Jim S Rothel

Abstract The in vivo dynamics of differentiated cells and interleukin (IL)-lβ, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ titres in afferent lymph were compared following orf virus reinfection and inactivated virus injection of previously infected sheep. The biphasic lymphoblast and cytokine response in t...

Military personnel are at risk for acquiring Mycobacterium tuberculosis infection because of activities in close quarters and in regions with a high prevalence of tuberculosis (TB). Accurate tests are needed to avoid unnecessary treatment because of false-positive results and to avoid TB because of false-negative results and failure to diagnose and...

To examine the hypothesis that results of the QuantiFERON-TB Gold assay (QFT-G), a whole-blood test for detection of tuberculosis infection, are more significantly related to known risk factors for tuberculosis infection in healthcare workers (HCWs) who have received bacille Calmette-Guerin vaccine than are results of the Mantoux tuberculin skin te...

Tuberculosis is responsible for more then 2 million deaths worldwide each year and vies with HIV as the world's most fatal infectious disease. In many developing countries, attempts to control the spread of infection rely solely on identification and treatment of those with active disease, ignoring subclinical infection. However, in developed count...

Nine university-affiliated chest clinics in Australia. To evaluate the sensitivity of a whole blood human gamma-interferon assay (HGIA, QuantiFERON-TB) for specific T lymphocyte responses and Tuberculin skin testing (TST) for the detection of Mycobacterium tuberculosis infection in subjects with culture-proven M. tuberculosis disease (TBCP). Prospe...

To determine the optimum cut-off level of a newly developed method for diagnosing tuberculosis infection based on whole-blood interferon-gamma measurement, and to study the basic characteristics of the method. 1) A total of 220 young, healthy individuals having no apparent exposure to tuberculosis infection, most of whom have had a vaccination with...

Identifying persons with latent tuberculosis infection (LTBI) is crucial to the goal of TB elimination. A whole-blood interferon gamma (IFN-gamma) assay, the QuantiFERON-TB test, is a promising in vitro diagnostic test for LTBI that has potential advantages over the tuberculin skin test (TST). To compare the IFN-gamma assay with the TST and to iden...

Public hospital, Victoria, Australia. To evaluate the effect of multidrug treatment and isoniazid (INH) chemoprophylaxis on the tuberculin interferon-y assay (QIFN) in 19 patients with culture-confirmed Mycobacterium tuberculosis and 119 health care workers (HCWs) with tuberculin skin tests (TST) > or =15 mm. Patients with M. tuberculosis were trea...

QuantiFERON-TB (QIFN) (CSL Limited) is a whole-blood assay for the recognition of infection with Mycobacterium tuberculosis. QIFN measures gamma interferon (IFN-gamma) production when purified protein derivatives (PPDs) of mycobacteria are incubated with venous blood samples. The specificity of QIFN in medical students before and after BCG immuniza...

Two peptides corresponding to putative protective regions located at the N- and C-termini of the host-protective T. ovis recombinant antigen, 45W, were synthesized. Antibodies raised against 45W and 45WB/X, a truncated form of 45W, were found to react strongly with the N-terminal peptide. When sheep were immunised with each peptide alone, the N-ter...

Two peptides corresponding to putative protective regions located at the N- and C-termini of the host-protectiveT. ovis recombinant antigen, 45W, were synthesized. Antibodies raised against 45W and 45WB/X, a truncated from of 45W, were found to react strongly with the N-terminal peptide. When sheep were immunised with each peptide alone, the N-term...

Serological testing for Lyme borreliosis using conventional enzyme-linked immunosorbent and immunoblot assays is ham- pered by poor correlation between test results and disease status. The high seroprevalence (around 10 to 20%) of Borrelia burgdorferi-specific antibodies in the general population in ar- eas of high endemicity can confound interpret...

A new test that measures interferon-γ (IFN-γ) release in whole blood following stimulation with tuberculin has the potential to detect tuberculosis infection using a single blood draw. The IFN-γ release assay was compared with the standard tuberculin skin test (TST) among 467 intravenous drug users at risk for tuberculosis in urban Baltimore. Among...

Sheep immunised with the Taenia ovis recombinant 45W antigen are protected from infection with the parasite. Two peptides were synthesised corresponding to putative host-protective regions at the N- and C-termini of 45W. Sera from sheep immunised with 45W or related recombinant proteins reacted strongly with the N-terminal peptide. Approximately 40...

The cannulated efferent lymph node in sheep was used to examine the effect of different adjuvants on the antibody and cytokine responses following sub-cutaneous vaccination with a recombinant Taenia ovis antigen (45 W). Vaccination with Quil A elicited relatively higher levels of IgM than did IFA or Al(OH)3. In general, 45 W specific IgG1 and IgG2...

In the present study we have investigated the use of recombinant ovine IL-1beta and TNF-alpha both alone and in combination, as natural adjuvants in vaccination trials in sheep. Initial experiments were conducted to investigate the physiological effects of the cytokines in vivo and determine what dose could be administered without adverse pyrogenic...

In the present study we have investigated the use of recombinant ovine IL-1 beta and TNF-alpha both alone and in combination, as natural adjuvants in vaccination trials in sheep. Initial experiments were conducted to investigate the physiological effects of the cytokines in vivo and determine what dose could be administered without adverse pyrogeni...

A monoclonal antibody (mAb) specific for ovine IL-1 beta was produced and, in conjunction with a polyclonal rabbit antiserum, used to develop a sensitive enzyme immunoassay (EIA) for ovine interleukin 1 beta (IL-1 beta). The mAb neutralised the activity of recombinant ovine IL-1 beta (rOvIL-1 beta) and native OvIL-1 in an ovine thymocyte proliferat...

The expression plasmids pGEX-2T and pT7-7 were used to express ovine (Ov) IL-2 cDNA in Escherichia coli. The pGEX-2T vector contained glutathione-S-transferase (GST) as the affinity handle and resulted in high level expression of the GST-IL-2 fusion protein. However, only a small proportion of this fusion protein was present in the soluble fraction...

Sheep were immunized with a protective recombinant antigen (45W) from the cestode parasite Taenia ovis using three different vaccine delivery systems, either alone or in different combinations. The DNA encoding 45W was cloned into the expression plasmid pcDNA 3 and an ovine adenovirus to create nucleic acid and recombinant viral vector vaccines, re...

Vaccination and challenge infection experiments were conducted in sheep using different forms of a recombinant protein (45W) from the cestode parasite Taenia ovis. 45W was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (45W-GST) and was produced as both soluble protein and insoluble inclusion bodies. Vaccination of...

The γ-interferon assay was evaluated in 203 M. bovis-infected cattle which had previously been identified by the single intradermal comparative tuberculin test, and in 923 cattle from 13 herds which were considered free of tuberculosis. Eight methods were used to interpret the results and the proportion of infected cattle giving a positive result r...

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis in sheep and goats. This disease is characterized by the development of pyogranulomas in the lymph nodes and lung tissue. To measure the cytokine gene expression in C pseudotuberculosis lesions, sheep were inoculated with two attenuated strains (Tox- and PLD-t) and a...

This report describes the use of a nucleic acid vaccine in a large outbred animal species both alone and in combination with a conventionally adjuvanted vaccine. The gene encoding a host-protective antigen (45W) from the sheep parasite Taenia ovis was cloned into the expression vector pcDNA3 and the resultant plasmid termed pcDNA3-45W. Eleven of 15...

To assess the possible rate of nosocomial transmission of TB to HCWs at one of our hospitals, we assessed the rate of strong Mantoux positivity (≥15mm) among HCWs, correlated this with prior BCG status and compared the results to those obtained from staff with no patient contact (non-HCWs). In addition, a subgroup of 109 of these HCWs, with Mantoux...

The epitheliotropic parapoxvirus, orf virus, can repeatedly infect sheep skin. A specific immune response is generated as reinfections induce smaller lesions with quicker resolution times than primary lesions. Cyclosporin-A treatment abrogates this partial immunity. Cytokine mRNAs detected in lesion biopsies include the transcripts for IL-1 beta, I...

The protective efficacy of a recombinant Taenia ovis vaccine antigen, 45W, was compared in sheep vaccine trials with antigen expressed by the full length 45W cDNA and by incomplete copies of the cDNA. Vaccine, trials were also carried out using antigen expressed by a cDNA (45S) having a sequence similar, but not identical, to 45W. Stability of the...

The dynamics and activation status of CD4+ and CD8+ T-cells differentially expressing the CD45R (220 kDa) antigen were studied in prefemoral efferent lymph draining the site of cutaneous reinfection with orf virus. CD4+, CD45R+ lymphoblasts preceded CD4+, CD45R- lymphoblasts during the first 48 h after reinfection. Thereafter, the output of both to...

Antigen-specific IgA-secreting cells were shown to be generated in peripheral lymph nodes following s.c. vaccination. The efferent duct of prefemoral lymph nodes of sheep were cannulated prior to vaccination at a site draining to the cannulated node. Vaccines were contained a recombinant protein (Taenia ovis 45W-GST) and either incomplete Freund's,...

This paper describes the evaluation of the protective antibody response of sheep to vaccination against Taenia ovis infection with a defined recombinant antigen (45W). Sera from 181 vaccinated sheep, collected prior to experimental challenge with T.ovis, were assessed for 45W specific IgA, IgG, IgG1, IgG2 and IgM levels and these results correlated...

This paper highlights problems associated with the quantitation of serum antibody levels to recombinant glutathione S-transferase (GST). Measurement of anti-GST antibodies in conventional immunoassays, where GST is bound directly to the ELISA plate, was found to substantially underestimate the amount of GST-specific antibody levels in test sera. Th...

Ovine tumour necrosis factor-alpha (OvTNF-alpha) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysaccharide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-alpha cDNA sequence. An expression vector carrying the coding sequence of the mature form of ovine TNF was constructed. The recom...

An expression vector bearing the gene segment encoding the mature form of ovine interleukin-1 beta (OvIL-1 beta) was constructed. This vector provided a rapid method for obtaining Escherichia coli derived recombinant OvIL-1 beta (rOvIL-1 beta) using the expression plasmid pGEX-2T. The level of expression of fusion protein in the soluble fraction wa...

Monoclonal antibodies (mAbs) and a polyclonal rabbit antiserum were raised against recombinant ovine tumor necrosis factor-alpha (rovTNF alpha). Ten mAbs specific for rovTNF alpha were isolated and designated TNF1-10. All mAbs were of the IgG1 isotype and reacted with rovTNF alpha in Western blot analysis. Eight of the ten mAbs, TNF1, TNF3-7 and TN...

The immune response to mycobacterial infections in cattle is predominantly cellular in nature and current diagnostic tests for M. bovis are based on the measurement of T cell responses. The low sensitivity of serological assays for tuberculosis is therefore not surprising and serological tests will at best be used to complement rather than replace...

The eradication of bovine tuberculosis is an ultimate aim of the beef industry and the development of accurate diagnostic tests will expedite eradication. Characterization of Mycobacterium bovis antigens, and a detailed understanding of their immune reactivity will aid in the development of more specific and sensitive diagnostic tests. With this ai...

Cellular responses to several purified antigens of Mycobacterium bovis were examined in experimentally infected cattle over a period of 36 months, using in vitro cellular proliferation and interferon-gamma assays. These antigens (12, 19, 22a, b, 24, 25, 30, 32, 39, 65 and 70 kDa) included the majority of M. bovis protein antigens described to date...

A cDNA encoding an ovine (ov) granulocyte colony-stimulating factor (G-CSF) was cloned by polymerase chain reaction, using primers based on the 5' and 3' nucleotide (nt) sequence of the bovine (bov) G-CSF cDNA. RNA was isolated from IFN-gamma and LPS-stimulated alveolar macrophages. The isolated ovG-CSF cDNA is 522 bp in length and does not include...

As the first step in the development of a cervine IFN-gamma assay for the diagnosis of tuberculosis in deer, cervine IFN-gamma, cDNA was amplified by polymerase chain reaction using primers based on the bovine IFN-y sequence. A high level of amino acid homology was found between the cervine and the ovine and bovine sequences (94% and 91% respective...

We have cloned a cDNA containing the complete coding sequence of ovine(ov) interleukin 4 (IL4) by the polymerase chain reaction using primers based on the 5' and 3' untranslated regions of the human IL4 gene. RNA was isolated from phorbol myristate acetate- and calcium ionphore A23187-stimulated mesenteric lymph node cells. The ovIL4 cDNA is 535 bp...

A monoclonal antibody-based sandwich enzyme immunoassay (EIA) for bovine interferon-gamma (IFN-gamma) has been developed and can be used in conjunction with a whole blood culture system to diagnose tuberculosis in cattle. During its development, normal bovine plasma samples were tested to establish background levels of circulatory IFN-gamma. Of 191...

The phospholipase D (PLD) gene (pld) has been deleted from the Corynebacterium pseudotuberculosis chromosome by using site-specific mutagenesis. Sheep infection trials indicate that the PLD-negative C. pseudotuberculosis strain (Toxminus) is incapable of inducing caseous lymphadentis (cheesy gland) even at doses two logs higher than that at which t...

The kinetics of interferon-gamma (IFN-gamma) production were studied in sheep mesenteric lymph node (MLN) cells at the molecular level using an ovine IFN-gamma cDNA probe and by bioassay which was verified by blocking antiviral activity with a monoclonal antibody (Mab) against recombinant bovine IFN-gamma IFN-gamma mRNA appeared in MLN cells within...

This paper describes the field evaluation of a serological test and a new in vitro assay for cell-mediated reactivity for the diagnosis of bovine tuberculosis. The use of a Mycobacterium bovis-specific antigen (MPB-70) in an ELISA to test the serological response to tuberculosis infection resulted in a specificity of 96.4% and a sensitivity of 18.1...

The recently developed gamma-interferon (IFN-gamma) assay system for the diagnosis of bovine tuberculosis in cattle has been accredited by the Standing Committee on Agriculture for use in Australia. In this test system, whole blood is incubated with tuberculin purified protein derivative (PPD) antigens for 16 to 24 h. The plasma is then collected a...

An extensive field comparison of the gamma interferon (IFN-gamma) assay and the single intradermal tuberculin test for the diagnosis of bovine tuberculosis was conducted in Australia. The specificity of the IFN-gamma assay was determined by testing more than 6000 cattle from tuberculosis-free herds and varied from 96.2% to 98.1%, depending on the c...

Cytokines are major modulators of the immune system of all animals. The cloning and expression of recombinant cytokine genes have permitted the analysis of their immune function and role in the control of the immune response to disease and vaccination. While human, murine, and bovine genes have been cloned and sequenced, the cloning of ovine cytoki...

Colony-stimulating factors (CSFs) are not only regulators of haemopoiesis but can also enhance the function of mature myeloid cells, and are therefore potential immune adjuvants. By use of the polymerase chain reaction (PCR) with primers based on the bovine granulocyte-macrophage CSF (GM-CSF) sequence, we have amplified the cDNA for ovine GM-CSF, p...

We have cloned and sequenced the cDNA for the coding region of ovine alveolar macrophage interleukin-1 beta. At the nucleotide level, the ovine cDNA shares 95, 74 and 71% homology with the bovine, human and murine cDNA equivalents or homologs. Comparison at the amino acid level revealed 95% homology with bovine IL-1 beta and approximately 57% with...

A clone coding for the entire gene for the Mycobacterium bovis protein antigen MPB70 was used to produce a series of overlapping subclones by making a series of deletions from the 3' end of the gene. The subclones expressed incomplete MPB70 proteins as fusions with glutathione-S-transferase. The insert DNA was sequenced to determine the extent of t...

SUMMARY The sensitivity of the abattoir inspection procedure introduced for Australian export beef in 1976 was compared to a detailed necropsy procedure for the detection of tuberculous lesions in cattle. In a sample of cattle that were reactors to the tuberculin test, abattoir inspection failed to detect an estimated 47% of cattle with lesions. Th...

Nine stable hybridoma cell lines were established which secreted specific monoclonal antibodies (MAbs) to bovine gamma-interferon (BoIFN-gamma). Specific binding of each of the MAbs to recombinant BoIFN-gamma (rBoIFN-gamma) was demonstrated in an indirect ELISA, whilst none of the MAbs bound to rBoIFN-alpha or rBoIFN-beta. In a Western blot the MAb...

The production of gamma-interferon (gamma-IFN) and antigen-specific immunoglobulin isotypes was monitored in sheep given primary and secondary inocula of protein and polysaccharide antigens with or without adjuvants. In efferent lymph from prefemoral nodes draining the site of inoculation, gamma-IFN levels increased within 24 h after injection of a...

An in vitro cellular assay for bovine tuberculosis has recently been developed. This assay detects gamma-interferon released in response to specific antigen in a whole blood culture system. The bio-assay previously described for the detection of bovine gamma-interferon (IFN-gamma) has now been replaced with a sandwich enzyme immunoassay (EIA) which...

Animals were identified from two sire lines as being homozygous for the class I bovine lymphocyte antigen (BoLA-A) w23. These animals were also shown to be homozygous for class II antigens (BoLA-D) which, however, differed between the two sire lines. Lymphocytes from these animals were then used either as stimulator cells in one-way mixed lymphocyt...

Peripheral blood leucocytes (PBL) from sheep immunized with pilus protein purified from Bacteroides nodosus serogroup A were cultivated in vitro and cloned in the presence of the specific antigen and autologous antigen-presenting cells (APC). The efficiency of cloning was enhanced by high proliferative responses to pili during the initial week of c...

Leucocidin from several strains of Fusobacterium necrophorum was partially purified by gel filtration on Fractogel HW55 (F), the majority of the activity being present in the 50 ml of filtrate collected after 1.1 void volumes had passed through the column (termed Fraction 1, or #1). The material also contained lipopolysaccharide in 12.5% SDS-PAGE g...