Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1994 Studies on immune responses to Bordetella avium in turkeys Poosala Suresh Iowa State University
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Studies on immune responses to Bordetella avium in turkeys
Suresh, Poosala, Ph.D.
Iowa State University, 1994
UMI 300 N. ZeebRd. Ann Arbor, MI 48106
Studies on immune responses to Bordetella avium in
turkeys
by
Poosala Suresh
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department: Veterinary Pathology Major: Veterinary Pathology
•— - — u — » • — -1 -
Signature was redacted for privacy.
Signature was redacted for privacy. For me Major Department
Signature was redacted for privacy. Fo/^é Graduate College
Iowa State University Ames, Iowa
1994 to
Anu, Vishy, Arvind, Anil, Nithin, Deepika,
my uncle
and
my beloved mom and dad Mi TABLE OF CONTENTS
GENERAL INTRODUCTION 1
LITERATURE REVIEW 5
Anatomy of the Avian Immune System 5
Primary Lymphoid Organs 5
Thymus 5
Bursa of Fabricius 6
Secondary Lymphoid Organs 7
Spleen 7
Mucosal Lymphoid Tissues 8
Gut Associated Lymphoid Tissue (GALT) 9
Bronchus Associated Lymphoid Tissue (BALT) 10
Paranasal and Paraocular Lymphoid Tissue 11
Conjunctiva Associated Lymphoid Tissue (CALT) 12
Lymph Nodes 1 3
Mucosal and Systemic Humoral Immune System of Poultry 13
Avian Immunoglobulins 13
Immunoglobulin G (IgG) 14
Immunoglobulin M (IgM) 14
Immunoglobulin A (IgA) 15
Antibody Response in Avian MALT 16 iv
Antigen Uptake and Presentation 16
Immune Response in MALT 18
Respiratory Immunity in Poultry 20
Potential role of MALT in Respiratory Immunity in Birds 20
Humoral Immunity 21
Cell mediated Immunity 22
Antibody Transport 24
Antibody Catabolism 27
Bordetellosis in Turkeys 29
Etiology 29
Growth Characteristics and Morphology 30
Pathology, Clinical Signs and Lesions 30
Immunity and Vaccination 32
Humoral Immunity 32
Vaccination 33
Diagnosis 34
Use of Serology for Diagnosis 34
SYSTEMIC AND MUCOSAL IMMUNE RESPONSES TO BORDETELLA AVIUM IN EXPERIMENTALLY INFECTED TURKEYS
Summary 36
Introduction 37 V Materials and Methods 38
Results 42
Discussion 50
Acknowledgements 52
References 52
A TIME COURSE STUDY OF THE TRANSFER OF IgG FROM THE BLOOD TO TRACHEAL AND LACRIMAL SECRETIONS IN YOUNG TURKEYS
Summary 55
Introduction 55
Materials and Methods 57
Results 61
Discussion 64
Acknowledgements 65
References 66
EFFECT OF PASSIVELY ADMINISTERED IgG IN COLONIZATION AND CLEARANCE OF BORDETELLA AVIUM IN TURKEYS
Summary 69
Introduction 70
Materials and Methods 72
Results 76
Discussion 77
Acknowledgements 87 vi References 87
GENERAL SUMMARY AND CONCLUSIONS 92
REFERENCES 96
ACKNOWLEDGEMENTS 120
APPENDIX. A MONOCLONAL ANTIBODY BASED LATEX BEAD AGGLUTINATION TEST FOR THE DETECTION OF BORDETELLA AVIUM 122 vii
ABSTRACT
Systemic and mucosal humoral immune responses to an experimental
B. avium infection were evaluated by using an ELISA technique. The three isotypes
of immunoglobulin, IgG, IgA and IgM, were detected in serum, tracheal washings
and lacrimal secretions in response to an intra-nasal and intraocular bacterial
challenge of 1-day-old turkeys. The IgM response was the earliest, starting at 2 weeks
and reaching a peak at 4-5 weeks after infection. The IgA response was early, steady
and lower in comparison. The IgG response was prominent and long lasting. The
IgG response started at 3 weeks, reached a peak at 5-6 weeks and slowly declined by
8 weeks. The total immune response declined by 8 weeks as the clearance of
6. avium approached completion. In this study, the cumulative antibody titers closely corresponded with decreasing numbers of bacteria.
The transfer of parenterally administered IgG was assayed in turkeys using
8. avium specific IgG and ELISA as a model. Purified IgG, specific to B. avium was injected and samples of serum and tracheal washings were collected and assayed by
ELISA at various time intervals. The IgG intravenously injected was detected in tracheal washings as early as 5 minutes PI, with peak levels occuring 10 minutes post-injection (PI). A rapid decline in serum IgG from 10 to 60. minutes PI was followed by a more gradual decline to baseline levels by 24 hours PI. The levels of
IgG in serum, tracheal washings and lacrimal secretions were closely corresponding after 10 minutes PI. These results indicate rapid transfer of IgG from the blood to viii mucosal secretions.
The role of parenterally administered IgG in the colonization and clearance of
B. avium from the tracheal surface was assayed. The IgG prevented colonization and also aided in the clearance of B. avium from the tracheal surface. This study showed that IgG, transferred from the bloodstream to mucosal surfaces, could inhibit colonization and promote clearance of infection.
A monoclonal antibody based-latex bead agglutination test was developed to differentiate B. avium from 6. branchiseptica and B. avium-iiiie bacteria. All 40 isolates of B. avium tested showed a positive reaction with this test. None of the 24
S. avium-Wke isolates showed a positive reaction. Out of 17 B. bronchiseptica isolates, two had minor cross reactions. 1
GENERAL INTRODUCTION
The disease bordetellosis in turkeys is a problem for turkey producers
throughout the United States and other countries (35,38,90). Although the disease
itself is not responsible for mortality in a flock, secondary infections may lead to
death. Due to the dearth of knowledge, in general, about humoral immunity in
turkeys exposed to Bordetella avium, there is no effective vaccine available. Even the
vaccines that are or have been used are not effective due to the age of the bird at
administration or the antigenic composition (92).
Many studies on immune responses to various respiratory pathogens in
mammals and birds have shown that antibodies are effective in either preventing
infection or promoting recovery from disease (13,55,62,139,197). There have been
few studies that clearly determined the role of each isotype of immunoglobulin in a respiratory mucosal disease. Several studies have been done to detect various antibodies in birds (13,55,62), but none of these gave a detailed picture of the role of each isotype. In birds, these kinds of studies have been sparse and information related to protective effects of antibody isotypes against a respiratory pathogen are even more sparse.
It has been known for at least three decades that birds are capable of producing all three isotypes - IgA, IgM and IgG- in response to a disease on the mucosal surfaces (116). Recent studies have shown the presence and importance of 2 mucosa-associated lymphoid tissue (MALT) at various locations (28,29, 86). Even
though, the role of these lymphoid tissues In the respiratory tract has not been fully
explored, present knowledge suggests that they might be very important for the
immune response. In response to an antigenic challenge, these lymphoid tissues
undergo marked hypertrophy, with increased cell numbers and probably germinal
centers, conveying a morphologic basis for the humoral immune response (68).
Similarly, previous studies have shown that the Harderian gland has increased
numbers of plasma cells and that it plays an important role in immunity against
diseases of the upper respiratory tract (176).
Current knowledge about the source of each antibody isotype in mammals may also apply to birds. The main sources of IgM and IgA seem to be local cell populations in the lamina propria of mucous membranes of certain tissues. The main source of IgG found at the mucosal surface seems to be serum (161). However, none of these sources are mutually exclusive, and there is no direct evidence for a single antibody being responsible for the immunity. A study in chickens demonstrated the transfer of IgG to lacrimal secretions (182). The relatively smaller size of IgG makes it capable of undergoing simple diffusion (114). However, IgM and IgA have secretory components which facilitate their transport across epithelial cells. Even though the role of IgA in mucosal infections is considered important, the role of IgM and IgG cannot be ignored as there is no direct evidence linking absence of IgA with susceptibility to disease. The overall picture with the currently available knowledge is that systemic and mucosal humoral immune responses together play an important 3
role in protecting an animal from respiratory infections.
An understanding of the immune response to Bordetella avium in turkeys is an
important step in understanding the role of each immunoglobulin isotype during
infection. Also, a knowledge of the source of each immunoglobulin should shed
light on mucosal immunology. A knowledge of the role of Immunoglobulin isotypes
in turkey bordetellosis may lead to better vaccination strategies and serologic
techniques.
Apart from understanding the immune aspects of this disease, there is also an urgent need for devising a simple diagnostic test to detect Bordetella avium and differentiate it from closely related bacteria like Bordetella bronchiseptica and B. av/um-like organisms. This kind of test could make the laboratory diagnosis of bordetellosis more rapid and efficient.
Objectives of Dissertation Research
The detection of a humoral immune response to Bordetella avium in turkeys suggests that antibodies may be important in the defense against this disease. Hence, the objectives of this research were to:
1) Determine the onset, duration, and quantities of immunoglobulin isotypes produced during infection, 2) characterize the temporal aspects of IgG transport from serum to mucosal surfaces, 3) analyze the importance of IgG in preventing colonization or clearing of 6. avium from the tracheal surface, and 4) devise a simple diagnostic test for identification of B. avium. 4 Dissertation Organization
This dissertation consists of three manuscripts; each one is presented in the format of the journal Avian Diseases. Proceeding the papers is a literature review; following the papers is a general summary and discussion. References cited in the general introduction and literature review follow the general summary and discussion. The first manuscript to be published in Avian Diseases is in press. The appendix is a complete research article published in Avian Diseases. Dr. Suresh was the principal investigator and first author for each phase of research and corresponding manuscript. 5
LITERATURE REVIEW
Anatomy of the Avian Immune System
The structure and organization of lymphoid tissue in birds differs significantly from that in mammals. Major differences are the absence of lymph nodes and the presence of a special lymphoid organ, the bursa of Fabrlcius in birds. The bursa is important in the development and differentiation of B-cells (163). In spite of the differences in structural organization of lymphoid tissues between mammals and birds, the functional aspects of lymphoid cells and peripheral lymphoid organs are similar (153). Small lymphocyte accumulations (0.5 mm in diameter) are present along lymphatic vessels in birds (153). Though these small nodules enlarge and develop germinal centers when stimulated by antigen, they lack the size and complexity to function as lymph nodes in mammals (130). However, the enormous number of these nodules in parenchymal tissues such as bone marrow, skin, liver, lung, kidney, pancreas, endocrine glands, larynx and trachea compensate for the lack of distinct lymph nodes (153).
Primary Lymphoid Organs
Thymus
The mature thymus is a 12- to 14- lobed structure located close to the jugular veins (163). The lobules of each lobe are further separated into cortical and 6
medullary zones. A dense epithelial cell network and Hassal's corpuscles are found
in the medulla. Also present in the avian thymus are lymphoid cells of various sizes,
epithelial cells, macrophages and myeloid cells (78). Evaluation with flow cytometric
analysis revealed that thymic cells are composed of large blast-type cells, lymphocyte
sized thymocytes and small thymocytes. In contrast to the mammalian thymus, the
avian thymus has afferent lymphatics and has the potential to act as a peripheral
lymphoid organ permitting B-cell traffic after hatching (121). Mature plasma cells are
found after 4 weeks (4,96). In response to antigenic stimulation, specific antibody presenting cells appear in the avian thymus (103). Chicken thymic extracts induce the specific T-cell allo-antigen Th-1 on embryonic bone marrow cells, suggesting the presence of thymic factors similar to those in the mammalian thymus (39).
This follicular epithelial lymphoid organ is located dorsal to the distal end of the cloaca (163). The horizontal infoldings and plicae formed by the developing epithelial cells are colonized by lymphoid cells which define cortical and medullary regions. Each bursal follicle is populated by approximately 10,000 lymphoid cells.
The bursal medulla contains small lymphocytes, plasma cells and macrophages (140).
The bursa of Fabricius, in addition to its role as a central immunological organ, also functions as a peripheral lymphoid organ. Antigenic material given by various routes can induce an antibody response from antigen presenting cells (APC) in the bursa
(190). The lymphoepithelium covering the bursal follicles is specialized for antigen 7
uptake (37). This lymphoepithelium Is capable of uptaking and transporting tracer
particles by pinocytosls. After localizing in bursal follicles, these tracer particles are
further distributed to other regions of the bursa by phagocytic cells (21). Also
observed in the bursa are T-cells (128) in special T-dependent areas with an APC
population (142). A dense lymph vessel network with lymphoid cells is present outside the follicles of the bursa (65). The lymph flow is away from the bursa, and it has been suggested that efferent lymph vessels are involved in the transport of cells from the bursa to peripheral tissues (65).
Secondary Lymphoid Organs
Spleen
The structure of the avian spleen is similar to its mammalian counterpart except for the ellipsoidal reticular cells arranged in sheaths around penicillary arteries
(144). Lymphoid cells colonize the spleen at 10 to 11 days of embryonic life in birds
(5). The red pulp and white pulp start to develop at 8 and 12 days of incubation, respectively (153,166). Periarterial tissue surrounding the central arteries and arterioles is dominated by T lymphocytes. Germinal centers are located within the periarterial lymphoid tissue located close to the central artery at a site where it branches into penicillary arterioles (179). B-cells are present at day 12 of incubation and IgG and IgA positive cells are present at the time of hatching (153). The lymphoid structure undergoes major development after hatching in response to antigenic stimulation. The spleen produces antibodies mainly against blood-borne 8
antigens (189). There is direct correlation between the generation of splenic germinal
centers and bursal development, both reaching peak maturity at 4 to 5 weeks of age.
The formation and presence of germinal centers requires cooperation between B- and
T- cells (179). B cells in the germinal centers are considered to represent a pool
rather than B cells differentiating into plasma cells (193).
Mucosal Lymphoid Tissues
Mucosa-associated lymphoid tissue (MALT) is present along mucous membranes throughout the body, particularly the digestive and respiratory systems.
The Harderian gland, conjunctiva-associated lymphoid tissue (CALT), lymphoid nodules associated with the nasolacrimal gland, bronchus-associated lymphoid tissue
(SALT), Peyer's patches, the cecal tonsil, and Meckel's diverticulum are the components of avian MALT (17,73). In birds, MALT is highly developed and may be functionally similar to mammalian lymph nodes in responding to antigen. Avian
MALT is strategically located at sites where there is high exposure to environmental antigens. Some component tissues of MALT, such as Harderian gland and Meckel's diverticulum contain large numbers of plasma cells primarily involved in local antibody production (138). Some other components of avian MALT, such as CALT have more lymphoid nodules and fewer plasma cells, presumably these tissues are involved in the initiation of immune responses, rather than local antibody production
(22). This kind of functional diversity in the immune responses of MALT makes it an important entity for the immunologist and even more important for the bird itself. 9
Gut Associated Lymphoid Tissue (GALT)
The presence of GALT has been demonstrated throughout the gastrointestinal
tract In mammals and birds. GALT refers to various lymphoid tissues in the oral
cavity, pharynx, small intestine, cecum, bursa and rectum. Hence, GALT contributes
substantially to the total amount of lymphoid tissue in the body. The morphologic changes associated with changes in cell populations in GALT in response to antigens
suggests their role in immune responses. The lymphoid nodules in the lamina propria of these organs contain germinal centers which are considered thymus dependent. The phagocytic cells and lymphoid cells considered to be bursa- dependent are present in the epithelial and sub-epithelial zones (153).
Avian Peyer's patches are structurally similar to those in mammals (22). The
Peyer's patches, located in the distal ileum, are characterized structurally by flattened intestinal epithelium, lack of goblet cells, and thickened villi. Both germinal centers and diffuse lymphoid tissue are present in Peyer's patches (43). Most B cells in
Peyer's patches have surface IgG with some IgM or IgA (43). The number of Peyer's patches increases up to 16 weeks of age and then decreases to a single residual patch
(22).
Meckel's diverticulum is the embryologie yolk sac remnant that separates the jejunum from the ileum (146). Infiltration of lymphoid cells begins at the age of 2 weeks and attains maturity, as indicated by functional germinal centers, at 5 to 7 weeks of age (179). Large numbers of plasma cells are present in the Meckel's diverticulum; like the Harderian gland it remains functional until 20 weeks of age 10 (146).
The germinal centers in the cecal tonsils can either be completely
encapsulated or incompletely encapsulated (143). The appearance and maturity of
the cecal tonsils is dependent on the antigenic stimulation in the intestine (141).
Bursectomy reduces the number of germinal centers in the cecal tonsils (102).
Bronchus Associated Lymphoid Tissue (BALT)
BALT is an important mucosal lymphoid tissue. It has been identified in several species including rabbits, rats, mice, guinea pigs, pigs, chickens, turkeys, dogs and humans (30,68). It is present at the bifurcation of airways where deposition of antigenic material occurs. BALT was first characterized 20 years ago (30,31). These authors mentioned that, chickens had more lymphoid tissue in the trachea compared to other species mentioned above. Also, in contrast to mammalian BALT, avian BALT occurs as prominent subepithelial lymphoid nodules, raising the surface epithelium and protruding into pulmonary bronchial lumina (30,68,187). A complete description of BALT in turkeys has been published (68,187). BALT of turkeys is present along the primary intrapulmonary bronchus. It is specifically associated with longitudinal mucosal folds and the openings of secondary bronchi (68). Light and electron microscopic studies of BALT in normal turkeys at various ages showed that it resembled other MALT. The lymphoepithelium of BALT in chickens varies from primarily squamous in 1 week old chicks to ciliated columnar cells in 6 week old chicks (68). In studies involving turkeys infected with Bordetella avium, BALT 11
nodules were more numerous and more widely distributed than in normal birds
(72,187). BALT nodules In all ages of turkeys have a dome-shaped ciliated epithelial
surface interrupted at the dome apex by patches of non-ciliated cells (68).
Ultrastructurally, the same study showed discontinuities in the basal lamina
associated with infiltrating lymphocytes. This shows that turkey BALT may be
specialized for antigen uptake and the associated immune response (68).
Paranasal and Paraocular Lymphoid Tissue
Birds have a closed, bony orbit, sinuses and paraocular glands. There are several accumulations of lymphoid tissue in the paraocular and paranasal areas (180).
Of the paraocular glands, the Harderian gland contains a dense infiltration of plasma cells. Some workers observed that these infiltrates along the ducts were primarily small lymphocyte nodules with germinal centers (19). The Harderian gland is a retrobulbar, tubulo-alveolar gland located in the orbit medial to the eyeball (163). Its secretions empty into the conjunctival space behind the nictitating membrane by a single duct. The Harderian gland is thought to contribute to the immune function of the orbital, nasal, and upper respiratory tract tissues.
The Harderian gland is divided into lobules composed of columnar epithelium supported by fibrovascular Interstitial septa. It is surrounded by a thin connective tissue capsule (194). Ultrastructurally, four types of epithelial cells are recognized
(164). The interstitium of the Harderian gland is infiltrated by lymphoid cells between 17 to 18 days of embryonic development. Lymphocytes, macrophages, 12
myoepithelial cells and heterophils are also present in the gland (19,167).
Plasmablasts forming desmosome-like junctional complexes with each other and
macrophages have been observed ultrastructurally (167). Plasma cell infiltration
begins at the time of hatching (194) and increases with age (176). Studies in
chickens have shown early infiltration of IgM-positive cells at 2 to 4 weeks (6). IgG-
and IgA-positive cells increased from 4 to 9 weeks in similar studies (6,176).
Mansikka et al., (1989), detected all three isotypes of immunoglobulins in the
Harderian gland plasma cells even in the absence of local stimulation, as determined by Northern hybridization, in situ hybridization and ELISA (123).
Numerous studies have shown antibody producing capability by the plasma cells in the Harderian gland. Conjunctival inoculation with Newcastle disease virus, infectious bronchitis virus (IBV) and Mycoplasma gallisepticum induced production of specific homologous antibody in the Harderian gland (158) in constrast to no detectable antibody responses after parenteral inoculation. One study showed lymphoid follicle formation at 14 to 21 days in the Harderian gland after parenteral inoculation with IBV (57).
Conjunctiva Associated Lymphoid Tissue (CALT)
CALT is morphologically similar to GALT and SALT (17,47,73) and contains multiple nodules of lymphocytes beneath the palpebral conjunctiva of the lower eyelid (17,47). CALT might function as a site of antigen uptake and initiation of a local immune response (17,47,48,72). Infection of turkeys with 6. avium increased 13 the number of germinal centers in CALT (72). Studies of CALT in rabbits (77),
humans (26,76) and birds (19,73,83,153) confirmed existence of plasma cells
producing secretory IgA. CALT in turkeys seems to resemble morphologically that in
rabbits. CALT in turkeys has been proposed to function as a site of antigen uptake, processing and presentation (74).
Lymph Nodes
Lymph nodes, classically present in mammals, are not present in most avian species including turkeys. Instead, there are regional concentrations of lymphoid tissue throughout the organs, tissues and lymphatic vessels (63). These lymphoid tissues are primarily made up of small lymphocytes. Lymphoid accumulations in chickens probably function like mammalian lymph nodes and have been observed along the posterior tibial, poplitial and lower femoral veins (145). These femoral lymph nodules have afferent and efferent lymphatics, peripheral germinal centers and internal sinuses equipped with valves to direct the flow of the lymph.
Mucosal and Systemic Humoral Immune System of Poultry
Avian Immunoglobulins
The major difference between avian and mammalian immunoglobulins is the presence of a major serum 7S immunoglobulin heavy chain that is about 10 Kd heavier than the mammalian gamma-chains. Immunoglobulin isotype diversity in 14
birds, is structurally limited to only three isotypes (7S IgG, IgM and IgA) with no
subclasses (25).
Immunoglobulin G (IgG)
The 75 IgG of chickens has several characteristics that resemble human IgG,; like susceptibility to proteolytic digestion, aggregation, and an H chain larger by one domain. This IgG has a total carbohydrate content of 6.0% (1,95), weighs 165 to
180 Kd (118) and has a sedimentation coefficient of 7.3 to 7.4 S (26). X-ray small- angle scattering studies have shown that the chicken IgG molecule is less flexible in comparison to mammalian IgG. The concentration of chicken IgG in normal serum ranges from 5-7 mg/ml (116). The metabolic half-life of chicken IgG is 3 days in neonatal (152) and 1.5-4.3 days in adult birds (119). In contrast to riiammalian species, IgG in the chicken is the predominant immunoglobulin in the tracheal secretions (48).
Immunoglobulin M (IgM)
Chicken IgM is similar to its mammalian counterpart in many respects.
Chicken IgM is found in serum as a pentamer and has a molecular weight of about
890 Kd and a sedimentation coefficient of 17 S (24,118). Monomeric IgM has also been reported in fresh serum treated with lO ^M iodoacetamide (116). The carbohydrate content is 6.6% (11). The metabolic half-life of chicken IgM is 1.7 days
(118). Mockett observed common IgM determinants in different avian species (134). 15
A secretory component associated with IgM has been detected as a free component
in egg white (157). Mockett (1986), detected IgM in bile and speculated that it might also be transferred from blood to bile by binding to secretory component (133).
Peppard et al. have shown the existence of a functional homologue of mammalian secretory component in chickens and biochemically characterized it (154,155). The concentration of IgM in normal chicken serum is 1-2 mg/ml (116).
Immunoglobulin A (IgA)
Birds have abundant polymeric IgA in bile and intestinal secretions (147, 116).
There is a relatively higher concentration of IgA in secretions on all mucosal surfaces, in comparison to the serum. In chickens, IgA forms less than 4% of total serum immunoglobulins (34). Polymeric IgA is present in bile and intestinal secretions, saliva, tears and tracheal washings of turkeys (60). Structurally, avian IgA resembles mammalian IgA, although this has not been rigourously tested by molecular methods
(115). It occurs in monomeric and polymeric forms, with the latter dominating in secretions and serum (34,120). Only 20% of serum IgA is monomeric, the other
80% exists in dimeric or multimeric forms (116). The molecular weight and sedimentation coefficients range from 350-900 Kd and 9-16 S, respectively (151,157).
Avian IgA has a J- chain (136) and a secretory component (25). Genome sequencing may be helpful in clarifying the degree of homology of avian IgA to other mammalian
IgA. The concentration of IgA in serum, intestine and bile is 0.3 to 0.645, 1.6 and
3.15 mg/ml, respectively (116). 16
Mammalian IgA has been functionally credited with inactivating bacterial
enzymes and toxins, antibody-dependent cell mediated cytotoxicity (ADCC),
preventing bacterial attachment to host cells and virus neutralization. Specific details
about the function of IgA in birds are lacking. Studies on the immune function of
avian IgA in experimental infections with Newcastle disease virus and infectious
bronchitis virus (IBV) were inconclusive (150). IgA was expected to be related to
viral neutralizing activity in tracheobronchial secretions after vaccination with
modified-live IBV, but a challenge infection provoked an IgG response in secretions with a minimal IgA response. Other immunoglobulin isotypes may substitute for the protective functions of IgA at mucosal surfaces in IgA deficient humans and birds as
IgM can also associate with secretory component (51,114).
Antibody Response in Avian MALT
Even though MALT occurs at various anatomic sites, the function is rather uniform. This function includes antigen uptake, processing, and antibody production at the mucosal surface. A number of papers have reviewed MALT functions
(28,29,33,86).
Antigen Uptake and Presentation
The first and most important step for generating an immune response is antigen uptake. This uptake mechanism has been evaluated primarily using carbon particles, latex beads or tracer macromolecules like ferritin, mycobacteria and horse 17 radish peroxidase (HRPO). The lymphoepithelium on the surface takes in the antigen by a process of pinocytosis (37). Carbon particles have been demonstrated histologically crossing the lymphoepithelium of rabbit appendix and mouse Peyer's patches followed by localization in the mesenteric lymph nodes (85,107). Latex beads have also been used in similar studies in rats, mice and dogs (117,192). Apical vesicles are present in lymphoepithelial cells of mammalian BALT, especially when an active immune response has been stimulated (160,188). The uptake of HRPO by
M-cells has been elucidated ultrastructurally in mouse Peyer's patches (148,164).
Studies have shown that tracer particles are taken up by BALT lymphoepithelium as well (188). Studies using ferritin and B. avium showed that both ciliated and non- ciliated cells of BALT are capable of uptake (70,72,75). These studies showed ultrastructurally that these particles appear in organelles associated with endocytosis.
Also, uptake of tracer particles like carbon, iron oxide and latex beads by lymphoepithelial cells in CALT was shown by a recent study (74).
Some studies have shown that both bronchial and nasal epithelial cells were able to present antigen to T cells and stimulate mixed lymphocyte reactions (MLR) in allogeneic systems (109). Also, studies on GALT have concluded that absorptive enterocytes constitutively express MHC-II (125) and are capable of presenting antigen and stimulating T cell proliferation, and stimulating IL-2 production (36,108,125). 18
Immune Response in MALT
Various immunocytochemical studies have shown that B lymphocytes and T lymphocytes of both T-helper and T-cytotoxic/suppressor subsets are present in BALT nodules, often in localized compartments (40,170). Distinct T- and B- lymphocyte regions have been demonstrated in the BALT of poultry (69,106). Antigen presenting accessory cells are of different kinds in the lymphoid regions of BALT and in the germinal centers of chicken BALT (106). Macrophages are distributed throughout the lung parenchyma and BALT in chickens (46,106). Several studies have demonstrated the appearance of germinal centers in the nasolacrimal duct, GALT, tonsils and BALT of chickens between 2 and 3 weeks of age (10,19,105,177). Germinal centers are prominent in the avian BALT, supporting the idea that this tissue may functionally compensate for the lack of lymph nodes in the bronchial region in birds. The presence of germinal centers might simplify the model for antigen initiated antibody response. This model indicates that virgin or naive B cells first encounter the antigen in T cell zones composed of T-helper lymphocytes, B lymphocytes and interdigitating dendritic cells (IDC). These antigen primed B cells migrate to primary follicles, where they enter the phase of exponential cell division. Affinity maturation also takes place at this step and only B cells with high affinity are allowed to proliferate.
Among these B cells, some proceed to small memory B cells while others differentiate into plasma cells producing immunoglobulins. Thus, the development of germinal centers seems to be an evolutionarily conserved plan as evidenced by its presence in several mammalian and avian species, like the model for cellular 19
interactions between T and B lymphocytes and APCs (186). The development of
BALT depends on the new antigens encountered everyday and on the genetic variability among animals (8).
Secretory IgA has been considered the primary humoral defense antibody on the mucosal surfaces. The dimeric or polymeric configuration associated with the secretory chain facilitates the mucosal transport of IgA (185). Studies in rabbits and humans have shown that B- immunoblasts in the mucosal lymphoid organs are mostly committed to the production of IgA (52,98). An effective presentation of processed antigen fragments to immature B cells turns on antibody production (113).
Isotype switching due to different interleukins might be one mechanism of secreting specific antibodies. The committed B cells leave the lymphoid tissue and migrate to various mucosal surfaces as immunoglobulin-producing plasma cells. The presence of processed antigen, APCs, interleukins, receptors and other vascular features contribute to the localization and distribution of antibody secreting cells at various mucosal surfaces (27,54). Also, it is known that the presence of secretory component in IgA permits transport of this antibody through the cytoplasm of the the epithelial cells
(162,181). Several studies have identified IgG and IgM in the secretions from mucosal surfaces.
Initiation of antibody responses at various sites other than the site of antigen administration is an effective component of mucosal and systemic immune responses.
This is made possible by the selective localization of T and B lymphocytes around the body (27,97,196). This selective localization is termed homing and has been 20
reviewed extensively. The high endothelial venules (HEVs) in lymph nodes act as
binding sites for the entry of lymphocytes expressing specific surface adhesion
molecules (196). This process of homing dictates the distribution of lymphocytes to
different tissues (50). The tissue origin of lymphocytes influences their subsequent
distribution to distant sites, thus lymphocytes from BALT tend to home to the lungs
and respiratory mucosa, lymphocytes from GALT tend to home to the gastrointestinal
tract (45,131). These homing patterns and subsequent interactions dictate the antibody response at different mucosal sites. These studies support the presence of a distinct humoral immune mechanism of MALT in response to antigens.
Respiratory Immunity in Poultry
Potential role of MALT in Respiratory Immunity in Birds
Because avian MALT develops at strategic sites of exposure to external antigens, it is likely that MALT has an important role in the immune response. In the respiratory tract, the BALT is located at the airway bifurcation points where there is optimal deposition of antigens due to changes in airflow direction and velocity (32).
This suggests that the lymphoepitheiium of BALT would be one of the initial contact points for airborne pathogens. The two main functions of BALT epithelium are uptake and transport of antigens. Additionally, lymphoepithelial cells may have a role in antigen presentation. Antigen transported across the lymphoepitheiium may interact with the underlying lymphoid cell population leading to a mucosal immune response. 21 Knowledge of avian BALT, suggests that both B- and T- lymphocyte compartments are present in SALT nodules and both T- helper and T-cytotoxic /
suppressor cells are involved (10,106). Also, accessory cells, including follicular dendritic cells (FDC) and macrophages, are present in avian BALT (46,106,188).
Unlike mammalian BALT, avian BALT has prominent germinal centers (31), in support for a humoral immune response function (122). Though there is little experimental documentation about high endothelial venules (HEV) and lymphocyte homing in BALT of chickens, they do occur in CALT and GALT (22,73). However,
HEV are located at the periphery of BALT nodules, mostly at the T and B lymphocyte region interface. Hence, the evidence points to a potential role of avian MALT in the humoral immune response and lymphocyte mediated immune help such as cytokine production, immune response enhancement, or immunoglobulin switch.
Humoral immunity
Antibody has a major role in protecting the host against infection with various pathogens. It can act in different ways; either by preventing attachment and colonization of pathogens to host cells or by neutralizing toxins and other harmful molecules. Indirectly, antibody aids in the clearance of bacteria by phagocytes and antibody-dependent cell mediated cytoxicity (ADCC). Several papers have suggested a role for immunoglobulins in protection against respiratory pathogens of poultry, such as in infectious bronchitis virus (IBV), Newcastle disease virus (NOV), influenza virus. Mycoplasma gallinarum, Escherichia coli and Bordetella avium (13,55, 22 62,139J 97).
The antibody in the respiratory tract, primarily detected in tracheal secretions,
may be secreted by the local plasma cell population or be transported to the
respiratory mucosal surface via the bloodstream (110). IgG diffuses most readily from
serum to the airway mucosa due to its small size (161). In contrast, IgA and IgM
found on respiratory mucosal surfaces are chiefly derived from the local plasma cell population rather than from serum. Transport of serum IgA and IgM to the respiratory mucosal surface has not been reported in avian species. Apart from these origins, antibody may also be found in mucosal secretions due to capillary permeability changes associated with local inflammation.
The potential role of each immunoglobulin isotype has not been discerned for the majority of mucosal diseases in mammals as well as in birds (49). However, IgA is considered to be the most important immunoglobulin isotype at mucosal surfaces in mammals. Since the absence of IgA does not result in profound susceptibility, other immunoglobulin isotypes must be important as well (114). The data for birds regarding the role of each immunoglobulin isotype is very limited.
Cell Mediated Immunity
Cytotoxic lymphocytes, macrophages, natural killer (NK) cells and granulocytes are the components of cell mediated immunity (CMI). In contrast to mammalian NK cells, in birds NK cell activity is not restricted to a single cell population. 23
In contrast to mammals, the lung lavage from birds is predominated by
heterophils with fewer lymphocytes and macrophages (80). Avian heterophils lack
myeloperoxidase and catalase enzymes and thus are incapable of killing several kinds
of bacteria, hence the birds may be more susceptible to respiratory disease (183).
However, lysozyme and cationic enzymes in heterophil granules are capable of
killing bacteria, and avian heterophils are capable of protecting birds against several
respiratory pathogens (183,184). In addition, the epithelial cells lining various gas
exchange regions are capable of phagocytosing foreign material.
The potential role of avian NK cells in respiratory defense has not been explored fully. However, some reports have indicated that NK cells may mediate cytotoxic activity against infectious bronchitis virus and Marek's disease (159). NK cells in birds act in a similar manner to mammalian counterparts; that is, they carry out their cytotoxic activity independently of major histocompatibility restriction (71).
In addition to non-specific cell mediated cytotoxicity, certain cells are capable of specifically killing cells (target cell) expressing surface antigens (tumor or viral).
Cytotoxic T lymphocytes (CTLs) kill cells expressing foreign antigens complexed with
MHC antigens of Class I. Also, cells such as NK cells, macrophages and granulocytes express Fc receptors and thus recognize antibody complexed with foreign agents
(ADCC). Avian macrophages and heterophils (61,178) express Fc receptors, but
ADCC has not been explored for non-respiratory pathogens (159,169). Lymphocyte blastogenesis and lymphocyte migration inhibition (LMI) tests indicate a potential role of CMI in protecting chickens against infectious bronchitis virus (IBV) (55). 24 Antibody Transport
In mammals, the three main immunoglobulin isotypes, IgG, IgA and IgM, are
capable of diffusing from serum into airways in small amounts (110). However, IgG,
in view of its relatively smaller size (161), is more abundant in airways than IgM and
IgA (126).
IgG transport in human and canine respiratory epithelium, has been shown to
occur by a receptor-mediated mechanism (132). This mechanism has not been fully
explored in avian respiratory epithelium. A major portion of polymeric IgA and very
little of IgM is transported by binding to polymeric Ig receptor (pIgR) on epithelial
cells (9). This transport mechanism is seen in bronchial epithelial cells of mammals
(32). No knowledge about such a transport mechanism is currently available for
avian species.
Immunoglobulins have been demonstrated in respiratory secretions of chickens
in response to agents such as Newcastle disease virus and avian paramyxovirus
(2,62,66). Several studies in birds showed that the amount of IgG in nasal, tracheal and tracheobronchial secretions exceeds that of IgA (23,48,67,82). These studies also speculated that there may be selective mechanisms for IgG transport in birds.
Paraocular fluids contain antibodies of different isotypes chiefly secreted from the plasma cells in regional glands (56). Plasma cells in the Harderian gland have been shown to secrete infectious bronchitis virus-specific IgA as demonstrated by immunofluorescence studies (56). Mucosal secretions contain both IgA and IgG, although IgA initially is the chief immunoglobulin (2,66). In virus infected chickens, 25
IgG overtakes other immunoglobulins and inhibits replication in the trachea for 4
weeks (66). In poultry, IgA is found in lesser quantities than IgG in tracheal
secretions, saliva and tears (48,116). IgA:IgG in serum and secretions indicate that
most of the IgA in the respiratory tract is locally produced (48). IgA is selectively
enriched in tracheal washings in comparison to serum, thus signifying an important
role for IgA, however, studies on the role of IgA in neutralizing Newcastle disease
virus or infectious bronchitis virus were inconclusive (67,82,150). In the IBV study, a
higher dose challenge elicited a prominent IgG response in secretions and little IgA
response.
Studies on the distribution of immunoglobulins in the respiratory tract of sheep showed that IgM was the main immunoglobulin seen by immunofluorescence and immunoperoxidase techniques in the nasal and bronchial glands of lambs before suckling (7). The same study showed that in adult sheep, IgA- and IgG- containing cells were in equal numbers in nasal mucosa and IgA cells predominant in the bronchial mucosa and lung. Cytoplasmic staining of bronchial epithelial cells for IgA and IgM was seen most frequently in areas of proliferating epithelium suggesting that immature cells may be engaged in immunoglobulin transport.
Pittard et al. (1977), proposed that the breastmilk macrophage is a potential vehicle for immunoglobulin transport (156). Burton and Smith (1977) showed that endocytosis plays a role in the immunoglobulin transport across the small intestine of new-born pig (44). Devenny and Wagner (1985) proved the ability of capillary endothelial vesicles to transport IgG across the capillary wall. Capillary endothelial 26
cells were found to endocytose fluorescent labeled IgG via a bulk fluid phase
mechanism. Transport of immunoglobulins and other proteins takes place in a
bidirectional manner across the peritoneal tissue (58).
Few studies have been conducted on the metabolism and passive transfer of
immunoglobulins in turkey hens. A study by Dohms and Salf (1978), showed that
IgG is the chief immunoglobulin transferred from hen to the egg. The transport of
IgM and IgA into the egg was considered negligible and unimportant (59). Chickens
have been passively protected against Eimeria tenella infection by parenterally
administered monoclonal antibody of IgCj isotype (53). Thus, serum antibody has
been shown to play an important role in immunity to coccidiosis.
Studies on kinetics of immunoglobulin transport into canine bronchial
secretions showed that a small percentage of IgG administered passively is recovered
in the secretions indicating transfer of humoral IgG into mucosal secretions (111).
Furthermore, the same study showed that IgM administered intravenously was not detected at all in these secretions. 1-125 labelled IgG has been shown to be transported across the small intestine of rats (137).
Toro et al. (1993), have shown that peak levels of 1-125 labelled IgG administered intravenously into the wing vein of chickens could be detected in the lacrimal secretions within 10 minutes (182). This study also suggested that the IgG level in serum declines rapidly from day one to day 20. Keeping these different interpretations of several studies in view, the role of IgG seems to be important even on mucosal surfaces. 27 Antibody Catabolism
Studies on the metabolism of immunoglobulins were largely conducted using
isotypes labelled with radioactive chemicals. The metabolism of immunoglobulins is
similar to other proteins, with increased catabolism occuring during hyperthermia
(18). The rate of immunoglobulin synthesis and degradation depends on the class of
immunoglobulins. In humans, an increase in the serum concentration of IgG
enhances its degradation rate, possibly due to an autoregulatory mechanism. The
same principle, however, does not apply to IgM and IgA, and, IgD and IgE are
catabolised rapidly even when their concentrations are low. Fab fragments and light
chains have very short half-lives while Fc fragments have a half-life equal to the
whole IgG molecule. It is useful to remember that more than half of the IgG and IgA
molecules are extravascular, while the IgM is largely intravascular (18). The catabolic
rate (percentage of intravascular pool broken down per day) of human
immunoglobulins were 6.7, 25, 18, 37 and 89 for IgG, IgA, IgM, IgD and IgE,
respectively (18). Similar data are not available for avian species.
There are different ways by which antibody concentrations in the body are
influenced. Antigen-antibody complexing, internalization and shedding of antibody leads to false interpretation of antibody catabolism studies. This happens in many viral and bacterial infectious diseases and tumors like Burkitt's lymphoma and human colon cancer (168,191). Also, the labelling method used in catabolism studies might complicate the results. In one study by Matzku et al., use of 67-Ga-phenolic aminocarboxylic acid chelate labelled monoclonal antibody demonstrated a major 28 difference from ^"-labelled monoclonal antibodies, probably due to longer retention of this conjugate in tissues physiologically involved in antibody catabolism (124).
Hereditary errors may also lead to faulty catabolism of immunoglobulins; in myotonic dystrophy an accelerated breakdown of IgG occurs (195).
Liver is the primary organ involved in antibody catabolism (79). However, a study expressing antibody on a concentration basis, showed that spleen accounted for
247% of the saturable compartment/kg; whereas, liver accounted for 25%/kg along with a minimal saturable uptake by bone marrow (64). This same study speculated that saturable uptake may relate to sinusoidal blood supply characteristic of liver, spleen and bone marrow.
A study on the kinetics of the decline phase of the primary response to human serum albumin in the chicken, showed that the antibody levels in the decline period
(7-17 days) manifested exponential decay and a constant t1/2 of 2.2 days, which according to the authors, approximated that observed with passively administered labelled 7S chicken immunoglobulin (88). These data suggest that antibody catabolism in general occurs in all animals and is similar to the catabolism of other plasma proteins. Interpretation of antibody kinetic studies is complicated by various factors influencing the distribution and catabolism of immunoglobulins. 29
Bordetellosis in Turkeys
Bordetellosis is a highly contagious upper respiratory tract disease of turkeys
characterized by sneezing, oculonasal mucus discharge, submandibular edema,
collapse of trachea and a predisposition to other infectious diseases (16). The
economic loss due to bordetellosis has not been assessed; but, losses due to impaired
growth and mortality due to colisepticemia and other secondary bacterial infections
might cause losses up to several million dollars annually to the turkey industry in the
USA. The disease has also been referred to as turkey coryza, alcaligenes
rhinotracheitis (ART), B. avium rhinotracheitis (BART) or turkey rhinotracheitis.
Etiology
The disease bordetellosis is caused by a gram negative bacterium called
Bordetella avium. The disease has been successfully transmitted to susceptible poults
(172.173). Bordetella avium is a motile, strictly aerobic, non-fermenting bacterium
(112.174). Morphological, nutritional, physiological, serological, electrophoretic and
DNA-RNA hybridization studies confirmed this bacterium as belonging to the
Bordetella genus. Infection with B. avium accompanied by stress is the primary cause of this disease. 30
Growth Characteristics and Colony Morphology
Bordetella avium produces small, compact, translucent, pearl-like colonies on
MacConkey's agar. Colonies reach a size of 1 to 2 mm after 24 to 48 hours of
incubation at 25°C (174).
Pathology, Clinical Signs and Lesions
Bordetella avium tends to be less pathogenic in chickens than in turkeys (135).
Natural infection in turkeys is typically recognized in 2 to 6 week old poults
(38,90,149). Bordetellosis is transmitted to susceptible turkey poults through close contact with infected birds or contaminated feed or water (172). The incubation period in natural infection ranges from 7-10 days (172); whereas, experimental intranasal and intraocular inoculation results in clinical signs within 4-6 days
(11,165).
The bacteria initially adhere to the ciliated epithelial cells of the upper respiratory tract. One review suggested that the pili of 8. avium are involved in the in vitro attachment of the bacteria to the mucosal surface of turkey tracheal expiants
(101). Colonization and expansion of the bacterial population leads to acute inflammation and mucus discharge from goblet cells causing sneezing and obstruction of the upper airways (84). The progressive sloughing of epithelial cells colonized by 6. avium leads to loss of cilia and prevents subsequent mucus clearance
(11). This damage to the ciliated epithelium is probably due to a tracheal cytotoxin
(81). Obstruction of the nasolacrimal ducts causes accumulation of foamy exudate in 31
the nares and medial canthus of the eye.
The clinical signs of bordetellosis seem to result from local and systemic
products of the inflammatory response, soluble bacterial toxins and physical
obstruction of the upper respiratory tract (16). The most prominent sign of
bordetellosis in a majority of 2 to 6 week old turkeys is sudden onset of sneezing.
The profuse mucus discharge from the eyes and nares gives the feathers of the head a
wet or dry matted appearance with isolated brownish plaques and stains. Some birds
develop submaxillary edema. Many birds show open mouth breathing, dyspnea,
altered vocal sounds, reduced intake of feed and water, and reduced activity. These
signs diminish after 2 to 4 weeks of infection (38,84,165), but the birds may become
more susceptible to other infectious agents.
The gross lesions in the upper respiratory tract are characterized by
generalized softening and distortion of the tracheal cartilage rings leading to dorso-
ventral collapse (11,187). The combination of tracheal collapse and accumulation of exudates seriously impinge on the tracheal lumen.
The histopathologic lesions of bordetellosis are characterized by cilia- associated bacterial colonies, loss of cilia, and desquamation of ciliated epithelial cells (11,12). The bacterial colonies are prominent features on the cilia 1 to 2 weeks after onset of clinical signs (11,99). Squamous metaplasia of tracheal epithelium Is not a feature in the early stages of infection though it may occur at a later stage.
Dysplastic changes of epithelium have been recognized in birds infected for 3 to 4 weeks. Goblet cells are depleted of mucus granules from the first to the third week 32
after onset of clinical symptoms. The lamina propria of the trachea contains multiple
aggregates of heterophils which are replaced by lymphocytes and plasma cells with
time (11,12). The plasma cells increase in numbers by the 3rd to 5th week of
disease and lymphoid nodules develop in the submucosa. BALT appears
hypertrophic and lymphoid nodules protrude into the bronchial lumen during the
course of the infection (187).
Immunity and Vaccination
Humoral Immunity
Antibodies are produced in most turkeys infected with B. avium (11,99).
Microtiter agglutination and ELISA have been used to detect serum antibodies within
2 weeks after the beginning of infection. Peak levels of antibody occur after 3 to 4
weeks of infection (11,99). Clinical signs of the disease subside within 1 week after
peak antibody titers. The presence of maternal immunoglobulins against B. avium
also suggests an important role for humoral immunity (13,91). Arp and Hellwig
successfully inhibited the adherence of B. avium to the tracheal mucosa by treatment
with convalescent serum and tracheal secretions from infected turkeys (87). This
inhibition was possible both with local and parenteral administration of convalescent
serum. The same authors, using Western immunoblots, identified at least eight
different bacterial proteins recognized by antibody in serum and tracheal secretions during a 4 week course of infection (87). The serum antibody response has been
shown to depend on the dosage of inoculum, age of the bird and factors influencing 33
colonization of the bacteria (15,42,89,92,94,100,104). Immunization studies using
vaccine strains show that birds less than 3 weeks of age develop a poor immune
response (92,100). Studies on the effect of 6. avium infection on cellular immunity
identified no effect on CMI (127-129), though one study by Simmons et al. (171)
showed thymic lesions and suppression of lymphocyte blastogenesis. Studies on
susceptibility of B. avium infected birds to live Pasteurella multocida and
hemorrhagic enteritis vaccines showed that these birds have reduced immunity to
these two agents.
Vaccination
The two vaccines that have been used for protection against bordetellosis
include a live temperature sensitive mutant (ts) of B. avium (ART-Vax^*^, American
Scientific Laboratories, Madison, Wl) and a whole-cell bacterin (ADJUVAC-ART, Ceva
Laboratories, Inc., Overland Park, KS). One more whole cell Bordetella avium bacterin is currently being used (Tri Bio Laboratories, Inc., Collegepark, PA). Original studies indicated that the live ts mutant vaccine could induce moderate serum antibody titers (41). However, further studies produced conflicting reports of its protective capability against the disease (89,92,94,100). Although the ts-mutant was able to adhere to respiratory epithelium and replicate slowly, it colonized poorly and induced limited immunity (14,15). The ts-mutant failed to prevent infection in day- old poults, but prevented the disease when used in poults 3 weeks of age or older.
This may be due to clearance of the inoculum by maternal antibody or to the 34
inherent inability of younger turkeys to mount a successful immune response or the
inherent resistance of 3-week old birds to resist infection.
Passive immunization with convalescent serum in 3-week old birds reduced
the adherence of 6. av/um to the tracheal mucosa (14). Also, vaccination with purified pili from 6. av/um protected the turkeys against B. av/um infection by preventing colonization (3).
It is inferred from these different studies that, the development of an effective vaccine against B. avium requires a better understanding of the immune responses, both maternal and acquired mucosal responses in turkeys, and better characterization of the bacterial antigens that are important in adhesion and colonization.
Diagnosis
Diagnosis of bordetellosis is based on clinical signs and lesions, isolation of B. avium, and serologic tests like ELISA, microagglutination test, biochemical tests and colony characteristics or a combination of these. Recently, a monoclonal antibody based latex bead agglutination test has been developed that can identify B, avium and differentiate B. avium from various B. awum-like strains (175).
Use of Serology for Diagnosis
Serological methods for the diagnosis of bordetellosis in turkeys have been previously described (20,93,99). An understanding of the levels of various antibody isotypes is helpful in assessing the stage of infection and the immune status in the 35 bird. A knowledge about the antibody isotypes in serum and secretions from mucosal surfaces could be helpful in employing easy methods of detecting antibodies, A knowledge of the serology will also help in studying immune responses to various vaccine preparations and assessing the value of each of these preparations in protecting the bird from bordetellosis. 36
MUCOSAL AND SYSTEMIC HUMORAL IMMUNE RESPONSE TO BORDETELLA AVIUM IN EXPERIMENTALLY INFECTED TURKEYS
Manuscript in press for publication in Avian Diseases, 38(2): 1994.
P. Suresh, L. H. Arp, and E. L. Huffman
Summary
Antibody response to Bordetella avium was measured in serum and mucosal secretions of experimentally infected turkeys. One-day-old turkeys were inoculated with 6. avium, and four inoculated turkeys and four uninoculated control birds per trial were euthanatized weekly from 1 through 8 weeks postinoculation (Weeks PI).
Maternal antibody of the IgG isotype, present in all 1-day-old birds sampled, decreased to background levels by 3 weeks of age. Antibody (IgG, IgM, IgA) was detected in serum, tracheal washings, and lacrimal secretions in response to 8. avium infection. Regardless of the sample site and isotype, antibody levels peaked at 4-6 weeks PI and then decreased rapidly from 6 to 8 weeks PI. In general, IgM and IgA peaked earlier (4-5 weeks PI) but declined more rapidly than IgG levels. Numbers of
B. avium in the trachea peaked at 2-3 weeks PI and then decreased rapidly from 4 to
8 weeks PI. Even though no direct causal relationship could be determined, the results indicate that an increasing level of antibody in serum, tracheal washings, and lacrimal secretions is temporally associated with clearance of B. avium from the trachea. 37
Introduction
Bordetella avium is a gram-negative, non-fermenting bacterium that causes a highly contagious upper-respiratory-tract infection in young turkeys (8,12,14). The clinical disease is characterized by oculonasal discharge, sneezing, dyspnea, tracheal collapse, and a reduced rate of weight gain (6,13). Cilia-associated bacterial colonies, progressive loss of ciliated epithelium, and depletion of mucus from goblet cells are distinctive characteristics of bordetellosis in turkeys (2,7). Colonization of the ciliated epithelium of the upper respiratory tract appears to be of singular importance in development of the disease.
Most turkeys develop a serum antibody response to infection with B, avium
(2,11). Serum antibodies, detected by microtiter agglutination, appear within 2 weeks after experimental exposure to B. avium and reach peak levels by 3 to 4 weeks postexposure (2,11). In one study, a rising serum antibody titer was associated with decreasing bacterial numbers in the trachea and recovery from clinical disease (11).
The serum antibody response, combined with evidence of maternal immunity, suggests an important role for humoral immunity in prevention of and recovery from infection with 8. avium (5,9). Convalescent serum and tracheal secretions from turkeys infected with 8. avium inhibit adherence of the bacteria to the tracheal mucosa in turkeys (3). In addition, adherence of B. avium is inhibited, whether convalescent serum is administered locally or parenterally.
Although several vaccination strategies have been developed for the prevention of bordetellosis in turkeys, our understanding of the essential components 38
of protective immunity remains incomplete. The present study was undertaken to
characterize the mucosal and systemic humoral immune response to 6. avium and to
study the relationship between antibody levels and bacterial numbers in the trachea.
Materials and Methods
Turkeys. Straight-run broad-breasted, white Nicholas turkeys were obtained
commercially (Willmar Poultry, Willmar, MN.) at 1 day of age; the turkeys used
were free of Mycoplasma meleagridis and Newcastle disease virus (NOV). Turkeys
were identified by wing-band and randomly assigned to one infected and one control
group for each trial. Forty to 45 turkeys were assigned to each group to compensate
for possible mortality. Turkeys in each group were placed in heated brooders (wire
cages) housed in separate identical environmentally controlled rooms with ample
ventilation at a temperature of 29-30 C. All turkeys were provided with antibiotic-
free commercial starter (Purina Mills Inc., St. Louis, MO.) and water ad libitum. The
birds were maintained in brooder batteries for the first 3 weeks and then moved to
wire grower cages.
Bacteria and Inocula. Bordetella avium strain 75 was originally isolated in Iowa from the trachea of a young turkey with respiratory disease (2). Bordetella avium strain F9000336 was similarly isolated in California and provided by Dr. Richard P.
Chin (Fresno, Calif.) (15). Bacteria were cultured aerobically with agitation for 24 hours in brain-heart infusion broth, and cultures were then diluted 1:100 with cold phosphate- buffered saline (PBS) to a final bacterial concentration of about 10^ 39
colony-forming units (CPU) per ml. Each inoculum was maintained on ice and
sampled for determination of CFU/ml immediately after inoculation. The inocula and
all subsequent tracheal isolates were verified as B. avium by reaction in a latex bead
agglutination test (15).
Experimental Design. Two identical inoculation trials were conducted. At 2 days of
age, four turkeys from each group were selected for collection of serum, tracheal
washings, and lacrimal secretions. In each trial, the remaining turkeys in one group
were inoculated with B. avium, and turkeys of the other group served as
uninoculated controls. Bordetella avium strain 75 was used as inoculum in the first
trial and strain F9000336 was used in the second trial. Each week, four infected
turkeys and four uninfected control turkeys were euthanatized for bacterial culture
and for collection of serum, tracheal washings, and lacrimal secretions. Clinical signs
of the disease were recorded weekly and gross lesions were recorded at necropsy.
Enzyme-linked immunosorbant assay (ELISA), was used to measure levels of IgA,
IgM, and IgG in serum and mucosal secretions.
Sample collection. Lacrimal secretions (3.5 //I) were collected from each
conjunctival sac of all sampled birds by absorption into a 3-mm disc of No. 42
Whatman filter paper, which was placed under the eyelid until saturated. This
procedure was standardized among birds by time and volume absorbed by the disc.
Discs from each eye were combined and placed with 600 //I of diluent (0.02 M PBS
with 0.05% Tween 20, pH 7.4) and stored at -70 C until required for the (ELISA).
To obtain tracheal washings, birds were euthanatized by intravenous injection of 40
pentobarbital sodium, and the cervical trachea was aseptically exposed. The trachea
was clamped midway from the larynx to the thoracic inlet, and 200 fj\ of diluent was
instilled into the upper tracheal segment. The trachea was gently massaged for 1 minute, and tracheal washings were collected with a syringe and catheter, further diluted to a total volume of 600//I, and stored at -70 C until required for ELISA.
For quantitative bacterial culture, a 1-cm segment of trachea was excised immediately below the clamp and placed in 9 ml of cold PBS with 1 % Triton X-100
(Eastman Kodak Co., Rochester, NY.) for 1 hour. For collection of serum 2 ml of blood was collected from the jugular or brachial vein of each bird, and serum was separated.
ELISA. The relative quantity and isotype of antibody in serum, lacrimal secretions, and tracheal washings were determined by an ELISA modeled after that described by
Hopkins et al. (10). Flat-bottomed 96-well Immulon 2 plates (Dynatech Laboratories,
Chantilly, VA.) were coated with whole-cell B. avium strain 75, washed, and blocked with 0.02 M PBS containing 5% normal goat serum (Bethyl Laboratories, Inc.,
Montgomery, Texas). Plates were stored in plastic bags at 4C and used within 3 months. Diluted tracheal washings and lacrimal secretions were thawed, mixed, and centrifuged to remove exudate and debris. Serum was mixed 1:100 in diluent. Test wells of each plate were filled with 100 //I of sample and incubated at 37 C for 1 hour. Plates were washed three times with 0.02 M PBS containing 0.1 % normal goat serum, emptied, and tapped dry. Two hundred (200//I) of horseradish peroxidase- conjugated goat anti-chicken (IgG, IgM, and IgA) antibody (Bethyl), diluted 1:800, 41
was added to each well as needed. Chicken antibodies used for immunization of
goats were purified by affinity chromatography (Bethyl Laboratories) and evaluated by
gel immunodiffusion (data not shown). Plates were incubated 2 hours at 37 C and
then washed three times as before. The ABTS substrate (Kirkegaard and Perry
Laboratories, Gaithersburg, Maryland) was prepared immediately before use by
mixing equal volumes of solutions A and B. One-hundred microliters of substrate
was added to each well, and the reaction was stopped after 9 minutes by the addition
of 50 //I of 0.2 M sulfuric acid. Absorbance (A) of each well was determined at 410
nm in a MR 700 microplate reader (Dynatech). Positive control wells on each plate
were loaded with pooled convalescent serum for IgG and IgM and pooled tracheal
washings for IgA collected from 6. avium-infected turkeys in previous studies.
Negative control wells were loaded with diluted serum from normal turkeys not
exposed to B. avium. Additional control wells lacked antigen, primary antibody,
secondary antibody, and substrate. All samples were tested in duplicate on different
plates.
Quantitative bacterial culture. Tracheal segments in cold PBS with Triton-X 100
were held at 4 C for 1 hour and then mixed 1 minute on a vortex mixer at the
maximum setting. All of these solutions were repeatedly diluted 1:10 in cold PBS
(pH 7.4) and plated immediately on blood agar as previously described (1). Plates were incubated 48 hours at 37 C, and colonies were counted.
Statistical analysis. The effects of treatment, bacterial strain, duplicate tests, and sample site were compared using A readings from ELISA for each of the three I;
42
immunoglobulin isotypes. Numerical data were evaluated by an analysis of variance
(ANOVA) with General Linear Models (PROC-GLM) as components of the SAS
software (Statistical Analysis Systems, Gary, N.G.). Split-plot analysis with treatment
as the main factor and source of isotype and time intervals as the subplot factors were
used to analyze the treatment effects. Significance levels of P ^ 0.05 were used
throughout unless indicated otherwise.
Results
Turkeys inoculated with either strain of B. avium developed a frothy ocular
and nasal discharge, matted feathers on the head, dyspnea, sneezing, and poor
weight gains from 1 to 4 weeks post-inoculation (weeks PI). Distortion and collapse
of the trachea was most obvious postmortem in turkeys euthanatized 2-4 weeks PI.
Clinical signs of disease and gross lesions tended to be slightly more severe in turkeys
inoculated with B. avium strain F9000336. Uninoculated control turkeys remained
clinically normal and free from infection throughout the studies. The B. avium
inocula used in both the trials contained 10^ GFU/ml. Bacterial isolates having
colony morphology suggestive of bordetella reacted positively in the latex bead
agglutination test for B. avium. Statistical comparisons of serological and
bacteriological data indicated no significant differences related to the strain of 8.
avium used, so, data were combined for the two trials.
Fig. 1 shows A values for antibody to B. avium in serum, tracheal washings,
and lacrimal secretions. No significant differences were seen between samples tested Fig 1. Antibody response to B. avium in serum, tracheal washings, and lacrimal
secretions of experimentally infected turkeys. Data are expressed as A from
ELISA conducted from 0 to 8 weeks PI. Vertical lines on the bars indicate SEM
(n -8, combined data, two trials). The A readings for IgM and IgA isotypes at
all locations in the control group were less than 0.1. 44
Serum
0 Trachea
• Lacrimal *« IS
2 3 4 5 6 Weeks Post-Inoculation IgG
0 Senim
0 Trachea
• Lacrimal % S i
0 1 2 3 4 5 6 Weeks Post-inoculation IgM 0.6 r
0 Serum E 0.5 - ë 0 Trachea 5? 0.4 - Tb • Lacrimal < % 03 I 0.2 - 0.1
2 3 4 5 6 7 Weeks Post-inoculation 45
on duplicate plates. Because antibody was collected from the tracheal mucosa by
washing, direct quantitative comparisons of antibody in serum, tracheal washings,
and lacrimal secretions were not made. Therefore, all comparisons are made only
with control turkeys of the same age. As all the ^ readings from the three collection
sites for immunoglobulin isotypes IgM and IgA in the control birds were less than
0.1, no graphical presentation or significant differences are needed for comparison.
A statistically significant (P ^ 0.01) elevation in IgA and IgM was detected in serum,
tracheal washings, and lacrimal secretions from 1 to 8 weeks PI. For each anatomical
site, IgA reached a peak at 3-5 weeks PI and then declined through 8 weeks PI (Fig.
1). Elevation of IgM reached a peak at 5 weeks PI and declined to near baseline
levels by 8 weeks PI, but was still significantly different (Fig. 1).
Maternal IgG against B. avium was detected in the serum of most turkeys sampled at 2 day of age. In turkeys of the control group, no antibody response to B. avium was detected during the study, except for maternal IgG, which went down rapidly in the first 3 weeks (Fig. 2) The IgG response to infection, first detected at 3 weeks PI, peaked at 5-6 weeks PI, and declined gradually through 8 weeks PI (Fig. 1).
Numbers of 6. avium colonizing the trachea increased steadily to 10'' ' CFU/cm at 3 weeks PI (Fig. 3) and then decreased exponentially to minimally detectable levels at 8 weeks PI in five of eight turkeys necropsied. Fig 2. Mean IgG Absorbance (6) reading at 410nm in control group serum
(n-8, combined data, two trials). Maternal IgG levels. 47
Control IgG In Serum 0.25
E O 0.2
^ 0.15 S 8 e 0.1 I a 0.05
0 1 2 3 4 5 6 7 8 Age in Weeks Fig 3. Colonization of the trachea by B. avium in trials 1 and 2. Each point
represents the mean (n-4) Log,o CFUs per linear cm of trachea expressed
in log,o. 49
Bacterial Count
7 Trial 1
Trial 2 6
o O)
1 2 3 4 5 6 7 8 Weeks Post-inoculation 50
Discussion
Results of these studies have suggested that antibody may play a significant
role in recovery of turkeys from and immunity to bordetellosis (5,9,17).
Administration of convalescent serum to turkeys inhibits adhesion of B. avium to the
tracheal mucosa (3). In the present study, we have demonstrated that IgA, IgM, and
IgG antibody isotypes are reliably produced in response to infection by B. avium.
Additionally, these isotypes are detectable in both serum and mucosal secretions.
Because B. avium specifically colonizes the ciliated epithelial cells of the upper
respiratory tract, the quantity and quality of antibody present on the tracheal mucosa
is of particular interest. Results from the present study indicate that all isotypes were present in tracheal washings, but IgA antibody may be detected in the trachea earlier
than IgM and IgG. The IgM response in the trachea reached a peak at about 4-5 weeks PI, roughly the time at which numbers of B. avium colonizing the trachea began to decline. In contrast, IgG levels in the trachea peaked after bacterial numbers had decreased substantially. Unfortunately, results from the present study do not allow us to accurately predict the relative importance of each antibody isotype in recovery from bordetellosis.
In general, levels of IgG and IgM in the serum correlated with levels in tracheal washings and lacrimal secretions at most times during the course of infection. This finding is consistent with the idea that these isotypes are capable of moving between the blood vascular system and mucosal surfaces. Recently, transfer of IgG from the serum to lacrimal fluids has been characterized in chickens (16). In 51
the current study, however, it is probable that IgG detected in mucosal secretions is
derived both from serum and local plasma cells. IgG was generally best detected in
serum, whereas IgM and IgA were at least as readily detected in tracheal washings
and lacrimal secretions.
The functional significance of anti-6. avium antibody in the lacrimal secretions
is unknown. Since the lacrimal secretions drain into the nasal cavity from the nasal
lacrimal ducts, there may be some benefit in clearing bacteria from the nasal mucosa
to reduce bacterial transmission and persistence. Use of lacrimal secretions for the
early serologic detection of B. avium infection is possible if one uses a detection
system for either IgA or IgM antibody.
Several authors have described serologic methods for the diagnosis of bordetellosis in turkeys (4,10,11). These authors have discussed the effectiveness of
ELISA and agglutination methods for detecting bordetellosis. Results of the present study indicate the importance of understanding when each antibody isotype is elevated during the course of disease. Serologic methods for detecting IgM may be most useful from the second to the fifth week of disease, whereas a method to detect
IgG will aid in the diagnosis only after the first 3 weeks of infection. Our results indicate that IgG can be expected to persist in the serum beyond the eighth week of disease.
The temporal relationship between increasing serum and mucosal antibody levels and the number of B. avium colonizing the tracheal mucosa suggests that antibody may contribute to recovery from infection. The critical time of infection 52
when numbers of bacteria begin to decline appears to be about 3-4 weeks after
exposure. All of the isotypes measured in this study are readily detectable in the
tracheal secretions during the third to fourth week of infection. Therefore, the
relative importance of each antibody isotype in resistance to bordetellosis remains
unknown. Additional studies will be needed to address this issue. The results of
the present study may serve as a reference for those attempting to stimulate immunity
to bordetellosis through either passive or active immunization.
Acknowledgements
The authors thank Mrs.Li Ling Wu, Department of Statistics, for her help in
designing the experiment and analysing the data.
References
1. Arp, L. H., and E. E. Brooks. An in vivo model for the study of Bordetella avium adherence to tracheal mucosa in turkeys. Am. J. Vet. Res. 47:2614-2617. 1986.
2. Arp, L. H., and N. F. Cheville. Tracheal lesions in young turkeys infected with
Bordetella avium. Am. J. Vet. Res. 45:2196-2200. 1984.
3. Arp, L. H., and D. H. Hellwig. Passive immunization versus adhesion of
Bordetella avium to the tracheal mucosa of turkeys. Avian Dis. 32:494-500. 1988.
4. Barbour, E. K., M. K. Brinton, S. D. Torkelson, J. B. Johnson, and P. E. Poss. An enzyme-linked immunosorbent assay for detection of Bordetella avium infection in turkey flocks: sensitivity, specificity, and reproducibility. Avian Dis. 35:308-314. 53 1991.
5. Barnes, H. J., and M. S. Hofstad. Susceptibility of turkey poults from vaccinated and unvaccinated hens to Alcaligenes rhinotracheitis (turkey coryza). Avian Dis.
27:378-392. 1983.
6. Berkhoff, H. A., H. J. Barnes, S. I. Ambrus, M. D. Kopp, G. D. Riddle, and D. C.
Kradel. Prevalence of Alcaligenes faecalis in North Carolina broiler flocks and its relationship to respiratory disease. Avian Dis. 28:912-920. 1984.
7. Gray, J. G., J. F. Roberts, R. C. Dillman, and D. G. Simmons. Pathogenesis of change in the upper respiratory tracts of turkeys experimentally infected with an
Alcaligenes faecalis isolate. Infect. Immun. 42:350-355. 1983.
8. Hinz, K.-H., G. Glunder, and H. Lunders. Acute respiratory disease in turkey poults caused by Bordetella bronchiseptica-like bacteria. Vet. Rec. 103:262-263.
1978.
9. Hinz, K.-H., G. Korthas, H. Luders, B. Stiburek, G. Glunder, H. E. Brozeit, and T.
Redmann. Passive immunisation of turkey poults against turkey coryza (Bordetellosis) by vaccination of parent breeders. Avian Pathol. 10:441-447. 1981.
10. Hopkins, B. A., J. K. Skeeles, G. E. Houghten, and J. D. Story. Development of an enzyme-linked immunosorbent assay for Bordetella avium. Avian Dis. 32:353-361.
1988.
11. Jackwood, D. J., and Y. M. Saif. Development and use of a microagglutination test to detect antibodies to Alcaligenes faecalis in turkeys. Avian Dis. 24:685-701.
1980. 54
12. Kersters, K., K.-H. Hinz, A. Hertle, P. Segers, A. Lievens, O. Siegmann, and J.
De Ley. Bordetella avium sp. nov. isolated from the respiratory tracts of turkeys and
other birds. Int. J. Syst. Bacteriol. 34:56-70. 1984.
13. Saif, Y. M., P. D. Moorhead, R. N. Dearth, and D. J. Jackwood. Observations on
Alcaligenes faecal is infection in turkeys. Avian Dis. 24:665-684. 1980.
14. Simmons, D. G., J. G. Gray, L. P. Rose, R. C. Dillman, and S. E. Miller.
Isolation of an etiologic agent of acute respiratory disease (rhinotracheitis) of turkey
poults. Avian Dis. 23:194-203. 1979.
15. Suresh, P., and L. H. Arp. A monoclonal antibody-based latex bead
agglutination test for the detection of Bordetella avium. Avian Dis. 37:767-772. 1993.
16. Toro, H., P. Lavaud, P. Vallejos, and A. Ferreira. Transfer of IgG from serum to
lachrymal fluid in chickens. Avian Dis. 37:60-66. 1993.
17. Tuomanen, E. I., L. A. Zapiain, P. Galvan, and E. L. Hewlett. Characterization of
antibody inhibiting adherence of Bordetella pertussis to human respiratory epithelial cells. J. Clin. Microbiol. 20:167-170. 1984. 55
A TIME-COURSE STUDY OF THE TRANSFER OF IGC FROM BLOOD TO TRACHEAL AND LACRIMAL SECRETIONS IN YOUNG TURKEYS
Manuscript for submission to Avian Diseases.
P. Suresh and L. H. Arp
Summary
Three-week-old turkeys were injected intravenously with Bordetella avium- specific IgC and absorbance readings were measured by an ELISA at various time intervals in blood, tracheal washings and lacrimal secretions. The IgG was detected in tracheal and lacrimal secretions as early as 5 minutes after injection and reached a peak 10 minutes after injection. Thereafter, the IgG absorbance declined rapidly reaching background levels by 24 hours. The absorbance of IgG reached an approximate equilibrium in all three locations by 60 minutes. The levels of IgG in all three sites were comparable at all times from 10 minutes to 24 hours after administration. The results of this study indicate that transfer of IgG from blood to mucosal surfaces in turkeys occurs rapidly.
Introduction
The importance of antibody in the defense of mucosal surfaces is well documented in poultry. A variety of avian respiratory pathogens such as infectious bronchitis virus (IBV), Newcastle disease virus, Mycoplasma gallisepticum, 56
Escherichia coli and B. avium stimulate the local and systemic production of antibody
(3,5,11J 8). The source of antibody in respiratory secretions is from the bloodstream
or from plasma cells present locally in the subepithelial tissues (7).
Of the three major antibody isotypes commonly detected in respiratory
secretions of mammals, IgA is frequently predominant. In contrast, IgG is frequently
the predominant isotype in respiratory secretions of avian species (2). In a
quantitative study of immunoglobulin isotypes in the lacrimal secretions of chickens,
Lebacq-Verhayden et al. (9) found the relative amounts of IgG, IgM, and IgA to be
0.87, 0.01 and 0.15 mg/ml, respectively. Of the three major immunoglobulin isotypes, IgG transfers most readily from the bloodstream to the mucosal suface of airways (10,12). In a recent study of the humoral immune response of young turkeys infected with B. avium, significant amounts of IgG, IgM, and IgA were measured by
ELISA in tracheal washings (15). The relative amount of IgG in the serum compared closely with amounts in the tracheal and lacrimal secretions over the course of infection. A recent study by Toro et a/. (17) using I'^^-labelled chicken IgG showed similar movement of parenterally injected IgG from the bloodstream to lacrimal secretions. Studies by these authors also showed a similar kinetic pattern of IgG in both lacrimal fluid and serum in response to IBV vaccination.
Few studies have been conducted on the metabolism and passive transfer of immunoglobulins in turkeys. Dohms and Saif (1978) showed that IgG is the primary immunoglobulin transferred from hen to the egg. These authors considered the movement of IgM and IgA into the egg as negligible and unimportant (4). However, 57
there has been no study of IgG movement from the bloodsteam to mucosal secretions
in turkeys. The purpose of this study was to document movement of IgG from the
bloodstream to the tracheal and lacrimal secretions in young turkeys.
Materials and Methods
Experimental Design. Young turkeys were injected intravenously with Bordetella
av/um-specific IgG and levels of specific antibody were determined in serum,
lacrimal secretions, and tracheal washings at time intervals from 5 minutes to 72
hours. Thirty-three turkeys were injected with 0.9 ml of B. av/um-specific IgG (8.7
mg/ml). Samples of blood, lacrimal secretions, and tracheal washings were collected
from three turkeys at 5, 10, 20, 40, and 60 minutes, 2, 4, 8, 24, 48 and 72 hours after administration of IgG. In addition, three turkeys, that were not injected with antibody, were euthanatized and sampled at the beginning and end of the study. The samples of serum, tracheal washings and lacrimal secretions were evaluated by ELISA for B. av/um-specific IgG.
Turkeys. Thirty-nine, one-day-old, straight run. Broad-breasted white Nicholas strain turkeys were obtained from Willmar Poultry, Willmar, MN. and maintained in brooder batteries for two weeks, and then moved to wire grower cages. Turkeys were individually identified with numbered wing-bands. Antibiotic-free commercial starter (Purina Mills Inc., St. Louis, MO.) and water were provided ad libitum. The turkeys used in this experiment were determined to be free of maternal antibody to
B. avium by ELISA. 58
Purification of immunoglobulin G. Several three-week-old turkeys were immunized subcutaneously with live B. avium twice at 2 week intervals. The blood from these turkeys was harvested one week after the last immunization and serum was separated and pooled with serum obtained from some birds used in an earlier study which had been experimentally infected with 6. avium by intra-nasal and intra-ocular routes
(16). IgG from this pooled serum was purified by a combination of Sephadex and
DEAE column chromatography as previously described (13). Briefly, the pooled sera were delipidated at 4 C using 4 ml of 5% dextran sulfate and 10 ml of 0.25 M MnClj per 40 ml of whole turkey serum. Precipitated lipids were removed by centrifugation at 8,500 X g for 15 minutes at 4 C. The supernatant was precipitated three times in
50% sodium sulfate in an ice bath. After each precipitation, the preparation was centrifuged at 3000 X g for 15 minutes at 4 C. The pellet was resuspended in one- half the original volume in PBS, pH 7.6. After the final step, the resuspended pellet was dialysed for 48 hours in PBS with constant stirring at 4 C. This preparation was then subjected to chromatography on a Sephadex G-200 (2.5 X 100cm) column and fractions were collected. The fractions of the second peak were concentrated and recycled on a Sephacryl HS-200 (2.5 X 45 cm) column and all fractions from a single symmetrical peak were pooled. These concentrated fractions were then subjected to
DEAE chromatography with a series of PBS buffer changes at pH 7.6, 6.4, 5.8, 5.4,
4.7 and 4.4. The IgG was eluted at 0.06 M PBS, pH 7.6 to a maximum concentration. These fractions were reconcentrated 12 fold by recycling in the same
PBS on DEAE three times. Further concentration of the specific IgG was achieved by 59
CNBr affinity chromatography on a B. avium-OMP bound CNBr activated Sephadex column (1 X 30 cm) as described (8J4). Purity of the IgG solution was assessed by agar gel diffusion and ELISA. For ELISA, plates were coated with B. avium followed by specific IgG. The secondary antibodies used were goat anti-chicken IgG, IgM and
IgA which were tested by agar gel diffusion and ELISA for specificity. The final concentration of the anti-B. avium IgG was 8.7 mg/ml.
Sample collection. At each time point, blood, tracheal washings and lacrimal secretions were collected from three turkeys. Blood (2ml) was collected from jugular or brachial vein and serum was separated. Lacrimal secretions (3.5 //I) were collected from each conjunctival sac by absorption into a 3-mm disc of No. 42
Whatman filter paper which was placed under the eyelid until saturated. This procedure was standardized among turkeys by time and volume absorbed. Discs from each eye were combined and placed with 600//I of diluent (0.02 M PBS with
0.05% Tween 20, pH 7.4) and stored at -70 C until required for the ELISA. To obtain tracheal washings, turkeys were euthanatized by intravenous injection of pentobarbital sodium, and the cervical trachea was aseptically exposed. The trachea was clamped midway from the larynx to the thoracic inlet, and 200//I of diluent was instilled into the upper tracheal segment. The trachea was gently massaged for 1 minute, and tracheal washings were collected with a syringe and catheter, further diluted to a total volume of 600 //I, and stored at -70 C until required for ELISA.
ELISA. The relative quantity of B. avium specific IgG in lacrimal secretions, serum and tracheal washings was determined by an enzyme-linked immunosorbent assay 60
modeled after that described by Hopkins et al., (6,16). Flat-bottomed 96-well
Immulon 2 plates (Dynatech Laboratories, Chantilly, Virginia.) were coated with
whole-cell B. avium (strain 75), washed, and blocked with 0.02 M PBS containing
5% normal goat serum (Bethyl Laboratories, Inc., Montgomery, Texas). Plates were
stored in plastic bags at 4C and used within 3 months. Diluted tracheal washings
and lacrimal secretions were thawed, mixed, and centrifuged to remove exudate and
debris. Serum was mixed 1:100 in diluent. Test wells of each plate were filled with
100 //I of sample and incubated at 37 C for 1 hour. Plates were washed three times
with 0.02 M PBS containing 0.1% normal goat serum, emptied, and tapped dry.
Next, 200 fj\ of horseradish peroxidase-conjugated goat anti-chicken IgG antibody
(Bethyl), diluted 1:800, was added to each well as needed. Chicken antibodies used
for immunization of goats were purified by affinity chromatography (Bethyl
Laboratories, Inc., Montgomery, Texas) and evaluated by gel immunodiffusion. Plates
were incubated 2 hours at 37 C and then washed three times as before. The ABTS
substrate (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland.) was prepared
according to the manufacturer's directions. One-hundred microliters of substrate was
added to each well, and the reaction was stopped after 9 minutes by the addition of
50 /j\ of 0.2 M sulfuric acid. Absorbance (A) of each well was determined at 410 nm in a MR 700 microplate reader (Dynatech Laboratories, Chantilly, Virginia). Positive control wells on each plate were loaded with pooled convalescent serum for IgG collected from B. av/um-infected turkeys in previous studies. Negative control wells were loaded with diluted serum from normal turkeys not exposed to 6. avium. All 61
samples were tested in duplicate on different plates.
Statistical Evaluation. All the numerical data were evaluated by analysis of variance
(ANOVA) with PROC-GLM as components of SAS software (Statistical Analysis
Systems, Inst., Gary, NC). The effects of the three locations, time intervals and
duplicate tests were determined by this model. The levels of significant differences
between means were always expressed at P < 0.05, unless indicated otherwise.
Results
High absorbance readings of S. av/um-specific igC were detected in tracheal
washings and lacrimal secretions 5 minutes after the intravenous administration of
IgG (Fig.1). Peak readings of specific IgG in the serum were detected at 5 and 10
minutes post-administration (PA). The absorbance readings of specific IgG reached a
peak in the lacrimal secretions at 5 minutes PA and in the tracheal washings at 10 minutes PA. From 10 to 40 minutes PA there was a rapid decline in specific IgG
from serum and mucosal sites. The rate of antibody decline in fluids sampled was much less from 60 minutes to 48 hours PA compared with the 10 to 40 minute time period PA. By 72 hours PA, levels of antibody detectable in all fluids were similar to levels in control turkeys. Absorbance readings of specific IgG were comparable in serum, tracheal washings, and lacrimal secretions from 60 minutes throughout the duration of the study. Fig. 1. Mean IgG absorbance at 410 nm. For each data pointy n - 3 birds. Serum Û , Tracheal washings —V-, Lacrimal secretions • 63
Serum ^Trac.wash. Lacr.secr.
I a I I I fi~\ I I / / I I I 0 5 10 20 40 60 2 4 8 24 48 72 Minutes Hours 64
Discussion
This study demonstrated the rapid movement of IgG from the blood to tracheal and lacrimal secretions in normal turkeys. Substantial movement occurred by the first sample time at 5 minutes PA. Our study differed from other studies on immunoglobulin kinetics in that we employed a specific ELISA system to detect the
IgG in contrast to radiolabelled immunoglobulins usually employed for similar studies. In a recent study in chickens using l"^-labelled IgG, a similar pattern of movement was noted from serum to lacrimal secretions (17).
The kinetics of IgG movement during the first 60 minutes PA could be due to one of several mechanisms including diffusion or active transport. We did not design our study to detect any specific mechanism of transfer of IgG. However, either of these two mechanisms could occur, as there might be specific receptor mediated mechanism favouring active transport which is highly functional or it could occur by a relatively simpler process such as diffusion. In contrast to IgM and IgA, IgG may diffuse across normal tissue barriers, since it has a relatively low molecular weight
(12). The nearly parallel clearance of IgG from serum, tracheal washings, and lacrimal secretions from 10 to 40 minutes PA suggests that levels of IgG had been reached in all the three fluids. Also the rapid clearance of the purified preparation of
IgG used in this study might partially be due to relatively shorter half-life (tl/2) in comparison to native IgG. We could not account for all the IgG that was injected in our study. Certainly, a significant amount of IgG could be lost through respiratory and lacrimal secretions over time, but other large mucosal surfaces such as the 65 gastrointestinal tract were not examined. Some loss may also be expected from the hepatobiliary and urinary systems.
If IgG has a role in bacterial clearance it would be in the initial few hours after parenteral administration. In our study, the levels of intravenously administered IgG reached baseline levels by 24 hours, which is more rapid than that reported in mammals (12). These results agree with a study by Arp and Hellwig (1), in which convalescent serum administered intravenously essentially diminished B. avium adherence to tracheal mucosa only during the initial 1 to 6 hours and not 24 hours after injection.
In conclusion, this study showed that IgG is readily transferred from the bloodstream to tracheal and lacrimal surfaces in healthy young turkeys.
Documentation of relatively high levels of IgG in respiratory secretions in birds (2,9) and evidence of efficient movement of IgG from the blood support a possible role for
IgG in defense against mucosal pathogens.
Acknowledgements
The authors are thankful to Mrs. Li Ling Wu, statistics consultant, Iowa State
University for her help in design of the experiment and analysis of data. We are also thankful to Mrs. Elise Huffman, Department of Veterinary Pathology, for her excellent technical help. 66
References
1. Arp, L. H., and D. H. Hellwig. Passive immunization versus adhesion of
Bordetella avium to the tracheal mucosa of turkeys. Avian Dis. 32:494-500. 1988.
2. Chhabra, P. C., and M. C. Goel. Normal profile of immunoglobulins in sera and
tracheal washings of chickens. Res. Vet. Sci. 29:148-152. 1980.
3. Darbyshire, J. H. Immunity to avian infectious bronchitis virus. In: "Avian
Immunology" M. E. Rose, L. N. Payne, and B. M. Freeman, Eds., British Poultry
Science Ltd., Edinburgh, Scotland., pp. 205-226. 1981.
4. Dohms, J. E., Y. M. Saif, and W. L. Bacon. Metabolism and passive transfer of
immunoglobulins in the turkey hen. Am. j. Vet. Res. 39:1472-1481. 1978.
5. Easterday, B. C. Immunity to Newcastle disease and avian influenza. In: "Avian
Immunology, Symp. 16" M. E. Rose, L. N. Payne, and B. M. Freeman, Eds., British
Poultry Science Ltd., Edinburgh, UK., pp. 179. 1981.
6. Hopkins, B. A., J. K. Skeeles, G. E. Houghten, and J. D. Story. Development of
an enzyme-linked immunosorbent assay for Bordetella avium. Avian Dis. 32:353-361.
1988.
7. Kaltreider, H. B. Local Immunity. In: "Immunology of the Lung and Upper
Respiratory Tract" J. Bienenstock, Ed., McGraw-Hill Book Company, New York, NY., pp. 191-215. 1984.
8. Kohn, J., and M. Wilchek. The use of cyanogen bromide and other novel cyanylating agents for the activation of polysaccharide resins
.Appl.Biochem.Biotechnol. 9:285-304. 1984. 67
9. Lebacq-Verheyden, A. M., J. P. Vaerman, and J. F. Heremans. Quantification and
distribution of chicken immunoglobulins IgA, IgM, and IgG in serum and secretions.
Immunol. 27:683. 1974.
10. Mazanec, M. B., J. G. Nedrud, X. Liang, and M. E. Lamm. Transport of serum
IgA into murine respiratory secretions and its implications for immunization strategies.
J. Immunol. 142:4275-4281. 1989.
11. Myers, R. K., and L. H. Arp. Pulmonary clearance and lesions of lung and air
sac in passively immunized and nonimmunized turkeys following exposure to aerosolized Escherichia coll. Avian Dis. 31:622-628. 1987.
12. Reynolds, H. Y. Immunoglobulin G and its function in the human respiratory tract. Mayo Clinic. Proc. 63:161-174. 1988.
13. Saif, Y. M., and J. E. Dohms. isolation and characterization of immunoglobulins
G and M ofthe turkey. Avian Dis. 20:79-95. 1976.
14. Stiller, J. M., and K. H. Nielson. Affinity purification of bovine antibodies to
Brucella abortus lipopolysaccharide. j. Clin. Microbiol. 17:323-326. 1983.
15. Suresh, P., and L. H. Arp. Serum and lacrimal antibody response to Bordetella avium infection in turkeys. Proceedings of the CRWAD, Chicago, IL. Abstract #261.
1990.
16. Suresh, P., L. H. Arp, and E. L. Huffman. Mucosal and systemic humoral immune response to Bordetella avium in experimentally infected turkeys. Avian Dis.
38(2). In Press. 1994.
17. Toro, H., P. Lavaud, P. Vallejos, and A. Ferreira. Transfer of IgG from serum to 68 lachrymal fluid in chickens. Avian Dis. 37:60-66. 1993.
18. Yagihaski, T., and M. Tajima. Antibody responses in sera and respiratory secretions from chickens infected with Mycoplasma gallisepticum. Avian Dis.
30:543-550. 1986. 69
EFFECT OF PASSIVELY ADMINISTERED IMMUNOGLOBULIN G ON THE COLONIZATION AND CLEARANCE OF BORDETELLA AVIUM IN TURKEYS
Manuscript for submission to Veterinary Immunology and Immunopathology.
P. Suresh, L. H. Arp and J. S. Haynes
Summary
This study was conducted to detect the effect of parenterally administered IgC on the colonization and clearance of Bordetella avium at the tracheal surface in young turkeys. In two separate experiments, three-week-old turkeys were infected with B. avium either after or before IgC administration. The comparisons were made between a control group which received a non-S. avium specific IgG (anti-KLH IgC) and the experimental group which received a specific IgG (anti-S. avium IgG). When given prior to bacteria, IgG reduced the numbers of colony forming units (CPUs).
Apart from non-specific immune mechanisms, specific IgG also seemed to have a role in preventing colonization. Similarly, in the second experiment to study the role of
IgG in bacterial clearance, when the IgG was given after bacterial inoculation, it had a significant effect in reducing the CPUs from the tracheal surface. Overall, the studies indicated a role for IgG in B. avium infection. These observations are further discussed in relation to the importance of IgG in such mucosal infections. 70
Introduction
Bordetella avium is the cause of a highly contagious, upper respiratory tract
disease of young turkeys (10,24). The disease is characterized by oculonasal
discharge, sneezing, dyspnea, tracheal collapse, and reduced weight gain (2,23,24).
The tracheal lesion is consistently characterized by colonization of ciliated respiratory
epithelium by 6. avium progressing to loss of ciliated cells and accumulation of
mucus and exudate in the trachea (2,9). Adherence of S. avium to the ciliated
epithelium is a prerequisite for colonization of the tracheal surface (2,9).
Antibodies have been shown to be important components in the defense of mucosal surfaces against viral and bacterial infections of humans and animals (18,20).
Antibodies have been shown to provide protection in the respiratory tract of chickens challenged with various respiratory pathogens, such as infectious bronchitis virus,
Newcastle disease virus, Mycoplasma gallisepticum, Escherichia coli, and B, avium
(5,6,19). Although information regarding the concentration of various immunoglobulins in tracheal secretions is incomplete, all three immunoglobulin isotypes (IgA, IgG and IgM) have been detected in tracheal secretions (15,16,28).
Antibody in the respiratory tract may be secreted by local plasma cells or transported from the bloodstream to the respiratory mucosal surface (12). Of the three isotypes, IgG diffuses most readily from the bloodstream to the respiratory mucosa due to its relatively small size (21). Transport of IgA and IgM to the respiratory mucosal surface from the bloodstream has not been reported in avian species. In addition to the transport of antibody across normal endothelial-epithelial 71
barriers, antibody of any isotype may cross the endothelial-epithelial barriers during
times of severe local inflammation and tissue injury.
Little is known of the role of each antibody isotype on the respiratory mucosa
of avian species. In a study of the relationship between serum antibody and the
adherence of Bordetella pertussis to human respiratory epithelial cells, the presence
of IgG and IgA in the convalescent serum was directly related to the antiadhesive
activity of the specific serum against the organism (30). Studies in our laboratory
have shown that parenterally administered convalescent serum effectively inhibited
the adherence and colonization of B. avium to the tracheal surface in turkeys (3). In a previous study we demonstrated the rapid and effective transfer of IgG from the bloodstream to tracheal secretions (27). Furthermore, in experimental bordetellosis of turkeys, IgG is the isotype that is present for the longest period of time compared with IgM or IgA (28). Although each of the three antibody isotypes may be important in 6. avium infection, only IgG lends itself to the time course-study of the earliest stages of infection by 8. avium. Preliminary studies documented that when convalescent serum (with IgG, IgM, and IgA) is administered intravenously to young turkeys, only B. av/um-specific IgG and small amounts of IgA are detectable in the tracheal secretions (data not shown). Therefore, the purpose of this study was to determine the effect of B. av/um-specific IgG on the colonization and clearance of B. avium in young turkeys. 72 Materials and Methods
Experimental design. In two separate experiments, 23-25 day-old turkeys were used.
The first experiment was designed to study the effect of 6. av/um-specific IgG on colonization by 6. avium. Fifty-four birds were randomly selected and divided into two groups of 27 each. Birds in the first group were injected intravenously into the wing vein with B. av/um-specific IgG. Birds in the second group were similarily injected with a non-6, avium specific IgG, an IgG specific for keyhole limpet hemocyanin. Ten minutes after injection of IgG, birds were inoculated intratracheally with 6. avium and samples were collected from 10 minutes to 48 hours.
The second experiment was designed to study the role of IgG in the clearance of 6. avium. Fifteen turkeys were inoculated with B. avium. Three of these turkeys were euthanatized at 24 hours post inoculation (PI) for quantification of bacteria in the trachea and anti-8. avium IgG in the serum and tracheal washings. The remaining 12 turkeys were divided into two groups of 6 each. One group was injected intravenously with anti-fi. avium IgG at 8 hour intervals and the other group was treated similarly with anti-KLH IgG at 8 hour intervals. Three birds from each group were euthanatized at 48 and 72 hours PI. Samples collected at the time of euthanasia from each turkey included: blood (serum), tracheal washings, and trachea for bacterial culture. ELISA was used to measure IgG specific for B. avium or KLH in the serum and tracheal washings.
Turkeys. Day-old, straight run, broad-breasted white Nicholas strain turkeys (n - 69) were obtained (Willmar Poultry, Willmar, Minn.). The birds were maintained in 73
brooder batteries for the first two weeks and then moved to wire grower cages.
Water and antibiotic-free commercial starter feed (Purina Mills Inc., St. Louis, MO.)
were provided ad libitum. All turkeys were individually identified by numbered
wing bands.
Bacteria and inoculum. Bordetella avium strain 75 originally isolated from the
trachea of a young turkey with respiratory disease was used in both experiments.
Bacteria were cultured aerobically with agitation for 24 hours in brain heart infusion
(BHD broth and transfered to BHI agar plates for an additional 24 hour incubation at
37 C. The bacterial growth was washed from the plates with cold phosphate-buffered
saline (PBS; pH 7.4) to make a bacterial suspension of approximately 10'° colony-
forming units (CFU)/ml of inoculum. For inoculation, a calginate swab dipped in
bacterial suspension was passed as gently as possible down the trachea three times.
The inoculum and representative colonies from tracheal cultures were verified as B. avium by the latex bead agglutination test (26).
Immunoglobulin G. After inoculation of several three-week-old turkeys subcutaneously at two week intervals with live B. avium, blood was harvested one week after the second inoculation, serum was separated and pooled with serum obtained from some birds used in the earlier study wherein birds were experimentally infected with 6. avium by intra-nasal and intra-ocular routes. For KLH, three-week- old turkeys were immunized subcutaneously twice at weekly intervals and blood was collected a week after the final injection and serum separated. The IgG was purified by a combination of Sephadex and DEAE column chromatography as previously 74
described (22). Further concentration of the specific IgG was done by CNBr affinity
column (13,25). The purity was assessed by agar gel diffusion and ELISA. For ELISA,
plates were coated with either B. avium or KLH followed by specific IgG. The
secondary antibodies were goat anti-chicken IgG, IgM and IgA. The final
concentration of the anti-B. avium IgG and anti-KLH IgG was 8.7 mg/ml and 9.4
mg/ml, respectively. In both experiments, turkeys were injected intravenously with
0.9 ml of concentrated anti-6. avium IgG or with 1.2 ml of anti-KLH IgG solution.
Sample collection. For experiment one, serum, tracheal washings, and tracheal
segments were collected from three turkeys at 0, 10, 30, and 60 minutes, and 2, 4, 8,
24 and 48 hours after bacterial inoculation. Two ml of blood was collected from the
jugular or brachial vein and serum was separated. To obtain tracheal washings, birds were euthanatized by intravenous injection of pentobarbital sodium and the cervical trachea was aseptically exposed. The trachea was clamped midway from the larynx to the thoracic inlet, and 200 //I of ELISA diluent was instilled into the upper trachea.
The trachea was gently massaged for one minute, and tracheal washings were collected with a syringe and catheter, further diluted in the ELISA diluent to a total volume of 600 //I, and stored at -70 C until required for ELISA. For quantitative bacterial culture, a 1-cm segment of trachea was excised immediately below the clamp and placed in 9 ml of cold PBS with 1 % Triton X-100 (Eastman Kodak Co.,
Rochester, NY.) for 1 hour.
ELISA. The relative quantity of IgG specific for either KLH or B. avium in serum and tracheal washings was determined by an enzyme-linked Immunosorbent assay 75
modeled after that described by Hopkins et al., (11,28). Flat-bottomed 96-well
Immulon 2 plates (Dynatech Laboratories, Chantilly, Virginia.) were coated with
whole-cell B. avium strain 75 antigen, washed, and blocked with 0.02 M PBS
containing 5% normal goat serum (Bethyl Laboratories, Inc., Montgomery, Texas).
Plates were stored in plastic bags at 4C and used within 3 months. Diluted tracheal
washings and lacrimal secretions were thawed, mixed, and centrifuged to remove
exudate and debris. Serum was mixed 1:100 in diluent. Test wells of each plate
were filled with 100 /j\ of sample and incubated at 37 C for 1 hour. Plates were
washed three times with 0.02 M PBS containing 0.1% normal goat serum, emptied,
and tapped dry. Next, 200 fj\ of horseradish peroxidase-conjugated goat anti-chicken
IgG antibody (Bethyl), diluted 1:800, was added to each well as needed. Chicken
antibodies used for immunization of goats were purified by affinity chromatography
(Bethyl Laboratories, Inc., Montgomery, Texas). Plates were incubated 2 hours at 37
C and then washed three times as before. The ABTS substrate (Kirkegaard and Perry
Laboratories, Gaithersburg, Maryland.) was prepared immediately before use by
mixing equal volumes of solutions A and B. One-hundred microliters of substrate
was added to each well, and the reaction was stopped after 9 minutes by the addition
of 50 fj\ of 0.2 M sulfuric acid. Absorbance (A) of each well was determined at 410
nm in a MR 700 microplate reader (Dynatech Laboratories, Chantilly, Virginia).
Positive control wells on each plate were loaded with pooled convalescent serum for
IgG from B. av/um-infected turkeys in previous studies. Negative control wells were
loaded with diluted serum from normal turkeys not exposed to 6. avium. All 76
samples were tested In duplicate on different plates.
The ELISA method for detecting the antl-KLH IgG was similar to that described
above except that each well on the plate was coated with KLH at a concentration of
1 microgram/ml and the secondary antibody goat antl-chlcken IgG (HRPO) was used
at 1:1000 dilution.
Quantitative bacterial culture. Tracheal segments, in cold PBS with Triton-X 100,
were held at 4 C for 1 hour and then mixed 1 minute on a vortex mixer at the
maximum setting. All of these solutions were serially diluted 1:10 In cold PBS (pH
7.4) and plated immediately on blood agar as previously described (1). Plates were
incubated 48 hours at 37 C, and colonies were counted.
Statistical analysis. The effects of treatment and duplicate tests were compared using
absorbance (A) readings from ELISA for the two locations, serum and tracheal
washings. Mean CPUs were compared at various times. All numerical data were evaluated by an analysis of variance (ANOVA) with General Linear Models (PROC-
GLM) as components of the SAS software (Statistical Analysis Systems, Inst., Gary,
N.C.). Split-plot analysis with treatment as the main factor and time intervals and sample source as the subplot factors were used to analyze the treatment effects.
Significance levels of P ^ 0.05 were used throughout unless indicated otherwise.
Results
Prior treatment of turkeys with anti-6. avium IgG in experiment one was associated with significantly fewer CPUs of B. avium bacteria in the trachea at various 77
times PI compared to turkeys treated with anti-KLH IgG (Fig. 1). A rapid decline in
CPUs in both treatment groups from 10 to 60 minutes PI was followed by a period of increasing CPUs from 60 minutes to 48 hours PI. Absorbance readings of IgG for both KLH and B. avium decreased rapidly from 0 minutes to 48 hours PI in both serum and tracheal washings (Fig. 2). By 24 hours PI, absorbance readings of IgG were similar to expected background levels in both groups. Absorbance readings of
IgG between groups and between serum and tracheal washings were closely comparable at all times.
In experiment two, there was a statistically significant (P ^ 0.05) difference in
CPUs of B. avium at 48 and 72 hours PI between the two groups (Fig. 3). In the anti-
KLH IgG treated birds, mean CPUs increased nearly two orders of magnitude (log,o); whereas, in the anti-6. avium IgG treated birds, mean CPUs remained unchanged or slightly decreased from 24 to 72 hours PI. Absorbance readings of IgG achieved by repeated intravenous Injections were significantly greater than control absorbance values (Fig. 4).
Discussion
This study indicated a possible effect of IgG on B. avium colonization. The parenterally administered single dose of IgG maintained reduced CPUs at specific times. Prevention of colonization by IgG may be explained by at least three different mechanisms: the IgG could bind to the bacteria and enhance clearance by ciliary flow, agglutinate bacteria and reduce the apparent CPUs, or it could prevent 1. Mean B. avium Log,o CPUs per linear cm of trachea plotted against time
after inoculation. Each data point represents n - 3 birds. Vertical bars at
each point indicate the standard deviation. B. avium LoglO CPUs/ cm trachea
ro CO oi Fig 2. Mean IgG absorbance readings (A) plotted against time after B. avium
inoculation. Each data point represents n - 3 birds. Absorbance (A) at 410 nm
p p p o o o o o^iocùl^çjïb)^
GO O' M i; ° 3 C _ 0> --
ro --
% o c 3 00 - Fig 3. Mean Log,o B. avium CPUs per linear cm of trachea plotted against time
after inoculation, each data point reprsents n - 3 birds. Vertical bars at
each point represent the standard deviation. 83
HBAigG •KLHIgG 1=1No IgG 5
24 48 72 Time series in hours Fig 4. Mean absorbance readings (^) of IgG after administration, each data point
indicates n - 3 birds. Vertical bars at each point represent the standard
deviation. 0.35 n
0.3;
0.25;
0.2;
0.15;
0.1 ;
0.05
0 86 bacterial motility and adhesion by blocking the necessary molecules (4,7,14).
The trend in CPUs in both groups during the course of experiment one indicates the importance of non-immune clearance mechanisms. The initially rapid fall in CPUs from 10 to 60 minutes is probably due to ciliary clearance (8). At 1-2 hours PI, the CPUs of bacteria seemed to stabilize, possibly due to an equilibrium reached between the rate of ciliary clearance and the rate of bacterial replication. It is speculated that the increase in numbers from 2-48 hrs is probably due to increased bacterial replication rate or a declining effect of antibody (17).
The rapid fall in IgG absorbance readings in this experiment was expected, as seen in our earlier study and also as observed in another study (29). The trend in
IgG absorbance in both serum and tracheal washings were comparable. A study by
Arp and Hellwig, 1988 (3), showed that parenterally administered convalescent serum against B. avium inhibited adherence at 1 and 6 hours but not at 24 hours.
This agrees with the similar effect of antibody on bacterial colonization and the associated decline of the intravenously administered IgG.
In the second experiment, the IgG absorbance was maintained at readings comparable to those seen in an experimental infection once colonization had been established. A significant difference in the CPUs between the two groups at 72 hours may indicate the effect of IgG in agglutinating and enhancing the clearance of bacteria. This effect of IgG on clearance was achieved by repeated injections of specific IgG. A single injection may not have produced a significant effect due to the rapid elimination of passively administered antibody. The CPUs in the KLH group 87
would probably have reached 10®°'' by one week, as observed in an experimental
infection.
In conclusion, this study demonstrated a possible role for IgG in both
colonization and clearance of B. avium. However, the number of colony forming
units determined in this experiment may be representing either individual or bacteria
agglutinated by IgG. In vivo agglutination by IgG may lead to enhanced ciliary
clearance of bacteria from the tracheal surface. In a natural infection, the initial IgM
response and IgA are probably important, and if they can act along with maternal
IgG, young birds may resist serious clinical disease.
Acknowledgements
The authors are very thankful to Dr. Susan J. Lamont, Professor, Department of
Animal Science, Iowa State University for help in design and review of experiment
and protocol. The authors also thank Mrs. Lie Ling Wu, statistics consultant.
Department of Statistics, Iowa State University for help in statistical design and
analysis of the data and Mrs. Elise Huffman, Department of Pathology for the
technical help during the experiment.
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GENERAL SUMMARY AND DISCUSSION
The purpose of these studies was to determine the importance of a humoral immune response in bordetellosis in turkeys. In the first study, antibodies of all three isotypes, IgG, IgA and IgM, were measured in serum and mucosal secretions (tracheal and lacrimal) by ELISA in experimentally infected turkeys. In all three fluids sampled, the antibody levels reached a peak around 4-6 weeks post-inoculation (PI) and then decreased rapidly from 6-8 weeks PI. The IgM and IgA responses peaked earlier (4-5 weeks PI) but declined more rapidly than IgG levels. The trend of increasing antibody levels correlated with a progressive decline in B. avium from 4 to 8 weeks of infection following maximum numbers at 2-3 weeks of infection. This clearly supports a role for antibodies in controlling a mucosal infection such as bordetellosis.
Even though no direct effect of each antibody isotype could be measured in this relationship, the results are clearly indicative of the general effect of any or all of the isotypes in clearing B. avium from the tracheal surface.
Of the three antibody isotypes, IgG is the isotype that persisted at all three anatomic sites. It has been shown by other researchers that IgG, at least in avian species, is present at high levels in mucosal secretions compared to other isotypes.
Our pilot studies indicated effective transfer of parenteral I y administered IgG, but not
IgM, to mucosal surfaces. Keeping all these interpretations in view, IgG seems to be an important and probably the major immunoglobulin isotype coming from the blood and passing readily to the mucosal surfaces, due to its smaller size. 93
Our second study effectively employed the 6. avium specific ELISA to detect
IgG transfer. This study showed that IgG given parenterally can be detected in the
tracheal or lacrimal secretions in as little as 5 minutes, reaching a peak around 10
minutes after injection. The corresponding levels of IgG at the three sites had a
similar trend at various time intervals. The IgG reached an almost undetectable level
by 24 hours at all three sites. Studies on the transport kinetics of immunoglobulins
are sparse in mammalian species and are even more rare in avian species. The IgA
and IgM might use specific receptor mediated mechanisms to cross the epithelial
barriers at the mucosal surface; however, IgG seems to diffuse effectively across the blood vascular and mucosal epithelial barriers. Apart from the specific mechanisms
involved, the molecular form in which the immunoglobulins exist could be important as well. In birds, IgM is present as a pentamer and IgA is multimeric, existing mostly as tetramers or dimers. Hence, IgM and IgA are probably being secreted mostly by plasma cells. However, IgG secreting plasma cells are also present at local sites.
Therefore, IgG might be coming from both local plasma cells and directly from the bloodstream; the latter probably being the major source. This study indirectly indicated the importance of IgG in mucosal infections in birds.
Our final study was designed to see if IgG could effectively prevent initial colonization and subsequent clearance of 6. avium from the tracheal surface of young turkeys. The first part showed that parenteral ly administered IgG could inhibit
B. avium numbers when given before bacterial inoculation, thus indicating inhibition of colonization. The mechanism of this effect on colonization can be attributed to 94
the ability of immunoglobulins to agglutinate the bacteria or to block adhesive
molecules on the surface of the bacteria. In the second part of this experiment,
repeated injections of IgG were used to maintain significant levels IgG at the tracheal
surface. The B. av/um-specific IgG caused a significant reduction in bacterial
numbers during the course of the experiment at 48 and 72 hours after infection. In
contrast, numbers of B. avium in the control group increased. Even though the
reduction was not great, it is suggestive of a role for IgG in clearing the bacteria.
Thus, IgG administered parenterally could exert its effect in vivo in the birds.
The roles of IgM and IgA on either colonization or clearance cannot be ruled out.
All three isotypes might have a synergistic effect in controlling the infection. Because
IgG is the maternal antibody transferred to the chick, it could be important during the
initial three weeks of life. Hence, vaccination strategies that enhance maternal
antibodies should be encouraged. Although, It might not be possible for IgG alone to
clear an infection, especially when it is overwhelming, it may reduce clinical disease.
A synergistic effect of the three immunoglobulin isotypes may be very important.
Being the immunoglobulin present for the longest duration at all three anatomic sites,
IgG may be useful for serologic diagnosis and evaluation of the immune status of
the flock.
Overall, these experiments have demonstrated the importance of humoral
immunity in avian respiratory tract infections and the transfer and protective effect of
IgG in controlling the infection. These kinds of studies of humoral immune responses may be useful for the evaluation of vaccines in future experiments. The 95 results of these experiments could be extended to studies concerned with immunity and infection of birds with various other respiratory pathogens in the design of related vaccines and evaluation of serologic responses.
Apart from these experiments conducted to study immune responses to B. avium in turkeys, an attempt was made to develop a diagnostic test for detecting B. avium. A monoclonal antibody (IgG isotype) was developed against this bacterium and effectively employed in a latex bead agglutination test. This test clearly differentiated B. avium from B. bronchiseptica and B. avium-like bacteria. The latter two are frequently isolated from the trachea of turkeys and are often confused with B. avium. This test was found to be simple, rapid and specific in distinguishing these related bacteria and is an important tool for researchers or diagnosticians dealing with respiratory infections in turkeys. 96
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ACKNOWLEDGEMENTS
I thank my parents for my education and for their confidence in my progress.
I am thankful to my wife, Puppy, for her tireless efforts in taking care of me and my
son; to my son, Nithin, for making this experience pleasant and adding meaning to
my efforts.
I sincerely thank my major advisor and mentor. Dr. Lawrence H. Arp. His
guidance was instrumental in guiding me to become a good student and researcher.
Under his supervision, I developed my scientific insight and writing qualities. My
thanks to Dr. J. S. Haynes for agreeing to be my co-major professor when Dr. Arp left
the university and for guiding me through the final phases of my program. I also
thank Dr. Ron Myers, who advised me in so many different ways and who made me
feel at home many times; to Dr. Prem Paul and Dr. Ron Griffith for their input
towards my research; to Dr. Susan Lamont for spending time with me designing the final two experiments and for the encouraging remarks during my tense schedule in this final phase.
I also thank Elise Huffman for her expert technical help and organization of the laboratory; Becky Smith and Diane Nelson for their technical services; Bettye
Danofsky, Mary Hull and Mikie Walker for their encouragement and help. I also wish to thank Drs. Greve, Niyo, Kluge, Miller, Hagemoser, Ledet, Andrews, and
Howard for their friendship, advice and encouragement during my tenure at Iowa
State. To my colleagues in the graduate office, Drs. Vahle, Hutto, Dyer and 121
Sirinarumitr, my thanks for sharing their time and knowledge; to Dr. Jim Eastep my
special thanks for his help in getting me acclimatized to the "computer atmosphere."
I thank my sister and her family for their encouragement. I appreciate my
brother Mr. Vishy, who with his own accomplishments has been my steady source of
inspiration.
Finally, I am grateful to my late uncle Sri. P. N. Ghatikachalam, for the encouragement he gave and the pride he took in my success. I thank God for making this all possible. 122
APPENDIX
A MONOCLONAL ANTIBODY-BASED LATEX BEAD AGGLUTINATION TEST
FOR THE DETECTION OF BORDETELLA AVIUM 123
A Monoclonal antibody-based latex bead agglutination test for the detection of
Bordetella avium
Poosala Suresh and Lawrence H. Arp'^
Department of Veterinary Pathology
College of Veterinary Medicine
Iowa State University
Ames, Iowa 50011
'^Department of Toxicology and Pathology
Hoffman La-Roche
Nutley, NJ 07110.
Published in Avian Diseases, 1993. 37: 767-772.
Supported by the California Turkey Industry Federation. Presented in part at the 1992 Conference of Research Workers in Animal Disease, Chicago, Illinois, November 9-10, 1992. 124
SUMMARY
The purpose of this study was to develop a rapid method to distinguish
Bordetella avium from closely related Bordetella avium-Wke and Bordetella bronchiseptica bacteria. A monoclonal antibody of the IgM isotype was produced in
BALB/c mice against live B. avium strain 75. The monoclonal antibody, in the form of ascites fluid, was added to a bovine serum albumin-glycine buffer (pH 8.6) and adsorbed to 3.03-A/m-diameter latex beads. Optimum concentrations of antibody, beads, and bacteria were determined. The latex bead conjugate was tested against
40 isolates of B. avium, 24 isolates of 6. avium-Wke bacteria, 17 isolates of B. bronchiseptica, two isolates of Alkaligenes faecalis, and several other common genera. Strong agglutination occurred with all 6. avium isolates and the 2 isolates of
A. faecalis. Weak agglutination occurred with Staphylococcus aureus and two isolates of B. bronchiseptica. There was no agglutination with any of the 6. avium-
Wke isolates. The latex bead agglutination test may be useful as an aid in the identification of B. avium when used in conjunction with other criteria.
INTRODUCTION
Bordetella avium is the causative agent of avian bordetellosis, a highly contagious upper respiratory tract disease of young turkeys (3,5,11). The diagnosis of bordetellosis is currently based on characteristic clinical signs, respiratory tract lesions, and isolation of B. avium from the trachea (2). B. avium and closely related 125
bordetella-like species produce pearl-colored, pinpoint (< 1 mm) colonies on
MacConkey's agar 24-48 hours after primary isolation from trachea. The rapid
differentiation of B. avium from B. bronchiseptica and B. avium-Wke bacteria has
been a diagnostic problem (3).
B. avium can be differentiated from 6. bronchiseptica by the urease reaction. B.
avium is urease negative and B. bronchiseptica is urease positive (4,8,12). The
greatest diagnostic dilemma comes with trying to distinguish B. avium from B. avium-
Wke bacteria (3,7). Bordetella avium-Wke bacteria are urease negative, but they are nonpathogenic and adhere poorly to the tracheal mucosa (1,7). Variations among strains and growth conditions make colony morphology an unreliable means for differentiating these closely related bacteria (3,6). The capacity of S. avium to cause hemagglutination of guinea pig erythrocytes provides a reliable method to distinguish between B. avium and B. avium-Wke bacteria, but the test is inconvenient and time- consuming for many laboratories (2,7). MAb and latex bead agglutination tests have been used effectively to diagnose many fungal and bacterial infections. A latex agglutination test was found to be sensitive and specific for identifying 200 strains of
Escherichia coli serotype 0157:H7 (9). The purpose of the present study was to design a simple diagnostic test to distinguish B. avium from closely related 6. avium-
Wke bacteria and B. bronchiseptica by employing a MAb against B. avium.
MATERIALS AND METHODS
Monoclonal antibody production. MAbs were produced using BALB/c mice against 126
immunized by intraperitoneal injection with a 0.2 ml suspension containing 10®
colony-forming units (CPU) of B. avium/m\ in phosphate-buffered saline (PBS). Mice
were reinjected 3 weeks later. Three days before cell fusion, mice were reinjected
intravenously. Spleen cells were harvested from the immunized mice and fused with
SP2/0 cells. Primary hybrids were cloned and screened by enzyme-linked
immunosorbent assay (ELISA) for production of antibody to live B. avium strain 75, B. avium-Wke bacteria, and Bordetella branchiseptica. Clones that produced antibody against B. avium were implanted in mice for ascites fluid production. Two MAbs were evaluated for use in a latex bead agglutination test. The isotype of each MAb was determined using an ELISA kit (Zymed Laboratories, South San Francisco, Calif.).
MAbs 3H4-G8 and 3G10-D2 were determined to be lgG3 and IgM, respectively.
Preliminary immunoblotting (performed by Dr. Vincent Collins, Washington
University, St. Louis, Missouri) has shown that 3G10-D2 reacts strongly with a
~20,000-molecular-weight protein of 6. avium. Minor cross-reactivity was also seen to outer-membrane proteins of B. branchiseptica and B. avium-Wke bacteria.
Coating of latex beads. The adsorption technique used to coat latex beads with MAb was modified from Masson et al. (10). A 0.1% solution of bovine serum albumin
(BSA) (Sigma Chemical Co., St. Louis, Missouri) was prepared in glycine buffer (GB pH 8.6) and 900 fj\ of the BSA-GB solution was transferred to an Eppendorf tube for mixing. The BSA-GB solution was mixed in a vortex mixer and incubated 1 minute at 37 C. MAb contained in 40 //I of mouse ascites fluid (1.97 mg protein/ml) was added to the BSA-GB solution and mixed twice while incubating for 5 minutes at 37 127
C. Next, 800//I of a 2.5% (wt/vol) suspension of 3.03 //m diameter latex beads
(Polysciences Inc., Warrington, Pa.) was added to the BSA-GB-ascites fluid solution,
mixed on a vortex mixer for 1 minute, and then incubated 15 minutes at 37 C. The
coated latex beads were stored at 4 C until used. Various incubation times,
concentrations of MAbs, and storage conditions were evaluated before selecting the
above method for producing a rapid, definitive reaction.
Bacterial isolates. A total of 40 isolates of B. avium, 24 isolates of B. avium-Wke
bacteria, and 17 isolates of B. bronchiseptica were evaluated for reactivity in the latex
bead agglutination test (Tables 1-3). Many of the isolates were used in previous studies by investigators throughout the United States during the past decade. All
Bordetella spp. were evaluated for growth characteristics on MacConkey's agar, urease reaction, and capacity to agglutinate guinea pig erythrocytes to ensure proper species grouping. In addition, two American Type Culture Collection isolates of
Alcaligenes faecalis and single isolates of Escherichia coli, Pseudornonas sp.,
Staphylococcus epidermidis, S. aureus and Proteus sp., were tested. Each isolate was streaked onto brain-heart-infusion agar or MacConkey's agar and incubated 24 hours at 35 C. Isolated colonies were picked from the plates for testing. In preliminary studies, a bacterial concentration of ~ 10'-10'° (CFU)/ml was found to produce a strong immediate agglutination reaction; however, bacterial concentrations as low as 10^ CFU/ml also produced detectable reactions.
Test procedure. A single drop (50 //I) of PBS was placed on a black porcelien or plastic surface. A single bacterial colony was picked from solid medium and stirred 128
into the PBS to form a turbid suspension. A single drop (50 fj\) of the coated latex
bead suspension was applied and stirred into the bacterial suspension with a wooden
applicator stick. The plate was rocked every 15 seconds and the agglutination
reaction was observed at and after 30 seconds and 2 minutes. The agglutination
reaction and time taken were scored. Typically, a 4+ reaction was observed as
spontaneous large floccules forming within 30 seconds, a 3+ reaction showed visible
flakes within 30 seconds, a 2+ reaction showed small flakes in between 30 seconds
and 2 minutes and a 1 + reaction showed small flakes after 2 minutes. A negative reaction (-) was always characterized by a uniform suspension with no visible flakes.
Various reaction volumes and incubation times were evaluated before selecting the procedures described above.
RESULTS
The latex bead agglutination test utilizing the MAb 3G10-D2 provided a rapid, simple means for distinguishing most isolates of 6. avium from B. av/um-like bacteria.
Of the 40 bacterial isolates identified as B. avium based on growth on MacConkey's agar, typical colony morphology, a negative urease reaction, and the capacity to agglutinate guinea pig erythrocytes, all isolates caused an obvious and rapid agglutination reaction (Fig. 1) (Table 1). None of the 6. av/um-like bacteria produced a detectable reaction (Table 2). Of the 17 6, bronchiseptica isolates, two produced a agglutination reaction after 2 minutes (Table.3). Of the other bacterial species, the two isolates of A. faecalis produced an obvious and rapid agglutination reaction Latex bead agglutination reaction. (I) A typical + + + + positive reaction to B. avium strain 75, (II) a + + reaction to Bordetella bronchiseptica strain 1188, and (III) a negative reaction to B. awum-like strain 001. See
Materials and Methods for explanation of scoring.
131
comparable to that produced by 6. avium isolates. 5. aureus caused a weak
agglutination reaction (Table.3).
Both MAbs provided easy-to-read latex agglutination reactions with most isolates
of B. avium. MAb 3G10-D2 gave a slight cross-reaction with two isolates
of B. bronchiseptica and MAb 3H4-G8 cross-reacted with 4 strains of B.
branchiseptica. Both 3HA-G8 and 3G10-D2 cross-reacted with both strains of A.
faecalis. MAb 3G10-D2, isotype IgM, produced a more rapid and obvious
agglutination reaction compared with MAb 3H4-G8. All subsequent experiments
were performed using MAb 3G10-D2. In preparation of the BSA-GB-ascites solution,
10 - 100 //I of ascites fluid was added to the BSA-GB solution. Volumes of less than
20 jj\ gave a delayed or indistinct reaction with B. avium, whereas volumes greater
than 80 //I caused autoagglutination of the latex bead conjugate. The time allowed
for adsorption of MAb to latex beads was varied from 3 to more than 15 minutes.
Incubation for only 3-4 minutes resulted in a preparation that agglutinated poorly with B. avium, whereas incubations of ^ 15 minutes provided satisfactory results.
The stability of the MAb-coated latex bead suspension was evaluated at 4 C and
37 C. The addition of 0.1% sodium azide had no effect on the agglutination reaction and therefore was used for all stability studies. When stored at 4 C, the latex bead suspension was stable and worked normally for at least 4 months. When stored at 37
C, the latex bead suspension had reduced agglutinating activity after 72 hours. 132
Table 1. Agglutination reactions of Bordetella avium with MAb 3G10D2-coated latex beads.
Isolate Year Agglutination identification SourceQrtiirr*o StateCfafo of originnrîoin isolatedicnlatarl reaction
75 L. H. Arp Iowa 1979 + + + + K8900955 B. R. Charlton California 1989 + + + + K8901019 B. R. Charlton California 1989 + + + + K8901090 B. R. Charlton California 1989 + + + + K8901260 B. R. Charlton California 1989 + + + + K8901341 B. R. Charlton California 1989 + + + + K8901437A B. R. Charlton California 1989 + + + + K891295A B. R. Charlton California 1989 + + + + K9000135 B. R. Charlton California 1990 + + + + F9000029 R. P. Chin California 1990 + + + F9000336 R. P. Chin California 1990 + + + + F9000947 R. P. Chin California 1990 + + + + 838 M. S. Hofstad Iowa 1980 + + + + 140 M. Matsumoto Oregon 1991 + + + + 009 Y. M. Saif Minnesota 1980 + + + 002/T7 Y. M. Saif Ohio 1979 + + + + 018 Y. M. Saif Ohio 1978 + + + + 045 Y. M. Saif Ohio 1981 + + + + 101 Y. M. Saif Ohio 1981 + + + + 124 Y. M. Saif Ohio 1982 + + + + 137 Y. M. Saif Ohio 1982 + + + + 140 Y. M. Saif Ohio 1982 + + + + 145 Y. M. Saif Ohio 1982 + + + + 162 Y. M. Saif Ohio 1982 + + + + 177 Y. M. Saif Ohio 1982 + + + + 178 Y. M. Saif Ohio 1982 + + + + 195 Y. M. Saif Ohio 1982 + + + 196 Y. M. Saif Ohio 1983 + + + + 017 Y. M. Saif Wisconsin 1981 + + + + 108/T5 Y. M. Saif Wisconsin 1981 + + + + 4671 D. G. Simmons North Carolina 1978 + + + + NCD D. G. Simmons North Carolina 1978 + + + + NCD1 D. G. Simmons North Carolina 1978 + + + + 1083 D. M. Wallis California 1990 + + + + 1090 D. M. Wallis California 1990 + + + + 1104 D. M. Wallis California 1990 + + + + 1211 D. M. Wallis California 1990 + + + + 1434 D. M. Wallis California 1990 + + + + 19 D. M. Wallis California 1990 + + + + 269 D. M. Wallis California 1990 + + + +
'^Note: Refer to Materials and Methods for scoring agglutination reaction. 133
Table 2. Agglutination reactions of Bordetella avium-Wke bacteria with MAb 3G10D2-coated latex beads.
Isolate Year Agglutination identification Source State of origin isolated reaction'^
234040 M. E. Carlson Wisconsin 1991
234041 M. E. Carlson Wisconsin 1991 -
234042 M. E. Carlson Wisconsin 1991 -
K8901437B B. R. Charlton California 1989 -
K891170B B. R. Charlton California 1989 -
K891295B B. R. Charlton California 1989 -
00611 M. W. Jack wood Indiana 1980 -
00711 M. W. Jackwood Wisconsin 1980 -
02311 M. W. Jackwood Ohio 1979 -
02811 M. W. Jackwood Ohio 1979 -
03011 M. W. Jackwood Ohio 1979 -
03111 M. W. Jackwood Ohio 1979 -
04411 M. W. Jackwood Ohio 1981 -
10111 M. W. Jackwood California 1981 -
11211 M. W. Jackwood Wisconsin 1981 -
15511 M. W. Jackwood Iowa 1982 -
15711 M. W. Jackwood Iowa 1982 -
16811 M. W. Jackwood Iowa 1982 -
001 L. D. Koehnk Iowa 1990 -
002 L. D. Koehnk Iowa 1990 -
003 L. D. Koehnk Iowa 1990 -
1370 D. M. Wallis California 1990 -
1384 D. M. Wallis California 1990 -
33 D. M. Wallis California 1990 -
Note: Refer to Materials and Methods for scoring agglutination reaction. 134
Table 3. Agglutination reactions of Bordetella bronchiseptica and other bacteria with MAb 3G10D2-coated latex beads.
Isolate Year Agglutinatior Identification Source State of origin isolated reaction'
S. bronchiseptica
K8901188 B. R. Charlton California 1989 + + K9000162 B. R. Charlton California 1990 + 55 L. D. Koehnk Iowa (unknown) - 056 Y. M. Saif Minnesota 1981 - oBsrrs Y. M. Saif Ohio 1982 - 095/r3 Y. M. Saif Ohio 1984 - 062/T5 Y. M. Saif Wisconsin 1981 - 063 Y. M. Saif Wisconsin 1981 - 064/r3 Y. M. Saif Wisconsin 1981 - 103 Y. M. Saif Wisconsin 1981 - 02 D. M. Wallis California 1990 - 1187 D. M. Wallis California 1990 - 1334 D. M. Wallis California 1990 - 135 D. M. Wallis California 1990 - 1370 D. M. Wallis California 1990 - 1384 D. M. Wallis California 1990 -
33 D. M. Wallis California 1990 -
Other bacteria
ATCC 8750 Alcaligenes faecalis + + + + ATCC 33585 Alcaligenes faecalis + + + + Escherichia coli - Pseudomonas sp. -
Staphylococcus epidermidis - Staphylococcus aureus + +
Proteus sp. -
Note: Refer to Materials and Methods for scoring agglutination reaction. 135
DISCUSSION
The MAb-based latex bead agglutination test proved to be useful for differentiating
B. avium from other closely related bacteria. Adsorption of the MAbs to latex beads
was not technically difficulty and the reaction was acceptable over a wide range of
concentrations of MAb, latex beads, and bacteria. The test is best suited for
distinguishing colonies of 6. avium from closely related bacteria on primary cultures
from turkey tracheas. The latex bead agglutination test meets the minimum criteria
established for the project.
Minor cross-reactivity with two isolates of B. branchiseptica was not unexpected,
as a previous study showed antigenic relatedness among the Bordetella spp. (8).
Unfortunately, the cross-reactivity with B. bronchiseptica was not detected when
primary hybrids were screened by ELISA. Although most isolates of 6. avium
produced a much more pronounced agglutination reaction compared with 6.
bronchiseptica, the urease reaction should be used to support the diagnosis. The
strong cross-reactivity of the MAbs with two isolates of A. faecalis is more of a
curiosity than a problem. We did not isolate A. faecalis from the tracheas of turkeys;
however, the test emphasizes the close antigenic relationship between S. avium and
A. faecalis. Colonies of A. faecalis produced a green hue on blood agar plates, but
they were indistinguishable from B. avium on MacConkey's agar. The reaction with
S. aureus was possibly mediated by the nonspecific binding of surface protein A with
the Fc portion of the MAbs. Staphylococcus spp. are readily differentiated from B. avium by colony morphology. 136
ACKNOWLEDGMENTS
We thank Dr. Richard Van Deusen, Iowa State University Hybridoma Service, for
his supervision of MAb production. We acknowledge the technical assistance of Elise
Huffman and the assistance of Dr. Mark Evans and Darl Pringle in the screening and
selection of MAb hybrids for use in subsequent studies.
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