isolation and pathogenic characterizations of ibdv isolate...

Isolation and pathogenic characterizations of IBDV isolate from an outbreak of IBD in a rural poultry unit in Bangladesh

M.Sc. thesis Abdul Ahad 1,2

1Department of Veterinary Microbiology and Network for Smallholder Poultry Devel-opmnet The Royal Veterinary and Agricultural University, Dyrlægevej 2, 1870 Frederiks-berg C., Denmark and 2 Sylhet Govt. Veterinary College, Tilagorh Sylhet-3100, Bangla-

desh.

Supervisors

Jens Peter Christensen, Associate Professor, Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Stigbøjlen 4, DK-1870 Fredriksberg C, Co-

penhagen.

Md. Rafiqul Islam, Professor, Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Mymensingh, Bangladesh.

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Summary Infectious bursal disease virus (IBDV), also popularly known as Gumboro disease is a conta gious disease of young chickens caused by a dsRNA virus belonging to the family Birnaviridae. It is an economically important pathogen of chickens with worldwide distribution. There are two distinct serotypes of IBDV. Virus strains belonging to serotype 1 are pathogenic, while serotype 2 viruses are avirulent for chickens. Clinical IBD is most commonly recognized in susceptible 3 to 6 week old chickens. The aim of this work was to describe outbreaks of IBD including different breeds of birds in a ru-ral area and in an urban area of Bangladesh and isolation of the field virus. Furthermore to evaluate the virulence of a local isolate of vvIBDV in vaccinated and unvaccinated local Sonali chickens against IBDV. Outbreaks of infectious bursal disease (IBD) in one chick shed at Mirpur Central Poultry Farm, Dhaka (urban area) and six chick rearing units at Madarganj, Jamalpur (rural area) were investi-gated during the period from July to October 2001. The number of birds in the shed was ap-proximately 1000-2000 (urban area) and 400-500 in the rural units. The age groups of the birds ranged from day old to two months. Detailed particulars of the outbreaks of IBD including history, age, breed of affected chicks, flock size, mortality and clinical signs were recorded. A local iso-late of vvIBDV from a recorded outbreak with the titre of 103.15 EID50 per 100µl was used for challenge experimental infection. Within the study period seven outbreaks were recorded. The highest and lowest mortality re-corded was 85% and 13% respectively. The birds were affected at the age between 20 and 54 days. Mixed infection and dehydration was found in the urban farm while helminth infection in the rural units. Highest and lowest morbidity and mortality recorded was 27% and 18% and 11% and 3% respectively after experimental infection in different groups of birds. It was found that 14 and 24 days after 2nd vaccination against IBD at day 35 and at day 45 there was a significant difference in antibody titre in unvaccinated and vaccinated groups of birds against IBD. In this study it was found that mortality was much higher in small flocks than in big flocks. More-over, it was found that the outbreaks in the small flocks almost affects all birds simultaneously. This might be due to only two persons giving the technical assistance and visiting all chick units and very poor bio-security management practice. Grouping of diseases on the basis of pathol-ogy revealed that dehydration was a common cause of death and it was only found in Govt. farm. However there was no helminth infection found in the urban unit but it was found in the ru-ral unit. This might be due to managemental problem. In Govt. farm helminth infection was not found because of regular deworming. In the rural units helminths poses a problem because de-worming does not take place. In addition the infection builds up in the village environment. Air-way infection was found also in the Govt. farm but not in the rural unit. This could be due to im-proper ventilation in the farm. It is clear from the present findings that IBD is prevalent in different production systems of poul-try in Bangladesh. It is one of the major poultry viral diseases causing very high mortality in chickens. In experimental infections the virus cause lower mortality than natural outbreaks sug-gesting that good biosecurity coupled with vaccination can prevent mortality in a great extent. However, the vaccine against IBDV produced from the Intervet company did not give full protec-tion to the challenged isolate. There was no significant difference in antibody titre until 7 days after 1st vaccination in unvaccinated birds compared to vaccinated birds but significant differ-ence in antibody titre was found 14 days and 24 days after 2nd vaccination of IBDV.

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It is highly recommended in order to control IBD in a rural environment biosecurity must be im-proved and training of the technical personnel should be provided.

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Acknowledgements The experimental work on which this M.Sc. thesis is based, was carried out at the Department of Pathology, Faculty of Veterinary Science at Bangladesh Agricultural University in Mymesingh, Bangladesh from July 2001 to April 2002. The study was financed by a grant from the Danish International Development Agency (DANIDA). I am indebted to a number of people for their help in the preparation of this thesis. First and foremost I wish to thank my supervisor Jens Peter Christensen for his guidance in designing the experiment, critically reviewing my articles and constant encouragements. I am also grateful to my supervisor Md. Rafiqul Islam for his constant supervision in my experimental work and in photomicrography. In addition I am very grateful to all people at the department of Pathology in-cluding teachers and staff for creating a congenial atmosphere to do my experimental work smoothly. Especially I would like to thank Jens Chrisitian Riise at the Network for Smallholder Poultry De-velopment for his technical support in handling of computers. Finally, I wish to thank my parents and my family for making the M.Sc. process possible and en-joyable. Abdul Ahad July 2002

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Table of contents Summary...................................................................................................................................... 1 Acknowledgements...................................................................................................................... 3 Table of contents ......................................................................................................................... 4

Chapter 1 ......................................................................................................................................... 6 Introduction .................................................................................................................................. 6

Chapter 2 ......................................................................................................................................... 7 Literature study ............................................................................................................................ 7

Chapter 3 ....................................................................................................................................... 14 Materials and methods .............................................................................................................. 14

Chapter 4 ....................................................................................................................................... 23 Results ....................................................................................................................................... 23 Economic analysis ..................................................................................................................... 30

Chapter 5 ....................................................................................................................................... 32 Discussion.................................................................................................................................. 32 Conclusion and future perspectives .......................................................................................... 34

Chapter 6 ....................................................................................................................................... 35 References................................................................................................................................. 35

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Chapter 1

Introduction Livestock plays an important role in the agricultural economy of Bangladesh. The magnitude of the contribution of the livestock sub-sector to the countrys gross domestic product (GDP) is 3.1 percent and to agricultural GDP it is about 11 per cent. Data generated by the Department of Livestock Service shows that the loss of livestock due to various diseases resulted in losses of approximately 140000 million taka per year (1$=58 taka) in the mid eighties (Hassan, 1985). Losses were calculated as 25% and 60% of the total value of livestock and poultry. The loss from livestock diseases in Bangladesh could be estimated between Taka 20.70 and 49.46 bil-lion. Since the country does not have an effective disease control program, the loss from dis-eases may be counted on the higher side (Nakamura, 1990). In 1997-98 the small-scale family poultry system in Bangladesh has been estimated to account for about 80% of the total poultry population (Huque, 1999). It is one of the most important income-generating activities for rural women, landless poor and marginal farmers. Bangladesh has a large potential to increase meat and egg production through improvement of indigenous practices in extensive family poultry production system. Infectious Bursal Disease (IBD) is a highly contagious, globally occurring viral poultry disease. The disease was first reported by Cosgrove, who in 1962 observed a disease, affecting chickens on farms in the neighborhood of Gumboro, Delware, USA (Cosgrove, 1962). Thus, Gumboro disease became synonymous for the condition. The virus causing IBD suppresses the immune system of affected birds by damaging organs of primarily the humoral cell defense, particulary Bursa Fabricii, which is why IBD has become the alternative name for the disease. During the 63rd General Session of the Office International des Epizooties (OIE, 1995), it was estimated that IBD has considerable socio-economic importance at the international level, as the disease is present in more than 95% of the Member Countries (Eterradossi, 1995). In this sur-vey, 80% of the countries reported the occurrence of acute clinical cases. The domesticated hen (Gallus gallus) is the only species for which IBD virus has been reported to induce clinical disease. However, serological surveys in wild birds (Wilcox et al., 1983; Gard-ner et al., 1997; Ogawa et al., 1998b) suggest their role as a reservoir. There is no indication that IBD virus infects humans. Infectious bursal disease virus (IBDV) is a non-enveloped icosahedral virus, approximately 58- 60 nm (Hirai and Shimakura, 1974) in diameter that is endemic in most poultry producing areas of the world. The virus is highly stable and has a tendency to persist in the environment despite thorough cleaning and disinfections. The virus can remain viable for up to 60 days in poultry house litter (Vindevogel et al., 1976). Landgraf et al. (1967) demonstrated that a bursal suspen-sion in nutrient broth retained viability after heating to 600 C for 30 minutes. There are two sero-types of IBDV: serotype 1 and 2. All viruses capable of causing disease in chickens belong to serotype1; serotype 2 viruses may infect chickens and turkeys and are considered non-pathogenic for both species. Viruses of both serotypes of IBDV share common group antigens that can be detected by fluorescent antibody test (FAT) and ELISA (Jackwood et al., 1982). The common (group) antigens for both serotypes have serotype-specific group antigens that induce VN antibodies (Azad et al., 1987; Becht et al., 1988) Genetic differences have been demonstrated between egg-laying strains with regard to suscep-tibility to IBDV. Bumstead et al. (1993) documented up to 80% mortality following infection with vvIBDV in selected, partially inbred, experimental lines. Differences among these lines were at-tributed to single gene, independent of the major histocompatibility complex. Various strains im-

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munized with inactivated vaccine differed in their ability to transfer maternal antibody to their progeny. Mortality is variable but can be as high as 50% dependent on the strain of the virus and con-comitant infections and mortality is usually higher in leghorn chickens compared to meat-type birds. In developing countries, IBD imposes a serious threat to profitable poultry production due to mortality and secondary losses due to immunosuppression. Infection with IBDV compromises the humoral and local immune systems. The effect of infection on both systems is more pro-nounced when chickens are infected early in life. The cellular immune system is also affected but that effect is transient and of lower magnitude (Lukert and Saif, 1997). Chickens are most susceptible to clinical infection from 3-6 weeks of age (Ley et al., 1983). Chickens less than two weeks of age are primarily sub-clinically affected leading to immunosuppression (Ley et al., 1979). In Bangladesh IBDV was first reported in 1993 (Chowdhury et al., 1996). Mortality was in broilers between 20-30%. In White Leghorn 40% mortality was observed and in Fayoumi chicks up to 80% was recorded. The size of the affected flocks was between 200 to 250 birds in layer chicks and 600 birds in broilers. The birds were affected at the age between 26 and 45 days. Experi-mentally the isolated virus can cause 100% mortality in 3 weeks old chicks (Chowdhury et al., 1996). In another outbreak it showed that the mortality due to IBDV and other mixed infection ranged from 7.2-16.73% (Rahman et al., 1996). Three isolates of IBDV were characterized and showed similarities to vvIBDV strains, first observed in Europe in the late 1980s (Islam et al., 2001). The present thesis focuses on the description of outbreaks of IBD including different breeds in a rural area and in an urban area of Bangladesh and the isolation of the field virus (Ar-ticle 1). In addition, investigation of the pathogenicity of a local isolate of IBDV in vaccinated and unvaccinated chickens was done and to see interaction between IBD and IBD and E. coli (Article 2). Chapter 2

Literature study

History Infectious bursal disease (IBD) also known as Gumboro disease was first recognized by Cosgrove (1962) as a clinical entity, in 1957, in southern Delware, USA. The etiological vial agent was isolated by Winterfield in 1962 (Lukert and Saif, 1997) who differentiated the disease from a previously established disease known as nephrotoxic infectious bronchitis viral infection of chickens. The term infectious bursal was proposed by Hitchner (1970). There are two serotypes of infectious bursal disease virus (IBDV) (McFerran et al., 1980). Sero-type 1 is pathogenic while serotype 2 is non pathogenic for chickens. Within serotype 1 many subtypes or pathotypes have evolved (Brown and Grieve, 1992). Clinical evidence suggests, that the standard or classical serotype 1 IBDV was predominant throughout the world until early 1980s (Brown and Grieve, 1992). In 1984/85 variant strains of IBDV started to appear in Del-marava peninsula, USA with increased mortality even in vaccinated flocks, and these new American strains were antigenically different from the classical strain (Snyder et al., 1988). These variant strains also differed from classical serotype 1 strains in that they produced a very rapid bursal atrophy with minimal inflammation. Vaccines prepared from classical strains did not give full protection against the variant IBDV strains (Snyder, 1990). Despite the high contagious nature, the mortality from infection with classical and variant strains of IBDV was very low. Most of the mortalities were due to immunosuppression and subsequent secondary infections (Cavanagh, 1992).

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In 1987 a highly pathogenic strain (849 VB) of type 1 IBDV emerged in Holland and Belgium (van den Berg et al., 1991). Mortality in exposed 3-14 weeks old layer replacement pullets at-tained 70% and 100% mortality in experimental infection. Gaudry (1993) reported outbreaks of vvIBDV (very virulent infectious bursal disease virus) in China and Russia in 1993, associated with 60% mortality in 10 days old Leghorn pullets. A virus responsible for outbreaks of vvIBDV in the UK designated the DV86 strain was characterized by Chettle et al. (1989), who confirmed that spontaneous enhancement of virulence had occurred without any major alteration in anti-genic structure. The acute forms of the disease were then described in Japan in the early 1990s (Nunoya et al.,1992; Lin et al., 1993), and they have rapidly spread all over Asia and to other countries. In Bangladesh first outbreaks of IBDV occurred in the early nineties (Article 1). Since then, they have been isolated in many countries including Central Europe (Savic et al., 1997), the Middle East, South America (Di Fabio et al., 1999) and Asia (Cao et al., 1998; Chen et at., 1998; To et al., 1999). On the other hand Australia, New Zealand, Canada and the US are so far unaffected (Snyder, 1990; Proffitt et al., 1999; Sapats & Ignjatovic, 2000). Moreover, only a spo-radic severe outbreak has been described in Finland (Nevalainen et al., 1999), where as the other northern European countries are still free (Czifra & Janson, 1999).

Incidence and distribution Infections with serotype 1 IBDV are of worldwide distribution, occurring in all major poultry-producing areas (Lukert and Saif, 1997). One exception to the ubiquitous nature of IBDV is New Zealand. It has been reported (Jones, 1986; With, 1985) that there is no evidence of IBDV infec-tions in that country. Because of vaccination programs carried out by most producers, all chick-ens eventually become seropositive to IBDV.

Epidemiology Infectious bursal disease is usually a disease of three to six week old chickens. An early sub-clinical infection before three weeks of age (Lukert and Saif, 1997), even in newly hatched chicks (Fadley and Nazerian, 1983), may occur. The disease has also been reported to occur up to 20 weeks of age in chickens (Okoye and Uzoukwu, 1981). All breeds are affected but severe reactions with highest mortality rate were observed in White Leghorn (Lukert and Saif, 1997). Chowdhury et al. (1996) observed higher mortality rate (70-80%) in the Fayoumi breed as com-pared to White Leghorn (40%) in a limited number of field outbreaks. Thirteen to 85% mortality due to IBDV was found in different breeds of chickens in field outbreaks (Article 1). Mortality due to IBD on various farms ranged from 1 to 40% in broilers and from 2 to 40% in layers (Kurade et al. 2000) and from 1.5 to 30% in native and broiler flocks respectively (Saif et al. 2000). How-ever, Meroz (1966) found that there was no difference in mortality between heavy or light breeds. Natural infections of turkeys and ducks have been reported (McFerran et al., 1980). The disease spreads rapidly by direct contact because of the highly contagious nature (Benton et al., 1967a). There is no report of egg transmission of IBDV. Infected birds have excreted the virus in their droppings for at least 14 days (Baxendale, 2002). Fishmeal in the feed contaminated with IBDV may act as a transmitter of the disease (Yongshan et al.,1994), while lesser mealworm as well as mosqito may act as a reservoir of IBDV (Snedeker et al., 1967; Howie and Thorson, 1981; McAllister et al., 1995).

Structure of the virus IBDV is a naked icosahedral, double-stranded RNA virus with a diameter of 55-60 nm (Hirai and Shimakura, 1974; Nick et al., 1976; Dobos et al., 1979; Jackwood et al., 1982) belonging to the family Biranviridae (Kibenge et al., 1988). The prototype of the family is infectious pancreatic ne-crosis of virus (IPNV) of fish. Other members of the family can affect insects and molluscs. The molecular weight of the virus ranged from 2.2 to 2.5 X 106 daltons (Nick et al., 1976; Müller et

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al., 1979) with the buyoant density of 1.34 g/ml (Hirai and Shimakura, 1974; Nick et al., 1976; Dobos et al., 1979; Jackwood et al., 1982). The virion has a single capsid shell composed of 32 capsomers and a diameter of 60 to 70 nm. The larger segment A (approximately 3400 base pairs) is monocistronic and encodes a polyprotein that is auto-processed after several steps into mature VP2, VP3 and VP4 (Müller & Becht, 1982; Azad et al., 1985; 1987; Hudson et al., 1986; Kibenge et al., 1997). Segment A can also encode VP5, a short 17kDa protein (Mundt et al., 1995). The smaller segment B (approximately 2800 bp) encodes VP1, the viral RNA polymerase of 90 kDa (Müller & Nitschke, 1987; Spies et al., 1987).

Viral proteins The smaller segment encodes the multifunctional protein VP1, which has RNA-dependent RNA polymerase activity (Spies et al., 1987) and capping enzyme activity (Spies and Müller, 1990). It is present in small amounts in the virion, both as a free polypeptide and as a genome-linked pro-tein (Müller & Nitschke, 1987; Kibenge & Dhama, 1997). VP2 builds up the external viral capsid (Böttcher et al., 1997) and contains a major conformational neutralizing antigenic domain, stretching from amino acid 206 to 350 (Azad et al., 1987; Becht et al., 1988; Schnitzler et al., 1993). This region has two major and two minor hydrophillic peaks (Bayliss et al., 1990; van den Berg et al., 1996), displays marked variations in the amino acid sequences among different strains of IBDV and is therefore designated as the variable domain (Bayliss et al., 1990). Amino acid changes in this variable domain have been found to be associated with antigenic drifts in IBDV (Heine et al., 1991; Schnitzler et al., 1993; Eterrradossi et al., 1998). Inoculation of the baculovirus-derived VP3 alone failed to induce neutralizing antibodies (Pitcovski et al., 1999). As suggested by Böttcher et al., (1997), VP3 would act as an intermediary, interacting with both VP2 and VP1, and the formation of VP1-VP3 complexes is likely to be an important step in the morphogenesis of IBDV particles (Lombardo et al., 1999; Tacken et al., 2000). VP4 is a non-structural polypeptide. It is involved in the auto-processing of the polyprotein as a virus-encoded protease of producing VP2a, VP3 and VP4 itself (Azad et al., 1987). VP4 has a specific prote-olytic activity which could be demonstrated (Hudson et al., 1986; Kibenge et al., 1997). VP5 was first described in IPNV (infectious pancreatic necrosis virus) particles (Havarstein et al., 1990) and has been identified recently in IBDV infected cells (Mundt et al., 1995). This viral protein more likely has a regulatory function and could play a key role in virus release and dissemination (Mundt et al., 1997).

Resistance to chemical and physical agents The virus is highly resistant to physical conditions and chemical agents. It can survive at 560C for 5 hr (Benton et al., 1967b), at 600C for 90 minutes, at room temperature 250C for 21 days (Cho and Edgar, 1969), viable for up to 60 days in poultry house litter (Vindevogel et al., 1976) and outside the host for at least four months (Baxendale, 2002). The agent is relatively refractory to ultraviolet irradiation and photodynamic inactivation (Petek et al., 1973). Virus retained their viability after heating to 600C for 30 minutes (Landgraf et al., 1967) and could withstand 560C for hours (Benton et al., 1967b). The IBDV can tolerate acidity as low as pH 2, but is inhibited in pH 12 (Benton et al., 1967b). It is refractory to 20% Ether and 5% chloroform for 18 hours at 40C (Benton et al., 1967b). Virus resists 1000 ppm concentration quaternary ammonium disinfectant, a 5% phenolic compound, and 1% phenol at 300C for 1 hour (Benton et al., 1967a). Chloramine was an effective inactivator of IBDV (Landgraf et al., 1967). Jackwood et al., (1996) described a successful inactivation of IBDV with phenol: chloform: isoamyl alcohol at a proportion 25 :24: 1 parts respectively. The virus can also be inactivated by 10% hydrogen peroxide (Neighbor et al., 1994) and invert soap with sodium hydroxide at 400C (Shirai et al., 1994).

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Pathogenesis Susceptibility of different breeds of chicken has been described with higher mortality rates in light than in heavier breeds (Bumstead et al., 1993; Nielsen et al., 1998). Inoculation of IBDV in other avian species fails to induce disease (McFerran, 1993). Bursectomy can prevent illness in chicks infected with virulent virus (Hiraga et al., 1994). The severity of the disease is directly re-lated to the number of susceptible cells present in the bursa of Fabricius. Therefore, the highest age of susceptibility is between 3 and 6 week, when the bursa of Fabricius is at its maximum development. This age susceptibility is broader in the case of vvIBDV strains (van den Berg et al., 1991; Nunoya et al., 1992). After oral infection or inhalation, the virus replicates primarily in the lymphocytes and macro-phages of the gut-associated tissue. Then virus travels to the bursa via the blood stream, where replication occur. By 13h post-inoculation (p.i.), most follicles are positive for virus and by 16h p.i., a second and pronounced viraemia occurs with secondary replication in other organs lead-ing to disease and death (Müller et al., 1979).

Clinical aspects of IBD The incubation period of IBD ranges from 2 to 4 days. Infection of susceptible broilers or layer pullet flocks is characterized by acute onset of depression. Birds are disinclined to move and peck at their vents (Cosgrove, 1962) and pericloacal feathers are stained with urates (Landgraf et al., 1967). Helmboldt and Garner (1964) detected histologic evidence of infection in cloacal bursa within 24 hours. Müller et al., (1979), using immunofluorescence techniques, observed infected gut-associated macrophages and lymphoid cells within 4-5 hr after oral exposure to IBDV. The European strains responsible for vvIBD produce clinical signs similar to conventional type 1 infection. The initial outbreaks were characterized by high morbidity (80%) and correspondingly significantly mortality, attaining 25% in broilers and 60% in pullets over a 7-day period (Chettle et al., 1989; van den Berg et al., 1991; Nunoya et al., 1992).

Gross Pathology Chickens which die acutely of primary IBD infection show dehydration of the subcuatneous facia and pectoral musculature (Cosgrove, 1962). Gross lesions of IBD have been well described (Cheville, 1967; Helmbodt & Garner; 1964; Landgraf et al., 1967; Ley et al., 1983; Skeeles et al., 1979a). The bursa of Fabricius is the principal diagnostic organ in which gross changes oc-cur following exposure to IBDV. Autopsy of birds dying in the acute phase 3-4 days after infec-tion, reveals dehydration and swelling of the bursa to about twice its normal size due to hyper-aemia and oedema. In severe cases, there is marked inflammation of the mucosa and a serous transudate giving the serosal surface a yellow appearance. Petechial hemorrhage on the muco-sal surface is common. Similar findings (Article 2) confirmed the challenge infection of IBD. By the 5th day after infection, the bursa has returned to normal size and by the 8th day it has atro-phied to about one third of its original weight. Swelling and white appearances of the kidneys and associated dilatation of the tubules with urates and cell debris are features encountered in some outbreaks but do not seem to be a con-sistent findings and increased mucus in the intestine (Baxendale, 2002; Cosgrove, 1962). The splenic enlargement was documented by Morales and Boclair (1993) who showed highly significant differences in bursa:spleen weight ratio of 2.4 for controls compared with 0.9 in chicks seven days after challenge. Very often small grey foci are uniformly dispersed on the surface of the spleen (Rianldi et al., 1965). Occasionally, hemorrhages are observed in the mucosa at the juncture of the proventriculus and gizzard (Lukert and Saif, 1997)

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Histopathology IBD affects primarily the lymphoid structures- cloacal bursa, spleen, thymus, harderian gland and cecal tonsil, gut-associated lymphoid tissue (GALT), head associated lymphoid tissues (HALT) (Lukert and Saif, 1997). All lymphoid follicles were affected by 3 or 4 days postinfection. Lymphocytes were soon replaced by heterophils, pyknotic debirs and hyperplastic reticuloendo-thelial cells (Article 2). Hemorrhages often appeared but were not a consistent lesions (Helmbodt and Garner, 1964; Cheville, 1967; Mandelli et al., 1967; Peters, 1967). Following lytic changes, follicles are replaced by cysts lined by columnar epithelium surrounded by a fibroplastic interfol-licular stroma (Okoye and Uzoukwu, 1990). Cystic cavity develops after subsiding the inflamma-tory reaction and there was a fibroplasia in interfollicular connective tissue (Cheville, 1967) (Arti-cle 2) Lukert and Saif (1997). One of the recent isolates (variant A) of IBDV was reported to cause extensive lesions in bursa but the inflammatory response was lacking (Sharma et al., 1989). In spleen following initial perivascular reticuloendothelial hyperplasia, lymphoid necrosis was ob-served in the germinal centres by the 3rd day after infection (Helmbodt and Garner 1964), Re-population commences by the fifth day and is complete in eight days (Okoye and Uzoukwu, 1990). Type 1 IBDV infection in 1-day-old broilers devoid of maternal IBD antibody was investi-gated by Dohms et al., (1981) who showed that plasma cells, which normally populated the Harderian gland by 3 weeks of age, were significantly reduced in numbers compared with non-infected controls. Histologic lesions of the kidney are nonspecific (Peters, 1967) and probably occur because of severe dehydration of affected chickens. Helmbodt and Garner, (1964) found kidney lesions in less than 5% of birds examined. The liver may have slight perivascular infiltration of monocytes (Peters, 1967).

Imunosuppression and interaction with other pathogens The first published description of the immunosuppressive effect of IBDV in the chicken demon-strated a diminished antibody response to Newcastle disease vaccination (Faragher et al., 1974). Pattison and Allan (1974) demonstrated the persistence of Newcastle disease virus in the respiratory tract of chickens which had earlier been exposed to IBD. There was moderate sup-pression when chicks were infected at 7 days and negligible effects when infection was at 14 or 21 days (Faragher et al., 1974). Hirai et al., (1974) demonstrated decreased humoral antibody response to other vaccine as well. Panigraphy et al., (1977) reported that IBDV infections at a young age caused a prolonged skin graft rejection. However, other workers (Giambrone et al., 1977 and Hudson et al., 1975) found no effect from early IBDV infections on skin graft rejection or tuberculin-delayed hypersensitivity reactions. Sivanandan and Maheswaran (1981) observed suppression of cell-mediated immune (CMI) responsiveness, using the lymphoblast transforma-tion assay. In a sequential study of peripheral blood lymphocytes from chickens inoculated with IBDV, a transient depression of mitogenic stimulation was reported (Confer et al., 1981). Sharma and Lee (1983) reported an inconsistent effect of IBDV infection on natural killer cell toxicity and a transient early depression of the blastogenic response of spleen cells to phytohemagglutinin. Depression in plasma cell activity in the Harderian gland is caused by IBDV (Pejkovski et al., 1979 and Dohms et al., 1981). Chickens infected with IBDV, day old at age, were completely deficient in serum IgG and pro-duced only a monomeric IgM (Ivanyi, 1975; Ivanyi and Morris, 1976). The number of B cells in peripheral blood was decreased following infection with IBDV but T cells were not appreciably affected (Hirai et al., 1979; Sivanandan and Maheswaran 1980). The virus appears to replicate primarily in B lymphocytes of chickens (Hirai and Calnek 1979; Ivanyi 1975; Yamaguchi et al.,

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1981). Apparently IBDV has a predilection for actively proliferating cells (Müller, 1986), and it was suggested that the virus affected "immature" or precursor B lymphocytes to a greater extent than mature B lymphocytes (Sivanandan and Maheswaran 1980). Chicks infected early with IBDV were more susceptible to inclusion body hepatitis (Fadley et al., 1976), coccidiosis (Anderson et al., 1977), Marek's disease (Cho, 1970; Sharma, 1984), hemor-rhagic-aplastic anemia and gangrenous dermatitis (Rosenberger et al., 1978), infectious laryngo tracheitis (Rosenberger et al., 1978), infectious bronchitis (Pejkovski, et al., 1979), chicken anemia agent (Yuasa et al., 1980), and salmonella and colibacillosis (Wyeth, 1975).

Diagnosis Classical IBD is characterized by acute onset, relatively high morbidity and low flock mortality in 3-6 weeks old broilers or replacement pullets. Diagnostic lesions include muscle haemorrhages and bursal enlargement (Hanson, 1967).

Isolation Hitchner (1970) demonstrated that chorio allantoic membrane (CAM) of 9 -11 days old embryos was the most sensitive route for isolation of the IBDV which could subsequently be adapted to the allantoic sac and yok sac route of inoculation. Hitchner (1970) observed that most mortality occurred between the 3rd and 5th days post inoculation. Affected embryos had edematous dis-tention of the abdomen, petechiae and congestion of the skin and occasionally ecchymotic hem-orrhages in the toe joints and cerebrum. Bursal samples from the infected ducklings were able to infected chick embryos that died in 96-120 hours after inoculation and the embryos showed the pathological lesions of infectious bursal disease (Bian et al., 1999). Similar findings were ob-served by the author (Article 1). Variant strains of IBD differ from standard viruses in that they induce splenomegaly and liver necrosis of embryos and produce little mortality (Rosenberger et al., 1985). McFerran et al., (1980) reported that three of seven chicken isolates of IBDV failed to grow in chicken embryo fibroblast (CEF) cells but propagated in embryonating eggs. Many IBDV isolates have been adapted to primary cell cultures of chicken embryo origin, includ-ing chicken embryo kidney (CEK) cells and chicken embryo fibroblasts (Lukert and Davis, 1974; McNulty et al., 1979). Because these cells produce low yields of virus (Lukert et al., 1975; Müller, and Becht 1982), there is a need for cell cultures that will produce higher yields of infec-tious virus required for experimental purposes. Cells susceptible to the virus other than cells of chicken origin include turkey and duck embryo cells (McNulty et al., 1979), mammalian cell lines derived from rabbit kidneys (RK-13) (Rianldi et al., 1972), Vero cells (Jackwood et al., 1987; Leonard, 1974; Lukert et al., 1975), derived from African green monkey kidneys; BGM-70 cells (Jackwood et al., 1987), from givet monkey kidneys; and MA-104 cells (Jackwood et al., 1987), from fetal rhesus monkey kidneys.

Serology The agar gel diffusion precipitin test (AGDP) was the original qualitative method to detect anti-body. Bursal homogenate is used as the antigen to demonstrate antibody 7 days after infection (Rosenberger, 1989; Article 1). The serum virus neutralization procedure is extremely sensitive (Weisman and Hitchner, 1978) and is sufficiently specific to differentiate between serotypes of IBD virus (Chin et al., 1984) The ELISA procedure (Engvall and Perlman, 1971) was adapted for IBDV serology and repre-sents a rapid, quantifiable, sensitive and reproducible procedure, which can be automated (Marquardt et al., 1980). The practice of sequential sampling of flocks to monitor antibody level

13

as influenced by vaccination, field exposure and time-related decay in titre was facilitated by the introduction of commercial ELISA test kits (Briggs et al., 1986, Article 2). Automated assays and computerized processing, storage and retrieval of data are the basis of flock profiling (Snyder et al., 1986). The relative advantages and applications for three methods of assaying IBDV anti-body titre have been summaized by Box (1988). Quantitative agar gel diffusion proved to be relatively insensitive, especially when monitoring sera from chicks to determine patterns of ma-ternal antibody decay and age of susceptibility (van den Berg et al., 1991).

Prevention of IBD Maintaining commercial flocks free of IBDV requires the application of sound biosecurity coupled with effective vaccination of parents and progeny (Lukert and Saif, 1997). Since decontamination alone is ineffective (Parkhurst, 1964), prevention of conventional type 1 strain IBDV is dependent on appropriate vaccination of parent breeders and broiler stock. The first vaccines to prevent IBD in broilers and replacement pullets were prepared by adaptation of field isolates in embryonated eggs (Edgar and Cho,1965; 1973). Attenuation of IBDV by passaging chick embryo kidney adapted cultures on a non-avian (VERO) tissue culture system (eight passages) resulted in a non-pathogenic virus. This candidate vac-cine was shown to be ineffective when administered orally but capable of stimulating high levels of antibody when injected subcutaneously (Lukert et al., 1975). Three commercial IBD vaccines were evaluated for pathogenicity and protective in specific pathogen free (SPF) chicks by Naqi et al., (1980). Wood et al., (1988) reported on the development of a candidate IBD vaccine strain designated 002-73 isolated in Australia. In vivo neutralization and passive protection suggested that the isolate would be effective against field strains prevalent in Europe. Selection of vaccines from the 'mild', 'intermediate', and low attenuation or 'hot' classification de-pends on managemental and stock-related factors, level and uniformity of maternal antibody transfer, virulence of field virus strains, and risk of challenge. High parental immunity was recog-nized as beneficial in protecting young chicks from field virus challenge during the critical first 2 weeks when the bursa is most vulnerable to damage induced by IBDV (Hitchner, 1976). In con-trast, high maternal antibody interferes with stimulation of IBD antibody induced by live attenu-ated vaccines (Wyeth and Cullen, 1978a). Propagation of virus on bursal tissue to produce inac-tivated oil emulsion vaccines (Wyeth and Cullen, 1979) produces a more immunogenic agent than virus prepared in specific pathogen free embryos (Wyeth and Chettle, 1982). Selection of vaccination programmes to protect broilers against vvIBDV strains that emerged in Europe in 1987 has required extensive evaluation of the dynamics of vaccine antigenicity, pathogenicity of virus, and maternal antibody. Challenge studies in SPF chicks conducted in Belgium evaluated intermediate and mild vaccines against the vvIBDV isolate designated 849VB (van den Berg et al., 1991). Chicks were vaccinated at 10 days of age and challenged 3-6 days later. Intermediate cloned D78 was mildly pathogenic to bursae, but was immunogenic and pro-vided 80% protection against mortality when challenged 4 days after vaccination. Mild IBD strain vaccines were ineffective in protecting against 849VB virus.

14

Chapter 3

Materials and methods

Cleaning and sterilization of glassware Used glassware were kept in 2% sodium hypochlorite solution for disinfections and kept over-night. All the glassware, except pipettes were soaked overnight in a household detergent. These were cleaned by brushing, washed thoroughly in running tap water three times and rinsed two times in distilled water. The cleaned glasswares were then dried on a bench at room tempera-ture or in an oven at 50-700C. The pipettes were washed repeatedly in tap water, rinsed two times in distilled water, and dried in an oven at 50-700C. Before sterilization the petridishes were either placed in a can or wrapped with a brown paper sealed with autoclavable tape. The glass-wares were usually sterilized by dry heat at 1600C for one and an half hour in an oven. However the bottles with plastic caps or rubber lined aluminum caps were sterilized by autoclaving for 20 minutes at 1210C under 15 lbs pressure per sq. inch. The caps were loosely fitted on the bottles during autoclaving. All autoclaved glasswares were immediately dried in an oven at 50-700C. The caps of the bottles were tightened after cooling.

Media and reagents Phosphate buffer saline (PBS)

Dulbecco's phosphate buffer saline solution A (PBS-A) was prepared as 10X concentration as follows Sodium chloride 80.0 g Potassium chloride 2.0 g Disodium hydrogen phosphate (Anhydrous) 11.5 g Potassium dihydrogen phosphate 2.0 g Glass distilled water 1000 ml All the ingredients were dissolved in distilled water and the pH was adjusted to 7.2 with 0.1(M) NaOH or 0.1(N) HCl. The prepared PBS was distributed in screw capped media bottle in 50 ml aliquots and sterilized by autoclaving for 15 minutes at 1210C under 15 lbs pressure per sq. inch. Before use 50 ml PBS (10X) was mixed with 450 ml sterile glass distilled water, to which one ml (40 mg) gentamicin was added to give a final concentration of 80 µg/ ml. Gentamicin

An injectable preparation of gentamicin containing 80 mg per 2 ml ampoule (Gentin, Opsonin) was procured and stored at -40C. 1% Agarose gel

Sodium chloride (NaCl) 8.0 gm Potassium chloride (KCl) 0.02 gm Disodium hydrogen phosphate (Na2HPO4) 1.15 gm Potassium dihydrogen phosphate (KH2PO4) 0.02 gm Distilled water 100 ml Agarose 1.0 gm Sodim azide (NaN3) 100 times strength 1.0 ml

15

Before adding agarose and sodium azide all the ingredients were dissolved in distilled water and the pH was adjusted to 7.2 with 0.1(M) NaOH or 0.1(N) HCl. Agarose was dissolved by heating in a microwave oven for 2 minutes and 1 ml of 100X sodium azide solution was given to 100 ml agar solution. The solution was dispensed in universal bottle at 15 ml aliquots and kept in chilling temperature for future use. Before using, one bottle was melt in sterilizer or in a microwave oven and poured on a 90 mm petridish to give a thickness of 3 mm.

Raising of chicks for experimental infection Before the arrival of chicks two poultry sheds were constructed, one was repaired and another was renovated. All the sheds were thoroughly cleaned with detergent and fumigated twice with potassium permanganate and formalin at the rate of 20 gm and 40 ml respectively for 100 square feet. Fumigation was done following termination of one experiment and a second fumiga-tion was carried out just three days before arrival of the chickens. Three attendants were em-ployed for rearing the birds. The first one reared the control birds, the second one reared the challenged birds with IBDV (Infectious bursal disease virus) and another attendant took care of the E. coli infected birds. The control birds were kept far away from the challenged birds, how-ever in the control groups, birds vaccinated against IBDV and unvaccinated against IBDV were kept in the same shed but in different compartments on litter system. Separate utensils were used in each group of birds. The birds were supplied with feed and clean water ad libitum.

Farms Outbreaks of infectious bursal disease (IBD) in five chick sheds at Mirpur Central Poultry Farm, Dhaka (urban area) and twelve chick rearing units at Madarganj, Jamalpur (rural area) were in-vestigated during the period from July to October 2001. The number of birds in each shed was approximately 1000-2000 (urban area) and 400-500 in the rural units. The age groups of the birds ranged from day old to two months. The study designs were cross sectional. Detailed par-ticulars of the outbreaks of IBD including history, age, breed of affected chicks, flock size, mortality and clinical signs were recorded. The disease was tentatively diagnosed on the basis of clinical history, signs and symptoms and necropsy findings. In each outbreak 4-5 bursal samples were preserved in sterile 50% buffered glycerol and preserved at -200C for serology and isolation of the virus. Diseases other than IBD were only diagnosed on the basis of post mortem lesion.

Criteria for grouping different diseases Infectious bursal disease : Hemorrhage / edematous swelling of bursa of Fabricius. Hemorrhage in the thigh / breast muscle. Agar gel precipitation. Omphalitis and or polyserositis: Presence or absence Gastro-intestinal disorders (G. I. disorders): Hepatomegaly with necrotic foci / hemorrhage or fresh blood in ceca / hyperemia and diffuse necrosis of mucosa with multifocal ulceration in small intestine. Dehydration : Difficult to removal of the skin Tubular kidney Air way infection : Air sacculitis

16

Exudation in the infraorbital sinus Rickets : Difficult to dislocate the hock joint. Rubbery joint. Rosette formation in costo-chondral junction. Helminth infection: Presence of adult helminth in the lumen of intestine Mixed infections: (Airway infection + Rickets) or (Airway infections + G. I. disorder)

Agar gel immuno diffusion test (AGID) Preparation of antigen

Each bursal sample in each outbreak was homogenized individually with a sterile pestle and mortar to make 100% (w/v) suspension in PBS. The suspension was clarified by centrifugation at 3000 rpm for 20 minutes and stored frozen in aliquots at -200C. Reference antigen and antiserum

Antigen- Strain F52/70, lyophilised bursal homogenate Antiserum- Lyophilised antiserum against F52/70 strain Source- Dr. N. Eterradossi, AFSSA, Ploufragan, France Preparation of agar plate

Wells were cut on agar plates with the head of a pasteur pipette in daisy pattern having a central well surrounded by six peripheral wells. The wells were 5 mm in diameter and 3 mm apart from each other. The agar plugs from cut wells were removed with a pasteur pipette attached to a suction pump. Test procedure

The individual samples and the positive control antigen were placed in the peripheral wells. The reference antiserum was placed centrally. The materials were placed in sufficient amount to fill the wells completely. The plates were incubated in a humidified box at 220C for 48 to 72 hours when the plates were examined for precipitation lines, between central and peripheral wells against reflected light with a home made viewer illuminated with a microscope illuminator.

Isolation of virus from field sample Source of sample

Virus isolation in chicken embryos was attempted from seven pooled samples in each outbreak. Three samples were tried with indigenous chicken embryo and four samples were tried with commercial hybrid chicken embryo .The preserved bursae of Fabricius in each outbreak were pooled and macerated in sterilized pestle and mortal to prepare a 10-20% (w/v) suspension in sterile PBS. The suspension was centrifuged at 3000 rpm for 30 minutes for clarification. The supernatant thus obtained was treated with a broad spectrum antibiotic (Gentamicin) @ 500 µg per ml for 30 minutes at room temperature and stored frozen at -200C. Prior to inoculation to the chicken embryo, the bursal homogenates were treated overnight with an equal volume of chloro-form to inactivate any enveloped virus if present (Islam et al. 2001).

17

Eggs

Twenty fertile indigenous chicken eggs and same number of commercial hybrid farm eggs were obtained from different owners and Bangladesh Agricultural University (BAU) farm respectively. The egg shell was cleaned and disinfected with 20% Savlon (ACI) and then eggs were incu-bated at 370C in a humidified incubator. The eggs were turned manually twice daily. Embryo inoculation and passage

Isolation of IBDV from seven samples were conducted in 10 days old embryonated chicken eggs using 3-4 embryos in each passage of individual sample. In case of initial embryo passage an amount of 0.2 ml of the tissue homogenate was inoculated on to the dropped chorio-allantoic membrane (CAM) following standard embryo inoculation technique (Senne, 1989). The embryos were candled twice daily and those dead within 24 hours of inoculation were dis-carded. The embryos, which died after 24 hours, were opened aseptically and examined for gross lesions. The CAM and whole embryo were washed repeatedly in sterile PBS and stored at -200C for next serial passage and other study necessary. For each sample, two serial embryo passages were performed. For this, the whole embryo and CAM were macerated in sterile PBS using pestle and mortar to prepare a 10-20% (w/v) suspension. The suspension was clarified by centrifugation, treated with antibiotic as done with original field samples. The embryo inoculation and observation were essentially the same as in the initial passage. Passage of virus in the chicken

Fifteen birds were randomly selected from the control group and they were kept in three sepa-rate cleaned fumigated cages. The cages were fumigated with potassium permanganate 20 gm and formalin 40 ml respectively per 100 square feet. In each cages five birds were placed. The age of the chickens were four weeks to get rid of any interference from maternal antibody to grow of the virus. The birds were passaged with positive IBDV field sample (A), embryo ho-mogenate (C) and embryo homogenate (BD3 wild type local isolate of IBDV) respectively at the rate of 100 µl intranasal, intraocular and 100 µl intracloacal route. The birds were sacrificed three days after passage. Collection of bursa

After sacrificing the birds the bursae were collected aseptically group wise into a sterile universal bottle. The bursae were preserved at a temperature of -200C for further study. The group that had the highest weight of the bursa was selected for experimental challenge infection. Titration of virus

A 20% (w/v) suspension was prepared in sterile PBS with the collected bursa which had been passaged with type C isolates. The suspension was diluted serially in sterile PBS to make seven 10-fold dilution. From each dilution 0.2 ml suspension was inoculated into five 10 days old in-digenous chicken embryo. The route of inoculation was dropped CAM route (Senne, 1989). The eggs were candled twice daily for seven days and the embryo mortality and infectivity was re-corded. The fifty percent end point dilution was calculated by the Reed and Muench method (1938) and 0.2 ml suspension of this dilution was considered to contain one 50% embryo infec-tive dose (EID50).

Birds Breed – Sonali Age – day-old

18

Total number of birds - 840 Number of groups - 28 Birds per group – 30 Parent stocks were vaccinated against different diseases at different days (Table 1.). Birds were reared in 4 different sheds. Challenged birds were kept far away from controlled birds, however controlled birds were kept on litter systems but challenged birds were kept on cage systems. All birds were supplied with feed and clean water ad libitum.

19

Table 1. Vaccinations schedule of parent stock Name of the vaccine Disease against Days BCRDV Newcastle Disease 7 BCRDV Newcastle Disease 21 Fowl Pox Fowl Pox 30 RDV Newcastle Disease 60 RDV Newcastle Disease 180

days interval

D78 Gumboro 14 D78 Gumboro 21

Table 2. Experimental groups Replicate 1 Group 1

(a) Replicate 2 Group 1

(b)

Unchallenged control

Replicate 3 Group 1 (c)

Replicate 1 Group 5 (a)

Replicate 2 Group 5 (b)

Challenged with IBDV at day 35


Challenged with E. coli at day 38

Group 9

Vit A Suf-ficient

Challenged with IBDV + E. coli at day 38

Group 10







Unvaccinated

Vit A De-ficient





Vaccinated against IBDV

Vit A Suf-ficient



20





Challenged with E. coli at day 38

Group 11

Challenged with IBDV + E. coli at day 38

Group 12







Vit A De-ficient



Virus A local isolate of IBDV with the titre of 103.15 EID50 per 100µl was used for challenge. The isolate had previously caused 78% mortality in a rural area in Nera breed of chicken where the flock size was 445. The isolate had been classified as vvIBDV (Veterinary Institute, Denmark, Kurt personal communication). Fifty microlitre was given intranasally and another fifty microlitre was given by intraocular route (Hoque et al., 2001).

Vaccine The vaccine used in this study was a commercial, locally manufactured modified live virus vac-cine, obtained directly from the manufacturer and stored at -200C until used. The vaccine was administered according to the manufacturers recommendations. D78: A freeze-dried culture of the nonpathogenic strain D78 of Gumboro Disease (Infectious Bursal Disease Virus), containing at least 104.5 PFU per bird dose (Intervet Company). BCRDV: A freeze-dried culture of the lentogenic F strain of Newcastle Disease virus. Source: Department of Livestock Service, Govt. People's Republic of Bangladesh

Morbidity and mortality estimation following challenge Following challenge with IBDV morbidity and mortality was recorded from day 0 p.i. to 15 days p.i. Clinical signs and symptoms were observed morning and evening twice daily following chal-lenge. Post mortem examinations in dead birds from unvaccinated against IBDV vitamin A (+) challenged, vaccinated against IBDV vitamin A(+) challenged, unvaccinated against IBDV vita-

21

min A(+) challenged + E. coli, vaccinated against IBDV challenged vitamin A(+) + E. coli, unvac-cinated against IBDV vitamin A(+) + only E. coli, vaccinated against IBDV vitamin A(+) + only E. coli, unvaccinated against IBDV vitamin A(+) control and vaccinated against IBDV vitamin A(+) control which were designated as 5,7,10,12,9,11,1 and 3 groups. Any gross changes found in different organs were recorded respectively.

Histopathology For histopathology the bursae were collected from birds at 3 days p.i (post infection) and 10 days p.i. Bursae from birds belonging to groups 1,5,3 & 7 were selected for this. Collected tissue were processed, sectioned and stained following standard histological technique (Luna, 1968). Formalin fixed tissues were washed overnight in running tap water, dehydrated in graded alco-hol, cleared in chloroform and embedded and blocked in paraffin. The sections were stained with Mayer's hematoxylin and eosin and studied under low and high power objectives of a light mi-croscope.

Sampling Bursa body weight ratio (At day 38 and day 45)

At day 38 and day 45 six birds from the group 1,2,3,4,5,6,7 & 8 were randomly selected and sac-rificed. Before sacrificing the weight of the individual birds were recorded. After sacrificing, the bursa was removed from individual birds and the weight was recorded. Subsequently they were kept in 50% neutral buffered formalin in a separate container. Bursa body weight ratio was cal-culated for individual birds. Collection of Serum (At day 14, 21, 35 and 45)

At day 14, 21, 35 and day 45 six birds were taken from groups 1 and 3. Each bird had an indi-vidual tag number. The birds were sacrificed by decapitation. Four to five ml blood was collected aseptically in clean, sterile vials from each of the bird. The collected blood was kept in a slanting position for clotting. After clotting the blood was scummed with a glass rod to loose the clot from the vial. Forty five minutes after scumming the clear serum appeared on the top of the vial. Se-rum was collected from the top of the vial by using a micropipette. Individual tips were used at the time of collection. Collected sera were kept in clean sterilized vials and kept in -200C for fur-ther use. Estimation of antibody titer by ELISA

The test was performed using the supplied Infectious Bursal Disease Antibody Test kit (IDEXX, USA) and the O.D. (optical density) values were calculated at Surgery & Obstetrics department of Bangladesh Agricultural University (BAU) using the ELISA reader (Spectra max 340 pc 384 Microplate Spectrophotometer, USA). Preparation of working antiserum against IBDV

The collected antisera were diluted at 1:500 dilutions using the supplied diluents with the kits. Ten µl serum were diluted with 2.5 ml (1:250) diluents and vortexed. After that 125µl of diluted serum were mixed with an equal volume of diluents (1:500) in a separate sterile polystrene mi-crotitre plate (flat bottom).

22

Protocol followed for determining the antibody titre against IBDV by ELISA method was done according to the manufacturer.

Statistical analysis The model used for analysis of the effects of vaccine, vitamin A, on bursa body weight ratio and titre of serum for each stage was Y= Intercept + age + vaccine + vitamin A + challenge + age*vaccine + age* vitamin A + age*challenge + vaccine*vitamin A + vitamin A*challenge + vaccine*challenge + residual Y= Intercept + age + vaccine + age*vaccine + residual. The analysis was conducted by using GLM process of SAS (SAS, 1987). Mortality in different challenged groups of birds were done by chi-square test.

Economic analysis This was done by calculating the mortality percentage of dead birds found in the outbreak of IBD in field investigation. Mortality in field investigation due to IBD was compared with the mortaltity in experimental condition. For the calculation value of feed cost, biosecurity cost like vaccination and other preventive measures and cost of day old chick were taken into account.

23

Chapter 4

Results

History and clinical manifestations of IBDV outbreaks There was a total of seven outbreaks occuring within the study period. One outbreak occured in one of the five sheds in the Govt. farm and the other six outbreaks occured in different rural poultry units. The size of the affected flocks was between 400 to 500 birds in the rural area but 1000 to 2000 in the govt. farm. The birds were affected at the age between 20 and 54 days (Ta-ble 3). Morbidity was almost 100% in each outbreak however mortality varied from 13% to 85% (Table 3). The clinical signs and symptoms included anorexia, depression, ruffled feathers, diar-rhoea and death. Mortality reached a peak within two to three days of onset and then declined.

Table 3. Outbreaks of IBDV in the rural units and the govt. farm Outbreak

Date Breed Flock size

Mortality (%)

Age in days

A (rural unit) 08/08/01 Nera 494 45 54 B (rural unit) 29/08/01 Nera 381 85 33 C (rural unit) 30/08/01 Nera 445 78 34 F (rural unit) 30/08/01 Fayoumi 435 73 33 E (rural unit) 05/09/01 Fayoumi 400 68 25 G (rural unit) 20/09/01 Fayoumi 257 13 42 D (Govt. farm) 30/09/01 Fayoumi 1755 36 20

Postmortem findings At necropsy, the carcasses were found either in good flesh or highly dehydrated and emaciated. Petechiae and ecchymotic hemorrhages were often observed in the leg and breast muscle. In most cases the bursa of Fabricius was swollen hemorrhagic and edematous with creamy or yel-lowish discoloration. Sometimes the bursa became atrophied which contained some cheesy mass in the lumen. Hepatomegaly and splenomegaly were observed in most case. A few chicks with swollen and pale kidneys were observed.

Agar gel diffusion test (AGID) All twenty nine samples were positive by AGID test showing clear cut precipitation line between unknown sample with known reference antiserum.

Virus isolation Virus isolation was attempted from 7 pooled IBDV positive samples. Mortality at day 1 (post in-fection) was considered non specific (Table 4) because IBDV cannot kill the embryo within one day. It was shown that rural chicken eggs are better for virus isolation than commercial farm eggs. Changes in dead embryos varied. In some cases there was severe congestion and hem-orrhages in the feather tracts of the skin and toes but some showed small congestion on head and toes. The embryos (A & C) having characteristic typical hemorrhages were selected for fur-ther passage.

24

Table 4. Virus isolation Embryo mortality at day p.i. Group Embryos

(N) D1 D2 D3 D4 D5 D6 D7 A 4* - - - - 2 - - B 3* 1 - - - - - 1 C 3* - - 2 - - - - D 3** - - 1 - - - - E 3** - - - - - - - F 3** - - 1 - - - - G 3** - - - - - - 1 * Eggs from rural chickens ** Eggs from commercial farm chickens

Passage of virus in chicken It was found that all the birds were affected showing the typical signs and symptoms of IBDV with passaged virus. Within three days one bird both from type C and BD3 wild type died from 5 inoculated birds (Table 5). The group which had the highest weight of the bursa (group C) was selected for experimental challenge infection. All the bursae were edematous, swollen with a gelatinous yellowish transudate covering the serosal surface.

Table 5. Passage of virus in 4 weeks old chicken Group of birds No. of birds No. of dead

birds Affected bird

Weight of bursa (gm)

A (field sample) 5 0 5 5.88 C (embryo homogenate) 5 1 4 6.90 BD3 wild type (Embryo homogenate)

5 1 4 5.16

25

Titration of viral inoculum It was found that at 10-4 dilution one embryo infected within five embryos but after 10-5 dilution and onwards there was no infectivity in embryos and at 10-1 and 10-2 (Table 6) dilution all the inoculated embryos were infected. The dead embryos were congested, with varying degree petechiae and hemorrhage along the feather tract, toe joint. Here the calculation of titre of viral inoculum is given below. % Mortality 50 - Next below 50% Proportionate Distance (PD) = % Mortality above 50 - Next below 50% 50-17 = 83-17 33 = 66 = 0.5 EID50 = (PD X Log of dilution factor) + Log of dilution with more than 50% mortality = (0.5 X 1) + 3 = 3.50 0.2 ml contains 103.5 EID50

Table 6.Titration of viral inoculum

Dilu

tion

Embr

yos

Day

0

Day

1

Day

2

Day

3

Day

4

Day

5

Day

6

Day

7

No.

of

infe

cted

em

bryo

s

No.

of h

ealth

y

Cum

ulat

ive

No.

of i

nfec

ted

. Cum

ulat

ive

No.

of h

ealth

y %

Mor

talit

y 10-1 5 0 0 0 1 2 0 1 1 5 0 15 0 100 10-2 5 0 0 0 1 2 0 2 0 5 0 10 0 100 10-3 5 0 0 0 0 0 1 0 3 4 1 5 1 83 10-4 5 0 0 0 0 0 0 0 1 1 4 1 5 17 10-5 5 0 0 0 0 0 0 0 0 0 5 0 10 0 10-6 5 0 0 0 0 0 0 0 0 0 5 0 15 0 10-7 5 0 0 0 0 0 0 0 0 0 5 0 20 0

Mortality percentage of different birds (estimated) The mortality percentage of different diseases is shown in Table 7. It shows that the mixed infec-tion is much higher in the urban farm than in the rural unit. Helminth infections were observed only in rural units but not found in the Govt. farm. However uneven flock and birds died from de-hydration is found only in Govt. farm.

26

Table 7. Mortality percentage of different birds (estimated) Name of the group of disease Govt. farm

(221 birds) Madarganj rural unit (167 birds)

Mixed infection (Air way infection + rickets) 05 00 Mixed infection (Air way infection + omphalitis) 04 00 Omphalitis and or polyserositis 35 28 Gastro-intestinal disorders 16 18 Dehydration 11 00 IBD 07 40 Air way infections 14 00 Rickets 08 03 Helminth infection 00 11

Clinical signs in chickens after experimental infection Following experimental infection, marked depression was the first clinical sign that appeared highest in 27% of chicks in the infected group on day 3 p.i. and the remaining birds of the group by day 4 p.i. Whitish diarrhea, ruffled feathers, reluctance to move, kept standing with head downwards and anorexia followed this. There was significant variation in mortality among vacci-nated and unvaccinated groups (p<0.05) (Figure 1). Highest mortality (18%) was found in un-vaccinated vitamin (A+) + E. coli group and lowest (3%) found in vaccinated vitamin A sufficient group (Table 8).

Table 8. Morbidity and mortality after challenge up to 14 day p.i. Name of the group No. of

birds No. of dead birds

% Mortal-ity

No. of diseased birds at 3 dpi

% Morbid- ity at 3 d.p.i.

Unvaccinated Vit. A suff. Control

79 0 0

Vaccinated Vit. A suff. Control

78 3 4

Unvaccinated Vit A. suff. Challenge

75 8 11 16 21

Vaccinated Vit A. suff. Challenge

75 2 3 8 11

Unvaccinated Vit A suff. challenge + E.coli

22 4 18 6 27

Vaccinated Vit A suff. challenge + E.coli

22 1 5 2 9

Unvaccinated Vit A suff + only E.coli

22 2 9

Vaccinated Vit A suff. + only E.coli

22 2 9

27

Fig 1. Mortality in challenged groups of birds

0

2

4

6

8

10

12

Unvaccinated Vaccinated Groups

Mor

talit

y ra

te

Vit A sufficient

Gross pathological changes in chickens after experimental infection The chicks which died on 3 and 4 day p.i. were in good flesh. Hemorrhage was seen in the thigh muscle. The bursa was swollen, edematous and congested. Occasionally severe hemorrhage in the bursa was observed. The thymus was swollen. The birds, which died between day 7 and 10 p.i. were severely dehydrated, urate deposition, black liver, hepatomegaly, caseous mass in the bursa, soft bone (rickets) and lungs congested.

Histopatholgy Histological evaluation of the bursa showed marked bursal lymphoid necrosis and depletion in the infected groups. There were no significant differences in the vaccinated and unvaccinated groups of birds against IBDV in the histopathological findings. At day 3 post inoculation, severe lymphoid necrosis and depletion occurred both in the medulla and cortex of the follicles. Severe edema with heavy infiltration of heterophils and macrophages was observed with many karyor-rhectic nuclei of lymphocytes. At day 10 post inoculation, the lymphoid cells in the cortex and medulla were severely reduced with vacoulation and cysts, which contain clear to pinkish fluids.

Bursa body weight ratio Bursa body weight ratio was calculated at day 3 p.i. and day 10 p.i. (Table 9). At day p.i. 3 hypertrophy of the bursa in the challenged groups of birds was demonstrated. However there was no variation within the vaccinated vitamin A deficient group of birds. At days 10 p.i. atrophy of the bursa were observed in the challenged groups of birds compared to the control groups. However atrophy was not observed in vaccinated vitamin A sufficient groups of birds in compari-son to the control group (Figure 2 and 3). Besides that, the bursa body weight index was also calculated at day 3 p.i. and day 10 p.i. by comparing the respective control groups of birds and showed hypertrophy and atrophy of the bursa respectively (p<0.05) (Figure 4).

28

Table 9. Bursa body weight ratio at day 3 and 10 post infection Name of the group Mean. ± SD at

day 3 p.i.

Mean ± SD at day 10 p.i.

Unvaccinated Vit. A sufficient control

2.73 ± 0.92 2.99 ± 2.46

Unvaccinated Vit. A sufficient challenged

4.05 ± 2.89 1.47 ± 0.57

Unvaccinated Vit. A sufficient control

3.86 ± 2.32 2.84 ± 0.66

Unvaccinated Vit. A deficient challenged

4.73 ± 2.22 2.51 ± 1.24

Vaccinated Vit. A sufficient control

2.44 ± 1.40 1.86 ± 1.08

Vaccinated Vit. A sufficient challenged

4.21 ± 3.52 3.30 ± 1.37

Vaccinated Vit. A deficient control

2.53 ± 1.07 2.04 ± 0.74

Vaccinated Vit. A deficient challenged

2.56 ± 0.64 1.76 ± 0.72

Fig 2. Bursa / Body Weight Ratio at Day 3 p.i.

00,5

11,5

22,5

33,5

44,5

5

Unvac. (VitA+)

Vac.( Vit A +) Unvac.(Vit A -) Vac.(Vit A -)

Treatment

Bur

sa /

Bod

y W

eigh

t Rat

io

ControlChallenged

29

Fig 3. Bursa / Body Weight Ratio at Day 10 p.i.

3.23.33.43.53.63.73.83.9

Day 14 Day 21 Day 35 Day 45

Titr

e of

ser

um

Unvac. (Vit A+)

Vac. (Vit A+)

Fig 4. Bursa / Body Weight Index at Day 3 pi. and Day 10 p.i.

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

Unvac. (Vit A+) Vac. (Vit A+) Unvac. (Vit A-) Vac. (Vit A-)

Bur

sa \

body

wei

ght i

ndex Day 3 p.i.

Day 10 p.i.

Serum titre (At day 14, 21, 35 and 45) At day 14 there was no significant difference in maternal antibody level against IBDV and even seven days after the first vaccination no significant rise in antibody level could be detected against IBDV (at day 21) (Table 10 and Figure 5) in the two groups of birds. However 14 days and 24 days after the second vaccination at day 35 and at day 45, respectively, a significant variation was found in the unvaccinated vitamin A sufficient groups of birds compared to the vaccinated vitamin A sufficient (p<0.0.5).

Table 10. Serum titer at different days from four different groups of birds Day Unvac. (Vit A+) Vac. Vit (A+) Max Min Mean ± SD Max Min Mean ± SD 14 3.64 3.42 3.54 ± 0.10 3.74 3.55 3.61 ± 0.07 21 3.56 3.42 3.47 ± 0.05 3.57 3.44 3.52 ± 0.05 35 3.68 3.27 3.43 ± 0.15 3.74 3.65 3.70 ± 0.03 45 3.82 3.36 3.60 ± 0.22 3.95 3.76 3.86 ± 0.08

30

Fig 5. Mean titre of serum in different days

3.23.33.43.53.63.73.83.9

Day 14 Day 21 Day 35 Day 45

Titr

e of

ser

um

Unvac. (Vit A+)

Vac. (Vit A+)

Economic analysis

Table 11. Cost of rearing a chick up to 2 months old

Rea

ring

perio

d

Feed

in

take

da

ily

in

gram

per

chi

ck

Feed

in

take

w

eekl

y in

gr

am p

er c

hick

Tota

l fe

ed i

ntak

e of

per

ch

ick

in

2 m

onth

s (in

gr

am)

Feed

cos

t per

kg

in ta

ka

Feed

cos

t per

gra

m

Feed

cos

t pe

r ch

icke

n fo

r 2 m

onth

s re

arin

g

Cos

t of d

ay o

ld c

hick

Sub

optim

al b

iose

cuiri

ty

cost

for p

er c

hick

Tota

l cos

t for

rear

ing

per

chic

k (S

ub o

ptim

al b

io-

secu

rity)

Opt

imal

bio

secu

rity

cost

pe

r chi

ck

Tota

l co

st

for

rear

ing

chic

k w

ith o

ptim

al b

io-

secu

rity

1st week 10 70 1442 11.0 0.011

15.86 8.0 4.0 27.86 7 30.86

2nd week 13 91 3rd week 18 126 4th week 23 161 5th week 28 196 6th week 33 231 7th week 38 266 8th week 43 301

Table 12. Mortality due to IBD in field investigation Flock size in numbers

% Mortality Number of dead birds

Total number of birds

Total number of dead birds

494 45 222 2412 1516 381 85 324 445 78 347 435 73 318 400 68 272 257 13 33

31

Table 13. Comparing the mortality between field condition and experimental condition Field condition Experimental condition Difference in mortality Average % mortality due to IBD

63 3 1444

Number of dead birds 1516 72

Table 14. Cost of saving the birds with optimal biosecurity Value of the bird reared in sub optimal biosecurity

Value of the bird reared in optimal bio-security

Net profit= Value of the bird reared in op-timal biosecurity- Value of the bird reared in sub optimal biosecurity

Profit per chick in 2 months

40229.84 44561.84 4332 1.80

32

Chapter 5

Discussion This study was conducted on seven outbreaks of suspected IBD. Clinical manifestation and post mortem findings were studied. Before isolation of the virus in chicken embryos the field sample containing virus was confirmed by AGID test. Virus was isolated from a representative sample, a selected isolate was chosen for pathogenic characterization. Although all birds had been vaccinated against IBD high morbidity and mortality was recorded. Chicks aged between 20 and 42 days were affected with 13 to 85% mortality. This observation is similar to those reported in other countries where the very virulent pathotype of IBDV had emerged (Meulemans et al., 1974; van den Berg et al., 1991; Nunoya et al., 1992; Chowdhury et al., 1996). Mortality due to IBD ranging from 1 to 40% was found in broilers (Kurade et al., 2000; Saif et al., 2000). In this study it was found that mortality was much higher in small flocks than in big flocks. Moreover, it was found that the outbreaks in the small flocks almost affects all birds simultaneously. This might be due to only two persons giving the technical assistance and visit-ing all chick units and very poor bio-security management practice. However the clinical manifestations and gross lesions observed in the present study are in general in accordance to these documented earlier and reviewed by Lasher and Shane (1994) and van den Berg (2000). Usually the disease affects chicken only from 3 to 6 weeks of age. However in one outbreak in a rural unit affecting the Nera breed the age of chickens was 54 days, which was exceptional. A similar finding was also observed by (Okoye and Uzoukwu, 1981) who found that chicken up to 20 weeks could be affected. The study also showed that in the investigated outbreaks of IBDV there was no significant difference in mortality among the breeds of chicken. The investigations clearly indicate that problems concerning the vaccination procedures have to be addressed. Six out of 7 pooled samples of bursal homogenate caused embryo mortality and one did not. The embryo lesions were similar to those usually observed with IBDV (Lukert and Saif, 1997). In another study it showed that three Bangladeshi IBDV isolates causes 100% mortality in SPF chicken embryos within 6 days (Islam et al., 2001). However in this experiment the mortality pat-tern and the lesions found in the embryos were not consistent. This might be due to use of farm embryonated eggs instead of using SPF (specific pathogen free) eggs. It also shows that indige-nous eggs are suitable for isolation of virus in comparison to commercial embryonated farm eggs. This is possibly due to the presence of high titres of maternal antibody present in commer-cial eggs. It is believed that indigenous chicken get less exposure of IBD than commercial chicken. Passaging the three different IBDV virus isolates in chickens show the typical clinical symptoms at the 2nd and 3rd day p.i. was demonstrated which is similar with findings Lukert and Saif, (1997). The bursae were edematous, gelatinios and almost doubling the size that of normal which corresponds with the results of Cheville (1967), Ley et al. (1979) and Ley et al. (1983). It is interesting that in passaging the virus the embryo homogenate cause mortality in inoculated chicken but no mortality was found in the positive field sample. This might be due to adaptation of the virus in growing in the chicken embryo. Differences in the pathological findings between chickens from the rural village conditions and the urban poultry farm were observed. Grouping of diseases on the basis of pathology revealed that dehydration was a common cause of death and it was only found in the Govt. farm. How-ever there was no helminth infection found in the urban unit but it was found in the rural unit. This might be due to managemental problem. In the Govt. farm there is crowding of the flocks, but no proper attention is given to the birds. Helminth infection was not found because of regular deworming. In the rural units helminth poses a problem because deworming does not take

33

place. In addition the infection builds up in the village environment. Airway infection was found in the Govt. farm but not in the rural unit. This could be due to improper ventilation in the farm. This results corresponds the findings of CRD (chronic respiratory disease) infection by David and Harry (1997). The clinical signs and symptoms, observed following experimental infection, were similar to those described by others (Cosgrove, 1962; Lukert and Saif, 1997). The highest mortality on 18% was recorded in the unvaccinated vitamin (A+) + E. coli group and the lowest on 3% was recorded in the vaccinated vitamin A sufficient groups of birds. Van Den Berg et al. (1991), Chettle et al. (1989) and Nunoya et al. (1992) reported 100, 90 and 80% mortality, respectively following infection with vvIBDV on SPF chicks. In this experiment the local indigenous hybrid Sonali chickens originated from vaccinated parent birds. It was interesting to note that the clini-copathological manifestations of experimental acute IBD varied with the course of the disease. Death of chickens after day 3 and day 4 p.i. resulted in diseases which was associated with hemorrhages in the bursa and in the thigh muscle, edematous swollen bursa, mucus in the in-testine. However, such lesions were not apparent in the birds, which survived for more than 4 days. Similar findings have been observed by others (Lukert and Saif, 1997; Baxendale, 2002; Islam et al., 1997 and Kurade et al, 2000). The birds, which died after day 4 p.i. showed severe dehydration and caseous mass in the bursa. This has also been reported by Lukert and Saif (1994) and Baxendale (2002). It was observed that the vaccine did not give full protection to the birds even when the birds were kept on balance ration and in good conditions. Eighteen percent mortality was recorded in unvaccinated vitamin A sufficient birds, which is similar with other pre-vious workers (Ley et al., 1983). There was also no significant variation in the mortality between the groups which were infected with IBD alone and IBD with subsequent E. coli infection. To evaluate the degree of bursal damage produced by the local Bangladeshi vvIBDV isolate histopathology was done. At day 3 p.i. there was severe depletion of lymphocytes from the bursa of Fabricius leaving many small vacoules both in the medulla and in the cortex. Lymphocytes were replaced by heterophil and pyknotic nuclei. Severe edema was also demonstrated in the intra follicular septa. These findings were also observed by Lasher and Shane (1994), Lukert and Saif (1997) and Islam et al. (1997). At day 10 p.i. the inflammatory reactions declined and cystic cavities developed in the medullay areas of the follicles. Loss of demarcation between the follicular septa indicating the severe atrophy of the bursa was seen. These findings are in accor-dance with Okoye and Uzoukwu (1990), Sharma et al. (1989) and Cheville (1967). Bursa-body weight ratios calculated after infection with a local isolate of vvIBDV resulted in swelling and hypertrophy of the bursa in the challenged groups and at day 3 p.i. Atrophy of the bursa was observed at day 10 p.i. This is in accordance with previous findings (Rosales et al., 1989; Singh et al., 1994; Vladimir et al., 1997). However the changes in the challenged group compared to the control group was not significant. This might be due to control birds. The birds were not uniform and had variation in the bursa body weight ratio because of keeping the vacci-nated and control birds together in the same place but in a different compartment. Besides that at day 3 p.i. less lesions were demonstrated in the vaccinated vitamin A deficient group. This might be due to deficiency in vitamin A which has an important function in the immune response of the host. In addition at day 10 p.i. no atrophy was observed in the vaccinated vitamin A suffi-cient group of birds. This could be due to the vaccination and/ or presence of sufficient amount of vitamin A that helps in producing sufficient amount of antibody in order not to regress the bursa. In the challenged groups of birds at day 3 p.i. the highest bursa body weight ratio was recorded in the unvaccinated vitamin A deficient group whereas the lowest ratio was recorded in the vaccinated vitamin A deficient group. This might be due to deficiency in vitamin A that gives a non-consistent reaction to the challenged infection. At day 10 p.i. the lowest bursa-body weight ratio was demonstrated in the unvaccinated vitamin A sufficient challenged groups of birds.

34

ELISA test is considered as an ideal serological test in the diagnostic virology all over the world due to its specifity, sensitivity, simplicity and minimum time requirement. Antibody titre can be calculated from a single dilution described by Snyder et al. (1983) and Van Loon et al. (1981). Maternal antibody titre against IBDV were calculated prior to first vaccination against IBDV at day 14 and showed that there was no variation in group 1 and 3 indicating any influence of vac-cination or not. At day 21, 7 days after the first vaccination the serum titre was declining from day 14 even in the vaccinated group. This is in accordance with previous findings (Cao et al., 1995). However 24 days after the second vaccination at day 45 an increase in the maternal anti-body level in the unvaccinated vitamin A sufficient groups of birds was observed. This could be due to the fact that unvaccinated birds and vaccinated birds were kept together in the same shed but in different compartments, subsequent shedding of the live vaccine virus from vacci-nated group of birds to the unvaccinated vitamin (A+) groups of birds might have taken place. It has been suggested that the adverse economic effects of subclinical IBD on flock performance is caused by immunosuppression and subsequent superimposed infections (McIlroy et al., 1989). From the economic analysis it showed that if the chicks are reared in optimal biosceurity it profits 1.80 taka per bird in 2 months. This means if a farmer in a chick-rearing unit rears 250 birds she can profit 450 taka within 2 months by adding extra efforts in biosecurity. Following measures can be taken in controlling outbreak of IBD: Fumigation of the chick rearing unit before and after termination of each rearing flock. Placing an antiseptic foot dip in the entrance of the shed. Wearing a separate clean apron while working in the shed. Not allowing any unnecessary visitors. Training of the technical personnel who advise the rural women.

Conclusion and future perspectives It is clear from the present findings that IBD is prevalent in different production systems of poul-try in Bangladesh. It is one of the major poultry viral diseases causing very high mortality in chickens. The virus can be readily isolated by using indigenous fertilized chicken embryo. In ex-perimental infection the virus cause lower mortality than natural outbreaks suggesting that good biosecurity coupled with vaccination can prevent mortality in a great extent. However, the vac-cine against IBDV produced from the Intervet company did not give full protection to the chal-lenged isolate. There was no significant difference in antibody titre 7 days after the first vaccina-tion in unvaccinated birds compared to vaccinated birds, but significant difference in antibody titre was found 14 days and 24 days after the second vaccination of IBDV. The isolate also cause severe damage to the bursal follicle. The virus cause severe lymphoid necrosis at day 3 p.i. and cyst and vacoulation formation in the follicle at day 10 p.i.

Future perspective In this study the pathogenicity of field isolate of IBDV was examined using the local cross bred Sonali (♂ Rhode Island Red + ♀ Fayoumi) chickens obtained from the parent stock vaccinated against IBDV. Pathogenicity of the isolate should be investigated in SPF chickens. Detailed histopathological examination of various lymphoid organs like bursa, thymus, Harderain gland of chicken should be included in such studies. Bursal lesion scores also should be determined at different time points. The isolate should be characterized at antigenic and molecular level. Fur-ther investigations on the efficacy of different vaccines used in Bangladesh are strongly recom-mended, as the vaccine used in the present study did not give full protection. Interaction be-tween maternal antibodies and vaccines should be investigated to design an optimum vaccina-tion schedule. Attempts should be made to adopt the IBDV isolate to grow in cell culture.

35

Chapter 6

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Brown, M. D., Green, P., and Skinner, M. A., 1994: VP2 sequences of recent European "very virulent' isolates of infectious bursal disease virus are closely related to each other but are dis-tinct from those of "classical' strains. J. Gen. Virol. 75, 403-410. Bumstead, N., Reece, R. L., and Cook, J. K. A., 1993: Genetic differences in susceptibility of chickens lives to infection with infectious bursal disease virus. Poult. Sci. 72, 403-410. Cao, Y. C., Bi, Y. Z., and Zhu, J. M., 1995: Application of enzyme-linked immunosorbent assay for evaluation of immunological efficiency of chicks against IBD. Chin. J. Vet. Med. 21(11), 9-10. Cao, Y. C., Yeung, W. S., Law, M., Bi, Y. Z., Leung, F. C., and Lim, B. L., 1998: Molecular char-acterization of seven Chinese isolates of infectious bursal disease virus: classical, very virulent, and variant strains. Avian Dis. 42, 340-351. Cavangh, D., 1992: Recent advances in avian virology. Br.Vet. J. 148, 199-222. Chen, H. Y., Zhou, Q., Zhang, M. F., and Giambrose, J. J., 1998: Sequence analysis of the VP2 hypervariable region of nine infectious bursal disease virus isolates from mainland China. Avian Dis. 42, 762-769. Chettle, N., Stuart, J. C., and Wyeth, P. J., 1989: Outbreak of virulent infectious bursal disease in East Anglia. Vet. Rec. 125, 271-272. Cheville N. F., 1967: Studies on the pathogenesis of Gumboro disease in the bursa of Fabricius, spleen and thymus of chicken. Am. J. Pathol. 51, 527. Chin, R. P., Yamamoto, R., Lin, W., Lam, K. M., and Farver, T. B., 1984: Serological survey of infectious bursal disease virus serotypes 1 and 2 in California turkeys. Avian Dis. 28, 1026-1036. Cho, Y., and Edgar, S. A., 1969: Characterization of the infectious bursal agent. Poult. Sci. 48, 2102-2109. Cho, B. R., 1970: Experimental dual infections of chickens with infectious bursal and Marek's disease agents. I. Preliminary observation on the effect of infectious bursal agent on Marek's disease. Avian Dis. 14, 665-675. Chowdhury, E. H., Islam, M. R., Das, P. M., Dewan, M. L., and Khan, M. S. R., 1996: Acute in-fectious bursal disease in chickens: pathological observation and virus isolation. Asian-Austrlasian J. Anim. Sci. 9, 465-469. Confer, A. W., Springer, W. T., Shane, S. M., and Conovan, J. F., 1981: Sequential mitogen stimulation of peripheral blood lymphocytes from chickens inoculated with infectious bursal dis-ease virus. Am. J. Vet. Res. 42, 2109-2113. Cosgrove, A. S., 1962: An apparently new disease of chickens- avian nephrosis. Avian Dis. 6, 385-389. Czifra, G., and Janson, D. S., 1999: Infectious Bursal Disease in Sweden. Proceedings of the First Working Group 1 meeting on Epidemiology, COST Action 839, 06-08/06/99, Ploufragan, France.

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