sac exudates also may be taken on sterile swabs. The swab is streaked on a blood agar plate, which is then cross-streaked with a Staphylococcus culture and incubated at 37 C in a large screw-cap jar in which a candle is allowed to burn out. Staphylococcus epidermidis (Page, 1962) or S. hyicus (Blackall et al., 1982), which are commonly used as feeders, should be pretested because not all strains actively produce the V factor. Terzolo et al. (Terzolo et al., 1993) have reported the successful use of an isolation medium that contains selective agents which inhibit the growth of grampositive bacteria. This medium has the advantage of not using either a feeder organism or additives such as NADH. At the simplest level, IC may be diagnosed on the basis of a history of a rapidly spreading disease in which coryza is the main manifestation, combined with the isolation of a catalase-negative bacterium showing satellitic growth. At this level, the sinus exudate or culture should be inoculated into two or three normal chickens by the intrasinus route. The production of a coryza in hours is diagnostic; however, the incubation period may be delayed up to 1 week if only a few organisms are present in the inoculum, such as in long-standing cases. Better equipped laboratories should attempt more complete biochemical identification as described earlier. Additional studies of this nature are essential when isolates of NAD-independent H. paragallinarum are suspected. To perform this biochemical testing, the suspect 18

31 isolates are best grown in pure culture on medium that does not require the addition of a nurse colony. Many different media have been developed to support the growth of H. paragallinarum (Kume et al., 1980; Otsuki, and Iritani. 1974; Rimler, 1979; Terzolo et al., 1993). The medium described by Terzolo et al. (Terzolo et al., 1993) is particularly suited for those laboratories that find the cost of such ingredients as NADH and albumin expensive. The carbohydrate fermentation tests shown in Table 20.1 can be done in either a phenol red broth base (Rimler, 1979) or in an agar plate format (4). The agar plate method can be performed in conventional petri dishes (9 cm), allowing multiple isolates to be tested at once, or in small petri dishes (2 cm), allowing one to three isolates to be economically characterized. The agar plate method (4) has also been modified to be performed as a tube method (Terzolo et al., 1993). A PCR test specific for H. paragallinarum has been developed (Chen et al., 1996). This test is rapid (results available within 6 hours compared with days for conventional techniques) and has been shown to recognize all H. paragallinarum isolates tested, including the NAD-independent H. paragallinarum from South Africa and the variant Page serovar A isolates and the unusual Page serovar B isolates from Argentina (Chen et al., 1996). The PCR, termed the HP-2 PCR, has been validated for use on colonies on agar or on mucus obtained from squeezing the sinus of live birds (Chen et al., 1996). When used directly on sinus swabs obtained from artificially infected chickens in pen trials performed in Australia, the HP-2 PCR has been shown to be the equivalent of culture but much more rapid (Chen et al., 1996). When used in China, direct PCR examination of sinus swabs outperformed traditional culture when used on routine diagnostic submissions (Chen et al., 1998). The problems of poor samples, delayed transport, and poor quality (but expensive) media mean that culture will have a higher failure rate in developing countries than in developed countries making the PCR an attractive diagnostic option. The HP-2 PCR is a robust test; sinus swabs stored for up to 180 days at 4 C or _20 C were positive in the PCR (Chen et al., 1998). In contrast, culture of known positive swabs failed to detect H. paragallinarum after 3 days of storage at 4 C or _20 C (Chen et al., 1998). The HP-2 PCR has proven very useful in South Africa where the 19

32 diagnosis of infectious coryza is complicated by the presence of NAD-independent H. paragallinarum, Ornithobacterium rhinotracheale, as well as the traditional form of NAD-dependent H. paragallinarum (Miflin et al., 1999) Serology No totally suitable serological test exists for the diagnosis of infectious coryza. However, despite this absence of a perfect test, serological results are often useful for retrospective/epidemiological studies in the local area. A review of the techniques that have been used in the past is presented by Blackall et al. (Blackall et al., 1997). At this time, the best available test methodology is the HI test. Although a range of HI tests have been described, three main forms of HI tests have been recognized these being termed simple, extracted, and treated HI tests (Blackall and Yamamoto, 1998). Full details of how to perform these tests are available elsewhere (Blackall and Yamamoto, 1998). In the following text, the advantages and disadvantages of the three HI tests are briefly and critically discussed. The simple HI is based on whole bacterial cells of Page serovar A H. paragallinarum and fresh chicken erythrocytes (Iritani et al., 1977). Although simple to perform, this HI test can detect antibodies only to serovar A. The test has been widely used to both detect infected as well as vaccinated chickens (Blackall et al., 1997). The extracted HI test is based on KSCNextracted and sonicated cells of H. aragallinarum and glutaraldehydefixed chicken erythrocytes (Sawata et al., 1982). This extracted HI test has been validated mainly for the detection of antibodies to Page serovar C rganisms. The test has been shown to be capable of detecting a serovar-specific antibody response in Page serovar C vaccinated chickens (Sawata et al., 1982). A major weakness with this assay is that, in chickens infected with serovar C, the majority of the birds remain seronegative (Yamaguchi et al., 1988). The treated HI test is based on hyaluronidase-treated whole bacterial cells of H. paragallinarum and formaldehyde-fixed chicken erythrocytes (Yamaguchi et al., 1989). The treated HI has not been widely used or evaluated. It has been used to detect antibodies to Page serovars A, B, and C in vaccinated chickens with only serovar A 20

33 and C vaccinated chickens yielding high titers (Yamaguchi et al., 1991). The test has been used to screen chicken sera in Indonesia for antibodies arising from infection with serovars A and C (Takagi et al., 1991). Vaccinated chickens with titers of 1:5 or greater in the simple or extracted HI tests have been found to be protected against subsequent challenge (Sawata et al., 1982). Enough knowledge or experience is not yet available to draw any sound conclusions on whether there is a correlation between titer and protection for the treated HI test. An alternative serological test is a monoclonal antibody-based blocking ELISA, the B-ELISA (Zhang et al., 1999). While having shown very good specificity and acceptable levels of sensitivity, this test has several drawbacks. As there are only monoclonal antibodies for Page serovar A and C, the assay can detect only antibodies to these two serovars. The monoclonal antibodies that form the heart of the assays are not commercially available, limiting access to the assays. Finally, some isolates of H. paragallinarum do not react with the monoclonal antibodies and, thus, infections associated with these isolates cannot be detected with these ELISAs (Zhang et al., 1999). This ELISA has not been widely evaluated, and there is no knowledge about any correlation between ELISA titer and protection. The reduced sensitivity of the ELISA for serovar C infections indicates that the test would have to be used as a flock test only (Zhang et al., 1999). A B- ELISA kit based on the preceding B-ELISA has been developed (Miao et al., 2000). Based on pen trial data, the serovar A B-ELISA kit had a sensitivity of 95% and a specificity of 100%. The serovar C B-ELISA kit had a sensitivity of 73% and a specificity of 100% (Miao et al., 2000). Overall, the serological test of choice remains either the simple HI test (Iritani et al., 1977) for either infections or vaccinations associated with serovar A, the extracted or treated HI tests (Sawata et al., 1982; Yamaguchi et al., 1989) for vaccinations associated with serovar C, and the treated HI test (Yamaguchi et al., 1989) for infections associated with serovar C. There has been so little work performed on serological assays for infections or vaccinations associated with serovar B that it is not possible to recommend any test. 21

34 CHAPTER III MATERIALS AND METHODS The present research work was undertaken in The Department of Pathology, Bangladesh Agricultural University, Mymensingh during January to May 2012 to study the experimental pathogenesis in chicks by local isolate of Avibacterium paragallinarum Preparation of experimental house The experimental poultry house was properly cleaned, washed and then dried up. The room was fumigated with formaldehyde and with ammonia before introduction of chicks. The feeder, waterer and cages were cleaned with water and then fumigated with ammonia Experimental Chicks Twenty four, Cobb 500 day old broiler chicks were included in this study. The chicks were collected from Kazi Hatchery, Gazipur. Vaccination of chicks with BCRDV was performed on day Feed The broiler chicks were provided with commercial broiler starter and grower feed (Champion Starter and Champion Grower, Quality Feeds Ltd.) according to the age. The starter feed was given from day 1 to day 10 and the grower feed was given from day 11 up to the end of the experiment. The feed was stored in a dry place Experimental Pathogenesis Study Groupings Chicks (n = 24) at the age of day 14 were divided into 2 groups (group A and B) each consisting of 12 birds. Chicks were reared in separate cages. The group A was maintained as a control group without induction of infection. Birds of group B was maintained for bacterial inoculation. 22

35 Inoculation of Bacteria Chicks of group A were inoculated with 1 ml of 2 days old nutrient broth per bird at 14 days of age. Chicks of group B were inoculated at the age of 14 days with 2 days old culture broth of Avibacterium paragallinarum at the dose rate of 1 ml/bird (Islam, 2010) through intranasal route (Figure 1). The bacterial inoculum of A. paragallinarum was taken from Akter (2012) who isolated and identified the bacteria from field samples. Figure 1. Intranasal inoculation of A. paragallinarum on day 14 of age Post-mortem of chicks and sample collection On day 3 rd, 5 th and 7 th day of inoculation, four birds from each group were sacrificed for post-mortem examination and samples collection (Table 4.)Nasal swab was collected prior to post-mortem examination for isolation and identification head (nasal passage), trachea, lungs, intestine and liver were examined for any changes and kept in10% neutral buffered formalin for histopathological studies. The gross lesions of different organs were graded as: almost no lesions ( ±), mild lesions (+) and moderate lesions (++). 23

36 Table 2. No. of chicks slaughtered for sample collection on different days of postinoculation Days of post-inoculation Group A Group B Day Day Day Histopathology Nasal passage tissue, trachea, heart, lungs, and liver of different experimental inoculated chicks were selected for histopathological study. The formalin fixed tissues were trimmed, processed, sectioned and stained following standard procedure (Luna, 1968). Specific samples containing lesions from each group were used in histopathological study. The histopathological lesions of different organs of chicks were graded as: almost no lesions (±), mild lesions (+), moderate lesions (++), severe lesions (+++) Processing of tracheal tissue 1. The tissue samples were trimmed properly and fixed for 72 hrs with three changes of fixative. 2. To remove the fixative, the tissues were kept in running tap water for overnight after being fixed properly. 3. The tissues were dehydrated in grades of alcohol starting from 50%, 70%, 80%, 95% and in absolute alcohol, the tissues were changed at every 1hour interval. 4. The tissues were cleared by two changes in chloroform, one and half an hour for each. 5. The tissues were embedded with molten wax at 56 o C: 2 changes, one and half an hour for each. 6. Paraffin blocks containing tissue pieces were made using templates. 24

37 7. The tissues were sectioned with a rotary microtome at 5µm thickness. Then the sections were allowed to spread on hot water bath (45 0 C) 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 Processing of nasal passage tissue 1. The samples were placed in 10% buffered formalin in a volume 20 times that of the specimens for 7 days. The solutions were changed three times during this period. 2. After removing the fixative, the tissues were kept in running tap water for overnight after being fixed properly. 3. Samples were fixing in decalcifying solution until decalcification completed. The solution was changes daily for the first three weeks followed by changes every other day for the remaining period. 4. End point of decalcification was determined by specimen flexibility method. 5. The tissues were dehydrated in grades of alcohol starting from 50%, 70%,80%,95%, and 100%, each were 12 hours interval in two changes. 6. The tissues were cleared by three changes in chloroform, 4 hours for each. 7. The tissues were embedded with molten wax at 56 0 C: 4 changes, 8 hours for each. 8. Paraffin blocks containing tissue pieces were made using templates. 9. The tissues were sectioned with a rotary microtome at 5µm thickness. Then the sections were allowed to spread on warn water bath (45 0 C) and taken on oiland 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. 25

38 Preparation of decalcifying solution: Formic Acid-sodium Citrate method: Solution A Solution B Ingredients Amount Ingredients Amount Sodium citrate 50gm Formic acid (90%) l25ml Distilled water 250m1 Distilled water 125ml Solution A and Solution B were mixed in equal amount for use Preparation of stains Preparation of Harris Hematoxylin solution Ingredients Amount Hematoxylia crystals Alcohol, 100% Ammonium or potassium alum Distilled water Mercuric oxide (red) 5.0gm 50.0ml 100.0gm ml 2.5gm The Hematoxylin crystals were dissolved in the absolute alcohol and alum was added and dissolved in water and heated. The two solutions were removed from heat and thoroughly mixed and boiled as rapidly as possible. After removal from heat, mercuric oxide was added slowly. It was reheated until it became dark purple and removed from heat immediately and placed into a basin of cold water until cool. Just before using, 2-4ml of glacial acetic acid was added per 100 ml of solution to increase the precision of the nuclear stain. Before using the prepared solution was filtered Preparation of eosin solution a) 1% Stock alcoholic eosin Ingredients Eosin Y, water soluble Distilled water Dissolved and add alcohol, 95% Amount 1.0gm 20.0ml 80.0ml 26

39 b) Working eosin solution Ingredients Eosin stock solution Alcohol, 80% Amount 1 part 3 parts Just before use 0.5 ml of glacial acetic acid was added to each 100 ml of stain and stirred Routine Hematoxyrlin eosin staining procedure 1. The sectioned tissues were deparaffinized in 3 changes of xylene (3 minutes in each) 2. Then the tissue sections were rehydrated through descending grades of alcohol. (3 changes in absolute alcohol, 3 minutes in each; 95% alcohol for 2 minutes; 80% alcohol for 2 minutes; 70% alcohol for 2 minutes) followed by tap water for 5 minutes. 3. The tissues (trachea) were stained with Harris Hematoxylin for 15 minutes and the tissues (nasal sinus) were stained with Harris Hematoxylin for 1 hours. 4. Washed in running tap water for 15 minutes. 5. After washing the tissues were differentiated in acid alcohol: 2 to 4 dips (l part HCI and99 parts 70% alcohol). 6. Then washed in tap water for 5 minutes followed by two to four dips in ammonia water until sections were bright blue. 7. Stained with eosin I minute (tracheal tissue) and l0 seconds ( nasal sinuses) tissue to visualize cytoplasmic componant. 8. Differentiated and dehydrated in alcohol: 95% alcohol: 3 changes, 3 dips each; absolute alcohol: 3 changes 3 minutes for each. 9. Cleaned in xylene: 3 changes (5 minutes for each). 10. Finally the sections were mounted with coverslip using DPX. 27

40 Photomicrography Photornicrography was taken at the Department of pathology using photomicrographic camera (olympus pm-c 35 Model) onto fitted with Olympus microscope (Olympus, Japan) Reisolation of Avibacterium paragallinarum in Bacteriological Media 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 Nutrient broth Nutrient broth was prepared by dissolving 13 grams of dehydrated nutrient broth (HiMedia, India) into 1000 ml of distilled water and was sterilized by autoclaving at 121 C under 15 lb pressure per square inch for 15 minutes. Then the broth was dispensed into tubes (10 ml/tube) and was incubated at 37 C for over night to cheek their sterility and stored at 4 C in the refrigerator until used. Ingredients (g/l) Peptone 5.0 Sodium chloride 5.0 Beef extract 1.5 Yeast extract 1.5 Final Ph (at 25 0 C) 7.4± Nutrient agar 2.3 gms of Bacto-NA (Difco) was suspended in 100 ml cold distilled water taken in a conical flask and heated to boiling to dissolved the medium completely. After sterilization by autoclaving, the medium was poured in 10 ml quantities in sterile glass petridishes (medium sized) and in 15 ml quantities in sterile glass petridishes (large 28

41 sized) to form a thick layer therein. To accomplish the surface be quite dry, the medium was allowed to solidify for about 2 hours with the covers of the petridishes partially removed. The sterility of the medium was judged by incubating overnight at 37 C and used for cultural characterization or stored at 4 C in refrigerator future use (Carter, 1979). Ingredients (g/l) Peptic digest of animal tissue 5.0 Sodium chloride 5.0 Beef extract 1.5 Yeast extract 1.5 Agar 15.0 Final Ph (at 25 0 C) 7.4± Blood agar Forty grams of blood agar base (HiMedia, India) was suspended in 1000 ml of distilled water and heated for boiling to dissolve completely. The base was then autoclaved and cooled at 50 C using water bath. Then sheep blood collected aseptically was added at the rate of 5-7% of base. The medium was then poured in 20 ml quantities in to 15 X 100 mm petridishes and allowed to solidify. After solidification of the medium in the plates, the plates were allowed for incubation at 37 C for over night to cheek their sterility. Ingredients (g/l) Aager 15.0 Peptone 10.0 Sodium chloride 5.0 Beef extract 10.0 Final Ph (at 25 0 C) 7.3±0.2 29

42 Isolation and identification of organisms Isolation and identification of Staphylococcus aureus Primary culture of Staphylococcus aureus Primary growth of all kinds of bacteria was performed in nutrient broth. 24 nasal swabs samples from live birds were collected with sterile cotton bud by gentle touch, and then inoculated into the nutrient broth, incubated over night at 37 C to obtain the primary culture Isolation of Staphylococcus aureus in pure culture After primary culture of the organism, a small amount of inoculum from nutrient broth was streaked onto mannitol salt agar showing characteristics morphology of Staphylococcus aureus were selected for subculture on nutrient agar, blood agar Isolation and identification of Avibacterium paragallinarum Primary culture of A. paragallinarum Twenty four nasal swabs samples from live birds were collected with sterile cotton bud by gentle touch, and then inoculated into the nutrient broth, incubated over night at 37 C to obtain the primary culture Isolation of A. paragallinarum in pure culture After primary culture of the organism, a small amount of inoculum from nutrient broth was streaked onto blood agar showing characteristics morphology of A. paragallinarum were selected for subculture. Tiny dewdrop colonies developed on blood agar media was considered positive for A. paragallinarum Study of colony morphology for identification The colony morphology of the isolates was studied as mentioned by Merchant and Packer (1967). Morphological characteristics (shape, size, surface texture, edge, elevation, color, opacity etc.) developed after 24hours of incubation were carefully studied and recorded. 30

43 Staining Preparation of Gram staining solution Crystal violet solution Stock crystal violet solution Ingredients Crystal violet Ethyl alcohol Stock oxalate solution Ingredients Ammonium oxalate Distilled water Working crystal violet solution Ingredients Stock crystal violet solution Stock oxalate solution Amount 10gm 1000ml Amount 1gm 1000ml Amount 20ml 80ml It was mixed and prepared when required. Lugol s iodine solution Ingredients Iodine crystal Potassiun iodide Amount 1gm 2gm These two were dissolved completely in 10 ml of distilled water and then distilled water was added to make 300 ml and store in amber bottle. Acetone alcohol Ingredients Ethyl alcohol Acetone Amount 250ml 250ml 31

44 Safranin (counterstain) solution Safranin stock solution Ingredients Safranin Ethyl alcohol (95%) Safranin working solution Amount 2.5ml 100ml The stock safranin was diluted 1:4 with distilled water Microscopic study of the suspected colonies Gram s staining was performed to determine the shape, arrangement and Gram reaction of the isolates as described by Merchant and Packer (1967). The procedure was as follows: 1. A small colony was picked up with a bacteriological loop, a drop of distilled water added then mixed and smeared on a glass slide and fixed by gentle heating. 2. Ammonium oxalate crystal violet was added on to the smear and allowed to react for ½ min. 3. Washed with running water. 4. Lugol s iodine was then added to act as mordant for one minute and then again washed with running water. 5. Acetone alcohol was then added, which act as a decolourizer, for 3-5 seconds. 6. Washed thoroughly in water. 7. After washing with water, safranine was added as counter stain and allowed to stain for two minutes. 8. The slide was then washed with water, blotted and dried in air and then examined under microscope with high power objectives (100X) using immersion oil Biochemical studies for the identification of organisms Several biochemical tests were performed for confirmation of the isolates. 32

45 Reagents for biochemical test i. Methyl Red and Voges-Proskauer broth (MR-VP broth) (Difco, USA) ii. Peptone water iii. Phosphate buffer solution Sugars i. Dextrose (LOBA Chemic Pvt. Ltd., India) ii. Sucrose (Wako, Japan) iii. Lactose (Merc, England) iv. Maltose (Techno Pharma., India) v. Manitol (Beximco Pharma., Germany) vi. Galactose (LOBA Chemic Pvt. Ltd., India) Carbohydrate fermentation test Preparation of Carbohydrate fermentation test reagents Bacteriological peptone Ingredients Bacteriological peptone Sodium chloride Distilled water Amount 10gm 5gm 1000ml Phenol red (0.2%) Ingredients Amount Phenol red 2gm Distilled water 1000ml Sugar (10%) Five basic sugar as dextrose, sucrose, maltose and mannitol were used for suger fermentation test. Ingredients Amount Specific sugar 1gm Distilled water 10ml 33

46 Preparation of sugar media and carbohydrate fermentation tests The medium consists of peptone water to which fermentable sugar was added to the proportion of 1 percent. Peptone water was prepared by adding one gram of Bacto peptone (Difco, USA) and 0.5 grams of sodium chloride in 100 m1 distilled water. The medium was boiled for 5 minutes, adjusted to ph 7.0, cooled and then filtered through filter paper: Phenol red, an indicator at the strength of 0.2 percent solution was added to peptone water and then dispensed in 5 m1 amount into cotton plugged test tubes containing a Durham's fermentation tubes, placed inversely. These were then sterilized in the autoclave machine. The sugars used for fermentation were prepared separately as 10 percent solutions in distilled water (10 grams sugar was dissolved in 100 ml of distilled water). A little heat was necessary to dissolve the sugar completely. The sugar solutions were sterilized in Arnold steam sterilizer at 100 C for 30 minutes for three successive days. An amount of 0.5 ml of sterile sugar solution was added aseptically in each culture tubes containing sterile peptone water and indicator. Before use, the sterility of the sugar media was examined by incubating it for 24 hours at 37 C. The carbohydrate fermentation test was performed by inoculating a loop full of nutrient broth culture of the organisms into the tubes containing five basic sugars e.g., galactose, maltose, sucrose, and mannitol, glucose and incubated for 24 hours at 37 C. Acid production was indicated by the color change reddish to yellow in the medium and presence of no of gas bubbles in the inverted Durham's tubes indicate no gas production. 34

47 Indole test Two ml of peptone water was inoculated with 5 ml of bacterial culture and incubated for 48 hours. 0.5 ml of Kovac s reagent was added, shaked well and examined after 1 minute, no development of red color. In positive case there is a red color in the reagent layer indicate indole Methyl-Red & Voges-Proskauer (MR-VP) test Composition of MR-VP medium (DIFCO Laboratories, USA) Ingredients Amount Buffered peptone 7.0gm Dextrose 5.0gm Dipotassium phosphate 5.0gm A quantity of 3.4 gm of Bacto MR-VP medium was dissolved in 250 m1 of distilled water dispensed in 2 ml amount in each test tube and then the tubes were autoclaved. After autoclaving, the tubes containing medium were incubated at 37 C for overnight to check their sterility and then stored in a refrigerator for future use. Two milliliters of sterile glucose phosphate peptone water were inoculated with the 5 ml of test organisms. It was inculated at 37 0 C for 48 hours. A very small amount (knife point) of creatine was added and mixed. Three milliliters of sodium hydroxide were added and shaked well. The bottle cap was removed and left for an hour at room temperature. It was observed closely for no development of pink color. In positive cases there was the slow development of a pink color. Methyl Red solution Ingredients Amount Methyl red 0.05gm Ethanol (absolute) 28ml Distilled water 22ml The indicator phenyl red solution was prepared by dissolving 0.1 gm of Bacto methylred in 300 ml of 95 percent alcohol and diluting to 500 ml with the addition of 200 ml of distilled water. 35

48 The test was performed by inoculating a colony of the test organism in 0.5 ml sterile glucose phosphate broth (as used in the VP test). After overnight incubation at 37 C, a drop of methyl red solution was added. A negative methyl red test was shown by a yellow or orange color. A positive test shown by the appearance of bright red color indicated the acidity Enzyme activity test Catalase test The organism was grown on a slope of nutrient agar or other suitable medium. One ml 3% H 2 O 2 was run down the slope and examined immediately and after 5 min for evaluation of gas. 36

49 CHAPTER IV RESULTS The present investigation was taken to study the pathogenesis of Avibacterium paragallinarum, the causal agent of IC in chicks. The isolation and identification of the causal agent from layer chickens was performed by Akter (2012). However, reisolation and identification of Avibacterium paragallinarum in experimental chicks was included in investigation Clinical signs of chickens Chicks of group A (inoculated with pure nutrient broth) did not show any remarkable clinical signs up to the end of the experimental period. However, chicks of group B (inoculated with A. paragallinarum) showed mild nasal discharge, conjunctivitis, depression and inability to move Gross study The results of gross study have been presented in Table 5 and Figures 2-5. Chicks of group A did not reveal any lesion related to the IC on day 3, 5 and 7. On the other hand, chicks of group B reveal lesions in different organs following bacterial inoculation which were progressively massive (Table 5). 37

50 Table 3. Results of gross study Groups Day after inoculation Gross lesions related to IC Group A (inoculated with pure nutrient broth) Group B (inoculated with A. paragallinarum) 3 ± 5 ± 7 ± 3 Mucus in nasal passage (+) 5 Mucus in nasal passage (+) Mild tracheal hemorrhage (+) (Fig. 5) 7 Mucus in nasal passage(+) Conjunctivitis (+) (Fig. 3) Swelling of sinuses and face(+) Congested lungs(++) (Fig. 4) ± = almost no lesions, + = mild lesions, ++ = moderate lesions Histopathological study The microscopic lesions have been presented in Tables 6 and 7 and illustrated in Figure Table 4. Results of histopathological studies of group A (inoculated with nutrient broth) Day of inoculation Nasal passage Lung Liver Heart Day 3 ± ± ± ± Day 5 ± + ± ± Day 7 ± + ± ± ± = almost no lesions, + = mild lesions 38

51 Table 5. Results of histopathological studies of group B (Inoculated with A. paragallinarum) Day of inoculation Nasal passage Lung Liver Heart Day 3 Acanthosis of nasal epithelium,congested blood vessel, hyperplasia of mucous gland (+) Mild pneumonic lesions (+) Lymphocytic infiltration(+) Fatty change and lipid nodule in macrophage (+) Day 5 Acanthosis of nasal epithelium,congested blood vessel, hyperplasia of mucous gland (+) Moderate pneumonic lesions (++) Lymphocytic infiltration (+) Fatty change and lipid nodule in macrophage (+) Day 7 Acanthosis of nasal stratified epithelium (Fig. 9), congested blood vessel (Fig. 9), hyperplasia of serous gland and necrotic tissue debris found in serous gland, presence of inflammatory cells (heterophils and lymphocytes) (++) Severe pneumonia(+ ++) (Fig. 15) Focal hepatitis(++) (Fig. 20) Fatty change and lipid nodule in macrophage (++) + = mild lesions, ++ = moderate lesions, +++ = severe lesions Reisolation of Avibacterium paragallinarum on day 7 Reisolation was performed only in tissues showing pathological lesions i.e. found on day 7 of post inoculation (PI). Only 4 samples were processed for reisolation. Reisolation procedures of Avibacterium paragallinarum have been illustrated in Figures Results of Gram's stain Tentatively diagnosed all samples from blood agar media were stained with Gram s stain. All of the suspected samples showed Gram- negative, red color, rod shaped bacilli arranged as single or paired (Fig. 8). 39

52 Results of biochemical tests Biochemical tests were performed from tentatively diagnosed 4 samples from blood agar media, a series of biochemical tests especially selective for A. paragallinarum were performed with the positive culture and Gram-negative rod shaped bacteria. The results are furnished below: Results of sugar fermentation test Four isolates fermented four basic sugars (maltose, sucrose, and mannitol, glucose) and produced acid and did not ferment galactose. Acid production was indicated by the color change from reddish to yellow (Table 8, Fig. 7) Results of other biochemical tests Four isolates were then subjected to different biochemical tests such as methyl-red test, VP test and Indole test. All the isolates were methyl-red negative; VP test negative and Indole test negative (Table 8). Isolates revealed the following pattern of biochemical reactions were regarded as A. paragallinarum. Table 6. Results of biochemical characteristics of A. paragallinarum Different Biochemical test Sample size Result Identification of bacteria Fermentation reaction with five basic sugars a. Glucose + A. paragallinarum b. Sucrose + A. paragallinarum c. Galactose 4 _ A. paragallinarum d. Maltose + A. paragallinarum e. Mannitol + A. paragallinarum Other biochemical test Indole _ A. paragallinarum MR 4 _ A. paragallinarum VP _ A. paragallinarum + = Positive; - = Negative; MR = Methyl red; VP = Voges proskauer 40

53 4.5. Enzymatic activity test Catalase activity test Twelve isolates from blood agar media were subjected to catalase test. All the isolates showed negative test (i.e. production of no buble) indicating A. paragallinarum. In summary, the probable experimental pathogenesis might be started with inoculation of A. paragallinarum through nasal passage and rhinitis was produced following reached to the various visceral organs via blood and finally showed lesions. The lesions that found in this experiment (rhinitis in association with focal hepatitis, progressive pneumonic lesions, fatty change in heart with lipid granuloma) are not normally present in adult and young chicks. Lesion was not found in control group but time dependently intensity of lesions was found in different organs in experimental inoculated group (inoculated with A. paragallinarum). This may be a latest finding of this disease. However, further investigation is needed. 41

54 Conjunctivitis Mild facial edema Figure 2. Depression of chicks of group B (Inoculated with A. paragallinarum) on day 7 of post inoculation. Figure 3. A chick of group B (Inoculated with A. paragallinarum) with conjunctivitis and mild facial edema on day 7 of post inoculation. Figure 4. Severely congested lung of a chicken of group B (Inoculated with A. paragallinarum) with on day 7 of post inoculation. Figure 5. Mild tracheal haemorrhage of a chicken of group B (Inoculated with A. paragallinarum) on day 3 of post inoculation. 42

55 Glac Glu Su Ml Mn Cont. Figure 6. Staphylococcus aureus produces golden yellow color colony on mannitol salt agar media. Figure 7. Fermentation of glucose, sucrose, mannitol, maltose with production of only acid and no galactose by A. paragallinarum. Figure 8. A. paragallinarum showing gram negative rod shaped bacilli ( Gram's staining. x830) Figure 9. A. paragallinarum produce smooth iridescent colonies with no hemolysis on blood agar media. 43

56 Figure 10. Nasal passage of group A (inoculated with nutrient broth) on day 7 of post inoculation showing no lesion. Figure 11. Nasal passage of group B (Inoculated with A. paragallinarum) on day 7 of post inoculation showing acanthosis (arrow). Figure 12. Nasal passage of group B (Inoculated with A. paragallinarum) on day 5 of post inoculation showing presence of reactive leukocytes (arrow). Figure 13. Nasal passage of group B (Inoculated with A. paragallinarum) on day 7 of post inoculation showing congestion of blood vessels (arrow) and acanthosis. Figure Figure Section Section of of lung lung of of a chicken a chicken of group A showing almost no of group A showing almost no lesion lesion (±). Figure 15. Lung of group B (Inoculated with A. paragallinarum) on day 3 of post inoculation showing mild pneumonic lesion. (+). 44

57 Figure 16. Lung of group B (Inoculated with A. paragallinarum) on day 5 of post inoculation showing moderate pneumonic lesion (++). Figure 17. Lung of of group group B on B day (Inoculated 7 of with post inoculation A. paragallinarum) showing severe on day 7 of post inoculation showing severe pneumonic lesion (+++). Figure 18. Fatty change, lipid nodules in macrophages and micronodules in heart on day 5 of post inoculation. Figure 19. Fatty change, lipid nodules in macrophages and micronodules in heart on day 7 of post inoculation. Figure 20. Liver of group A (inoculated with nutrient broth) on day 7 of post inoculation. Figure 21. Liver of group B (Inoculated with A. paragallinarum) on day 7 of post inoculation showing focal hepatitis. 45