I= DISEASE SPECIFIC DIAGNOSTIC METHODS AND LYMPHOKINES IN HUMAN BRUCILLOSIS
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Title of Thesis
DISEASE SPECIFIC DIAGNOSTIC METHODS AND LYMPHOKINES IN HUMAN BRUCILLOSIS

Author(s)
Hamida Nusrat
Institute/University/Department Details
Department of Microbiology/ University of Karachi
Session
2004
Subject
Microbiology
Number of Pages
206
Keywords (Extracted from title, table of contents and abstract of thesis)
lymphokines, human brucillosis, bacterial zoonoses, genus brucella, brucella melitensis, secreted cytokines

Abstract
Brucellosis is one of the five common bacterial zoonoses in the world caused by organisms belonging to the genus Brucella, which arc gram-negative, non-sporing, facultative intracellular bacteria. Brucellosis in animals and humans is endemic in many developing countries including Pakistan. Control of brucellosis in agricultural animals is a pre-requisite for the prevention of this disease in human beings. Recently, Brucella melitensis has been declared by the Centers for Disease Control and Prevention to be one of the three bio-terrorist agents due to the expense required for the treatment of human brucellosis patients. Also, the economic and agricultural loss caused by bovine brucellosis emphasizes the financial impact of brucellosis in the society. Human brucellosis is significant public health problem in an agricultural country like Pakistan, where the vast majority of the population is involved in land cultivation and livestock farming .

Currently, B. RB51 vaccine to immunize cattle and B. melitensis Rev.1 vaccine to immunize goats and sheep are used in many countries. However. These genetically undefined strains still induce abortion and persistent infection. Raising questions of safety and efficacy. Moreover, mechanism of pathogenesis of Brucella is complex and poorly understood. Brucella organisms not only resist killing by phagocytosis, but also proliferate inside macrophages. Investigation of various immunological and molecular aspects of pathogenesis in human brucellosis in Pakistan is the main. theme behind this study. Study of cytokine profile by ELISA, analysis of cytokine gene activation by RT-PCR, determination of disease-specific proteins of Brucella species by EUSA and Western blotting, and their possible role in disease chronicity and candidature for a future vaccine, are the various issues studied and discussed in the present research work

Depending on the outcome of secreted cytokine profile and cytokine gene expression in acute and chronic brucellosis patients as well as healthy subjects, we conclude that there is induction of both Th 1 and Th 2 cytokine subsets during human brucellosis. Thus. each subset amplifies itself and cross-regulates the reciprocal subset. Despite the ;significant production of protective Th l cytokines, IL-12, IFN-γ and TNF-α, macrophages' mechanism of killing Brucella seems to be defective leading to persistent infection in chronic cases. It can be inferred that the strong and combined inhibitory effects of IL-6, IL-10 and TGF-β, override the protective effects of Th 1 cytokines an.! thus, prolong the course of infection in human brucellosis. Our findings further suggest that the interaction between Brucellae, macrophages and cytokines may determine the acute or chronic outcome of the infection. It is reasonable to assume that optimal development and main1-enance of a protective response against the Brucella infection relies on a finely regulated balance of cytokines rather than upon the level of a single cytokine. Thus, protection may be more dependent upon the timing of the cytokine response rather than on the absolute level of cytokine expression.

The commercial LLISA kits containing LPS-O antigen may differentiate en acute and chronic patients by estimation or anti-Brucella LgM, IgG and IgA antibodies. However these ELISAs do not differentiate between chronic patients, treated and cured patients and Brucella-exposed subjects either healthy or suffering from other diseases with clinical signs and Symptoms overlapping with brucellosis; in all these individuals anti-Brucella IgG antibodies may be elevated. This is because the antibodies to LPS.O antigen may persist for a very long time after infection. In contrast anti-protein antibodies are relative]y short-lived and hence, a better indicator of active disease. We, therefore proposed to study antibodies to protein antigens of LPS-O deficient strain, B. abortus RB 51 by ELISA and Western immunoblotting to identify antigens, which may be useful indicators of active disease. In addition, anti Brucella IgG subclasses (IgG 1, IgG2, IgG3 and IgG4) were also studied to determine If any of the subclasses could be a better indicator of active disease and also to monitor the immunological effect of chemotherapy in human brucellosis.

In homemade ELISA with protein antigens of B. abortus RB51, the reactivity of anti-Brucella IgG and IgG I was higher in the sera of chronic patients and group-Il acute patients (positive for anti-Brucella IgM and IgG by commercial kit) than group I acute patients (positive for anti-Brucella IgM by commercial kit) The major IgG subclass antibodies detected against protein antigens were IgG1 and IgG4 for group-I acute patients and IgG1 and IgG3 for group-II acute as wcll as chronic patients of' brucellosis For healthy but exposed slaughtcrhouse workers, the frequency of reactivity of all the four IgG subclasses was of similar magnitude.

Immunoblot analysis for identifying dominant protein antigens of' B. abortus RB51 revealed that anti-Brucella antibodies were most frequently detected to the proteins of 200-250 kDa and 15-80 kDa in the sera of brucellosis patients. The IgG antibodies were directed against these proteins in the sera of all patients, while in few cases IgM and IgA antibodies also reacted with these proteins. In contrast, the sera of healthy but exposed slaughterhouse workers reacted with 30-70 kDa proteins but not with 200-250 kDa ones, suggesting that the antigenic fractions of diagnostic importance for active disease lie in the range of 200-250 kDa. Regarding the reactivity or anti-Brucella IgG subclasses, our results showed that IgG1 and IgG4 were mainly activated in brucellosis patients and IgG2 and IgG4. in the healthy slaughterhouse workers. with respect to specific antigens. Anti .BruceIla IgG1 I was primarily Induced against both high (200-250 kDa) and low (20-80 kDa) molecular weight proteins, whereas' IgG4 mainly against low (20-80 kDa) molecular weight proteins. It is known that Th2 and Th1 cytokines help production of IgG1 and IgG4 by human B lymphocytes, respectively. Our results suggest that high molecular weight (HMW) proteins of Brucella may be preferentially activating Th2 and 1ow molecular weight (LMW) proteins the Th1 cytokines. Thus, HMW proteins may be useful in the diagnosis of active disease by detecting anti-Brucella IgG/IgG,1 and LMW proteins In developing vaccines against brucellosis. Moreover, with continuation of chemotherapy for six months, colour intensity of antibodies reacting to HMW proteins decreased and the antibodies reacting to LMW proteins were not detected in the follow-up sera of patients.

In future, the disease-specific protein antigens of Brucella can be further investigated in establishing the next generation of ELISA tests as well as in designing vaccine candidates which can activate both protective cell-mediated and humeral immunity in human brucellosis.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
182.06 KB
2 1 Introduction 1
344.66 KB
  1.2 Etiology of Brucellosis 2
  1.3 Cell morphology and Culture 3
  1.4 Antigens of Brucella 4
  1.5 Epidemiology of Brucellosis 6
  1.6 Modes of Transmission of Brucella 7
  1.7 Mechanism of Pathogenesis in Brucellosis 9
  1.8 Clinical Manifestations in Brucellosis 12
  1.9 Diagnosis of Brucellosis 15
  1.10 Prognosis of Brucellosis 19
  1.11 Treatment of Brucellosis 20
  1.12 Prevention and Control of Brucellosis 20
  1.13 Immune Response to Brucellosis 21
  1.14 Brucellosis in Pakistan and South-East Asian Countries 30
3 2 Materials and Methods 34
216.77 KB
  2.1 Specimen Selection Criteria and Case Definition 34
  2.2 Bacterial Strains 35
  2.3 Quantitation of Secreted Cytokines in Sera and Cell Culture Supernatants 36
  2.4 RT-PCR for the Detection of Cytokine Gene Expression 38
  2.5 Commercial and In-House Enzyme-Linked Immunosorbent Assay (ELISA) for Estimation of Anti-Brucella IgM , IgA and IgG , and its Subclasses IgG1, IgG2, IgG3 and IgG4 44
  2.6 Study of Disease- Specific Brucella Proteins by estern Immunoblotting in the Diagnosis of Brucellosis 48
4 3 Results 58
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  3.1 Measurement of Cytokine Titer in Tissue Culture Supernatant and Sera Samples 58
  3.2 Study of Cytokine Gene Expression by RT-PCR 67
  3.3 Study of Anti-Burcella Antibodies with Commercial and Home Made Elisa 75
  3.4 Study of Disease-Specific Brucella proteins by Western Immunoblotting 90
5 4 Discussion 99
1424.85 KB
  4.1 Bibliography 112
  4.2 Appendix A- Tables 131