Sven D.C. Parsons

Zoonotic Mycobacterium bovis- induced Tuberculosis in Humans

We aimed to estimate the global occurrence of zoonotic tuberculosis (TB) caused by Mycobacterium bovis or M. caprae infections in humans by performing a multilingual, systematic review and analysis of relevant scientific literature of the last 2 decades. Although information from many parts of the world was not available, data from 61 countries suggested a low global disease incidence. In regions outside Africa included in this study, overall median proportions of zoonotic TB of ≤1.4% in connection with overall TB incidence rates ≤71/100,000 population/year suggested low incidence rates. For countries of Africa included in the study, we multiplied the observed median proportion of zoonotic TB cases of 2.8% with the continental average overall TB incidence rate of 264/100,000 population/year, which resulted in a crude estimate of 7 zoonotic TB cases/100,000 population/year. These generally low incidence rates notwithstanding, available data indicated substantial consequences of this disease for some population groups and settings.

Chronic pulmonary cavitary tuberculosis in rabbits: a failed host immune response.

The molecular determinants of the immune response to Mycobacterium tuberculosis HN878 infection in a rabbit model of pulmonary cavitary tuberculosis were studied. Aerosol infection of rabbits resulted in a highly differentially expressed global transcriptome in the lungs at 2 weeks, which dropped at 4 weeks and then gradually increased. While IFNγ was progressively upregulated throughout the infection, several other genes in the IFNγ network were not. T-cell activation network genes were gradually upregulated and maximally induced at 12 weeks. Similarly, the IL4 and B-cell activation networks were progressively upregulated, many reaching high levels between 12 and 16 weeks. Delayed peak expression of genes associated with macrophage activation and Th1 type immunity was noted. Although spleen CD4+ and CD8+ T cells showed maximal tuberculosis antigen-specific activation by 8 weeks, macrophage activation in lungs, lymph nodes and spleen did not peak until 12 weeks. In the lungs, infecting bacilli grew exponentially up to 4 weeks, followed by a steady-state high bacillary load to 12 weeks that moderately increased during cavitation at 16 weeks. Thus, the outcome of HN878 infection of rabbits was determined early during infection by a suboptimal activation of innate immunity and delayed T-cell activation.

Novel Cause of Tuberculosis in Meerkats, South Africa

The organism that causes tuberculosis in meerkats (Suricata suricatta) has been poorly characterized. Our genetic analysis showed it to be a novel member of the Mycobacterium tuberculosis complex and closely related to the dassie bacillus. We have named this epidemiologically and genetically unique strain M. suricattae.

Spontaneous Latency in a Rabbit Model of Pulmonary Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is an exquisitely adapted human pathogen capable of surviving for decades in the lungs of immune-competent individuals in the absence of disease. The World Health Organization estimates that 2 billion people have latent TB infection (LTBI), defined by a positive immunological response to Mtb antigens, with no clinical signs of disease. A better understanding of host and pathogen determinants of LTBI and subsequent reactivation would benefit TB control efforts. Animal models of LTBI have been hampered generally by an inability to achieve complete bacillary clearance. Herein, we have characterized a rabbit model of LTBI in which, similar to most humans, complete clearance of pulmonary Mtb infection and pathological characteristics occurs spontaneously. The evidence that Mtb-CDC1551-infected rabbits achieve LTBI, rather than sterilization, is based on the ability of the bacilli to be reactivated after immune suppression. These rabbits showed early activation of T cells and macrophages and an early peak in the TNFα level, which decreased in association with clearance of bacilli from the lungs. In the absence of sustained tumor necrosis factor-α production, no necrosis was seen in the evolving lung granulomas. In addition, bacillary control was associated with down-regulation of several metalloprotease genes and an absence of lung fibrosis. This model will be used to characterize molecular markers of protective immunity and reactivation.

Mixed infections of Corynebacterium pseudotuberculosis and non-tuberculous mycobacteria in South African antelopes presenting with tuberculosis-like lesions.

Routine meat inspection of antelope carcasses from a South African game reserve revealed a high prevalence of tuberculosis-like lesions. This study aimed to identify the causative agent of this disease and to describe its pathological features. In total, 139 antelopes were randomly harvested from the game reserve and subjected to meat inspection. Of these animals, 46 (33%) showed gross visible, tuberculosis-like lesions. Histopathological examination revealed the presence of encapsulated necrogranulomas in organs and/or lymph nodes of 22 of 27 animals tested. Tissue samples from lesions were processed for both non-selective bacterial culture and mycobacterial culture following decontamination. In non-selective cultures of lesions from 25 of 31 animals tested, Corynebacterium pseudotuberculosis was detected. Isolation of C. pseudotuberculosis was closely associated with the presence of necrogranulomas. In mycobacterial cultures of lesions from 9 of 41 animals tested, different species of non-tuberculous mycobacteria (NTMs) were detected. In 5 instances, depending on the culture procedure that was applied, either C. pseudotuberculosis or NTMs were isolated from the same tissue sample. Our results suggest that the disease has been caused by infections with C. pseudotuberculosis. In sub-Saharan Africa, the role of pathogens other than Mycobacterium bovis may be underestimated in causing tuberculosis-like lesions. In cases where potentially pathogenic NTMs are isolated from mycobacterial cultures of tuberculosis-like lesions, the non-use of additional non-selective culture techniques could lead to misinterpretations of the diagnostic test results.

Detection of Mycobacterium tuberculosis infection in dogs in a high-risk setting.

Dogs infected with Mycobacterium tuberculosis can develop clinical tuberculosis (TB) but there are currently no validated immunological assays for diagnosing this infection in this species. Using a post mortem survey we investigated the prevalence of non-clinical M. tuberculosis infection and clinical TB disease in a high-risk population of dogs and developed and utilised a novel interferon-gamma release assay to determine the risk of transmission of M. tuberculosis from TB patients to contact dogs. The prevalence of clinical TB in dogs from a high-risk setting was 1% (95% CI: 0-5%) while the prevalence of immunological sensitization to M. tuberculosis antigens in dogs living in contact with sputum smear-positive TB patients was 50%. The IGRA proved a useful test of M. tuberculosis infection in dogs and the high levels of transmission of this pathogen from humans to companion dogs should be considered when assessing the zoonotic risks associated with such animals.

Modification of the QuantiFERON-TB Gold (In-Tube) assay for the diagnosis of Mycobacterium bovis infection in African buffaloes (Syncerus caffer).

African buffaloes (Syncerus caffer) are the most significant wildlife maintenance hosts of Mycobacterium bovis, the causative organism of bovine tuberculosis (BTB). Current diagnostic tests for the detection of M. bovis infection in free-ranging buffaloes have numerous limitations and we wished to evaluate a modification to a human TB assay, the QuantiFERON-TB Gold (In-Tube) assay (QFT), as a practical diagnostic test for BTB in buffaloes. One hundred and seventy-five buffaloes were tested using the single intradermal comparative tuberculin test (SICTT) and a modified QFT (mQFT). An appropriate cut-off point for the mQFT was derived from SICTT results using receiver operator characteristic curve analysis. Twenty-six SICTT-positive buffaloes were killed and subjected to necropsy, and selected tissues were processed for mycobacterial culture and speciation. An optimal cut-off point for the mQFT was calculated as 66pg/ml. The assay correctly detected 39/40 SICTT-positive buffaloes and 129/134 TST-negative buffaloes and M. bovis was cultured from 21/26 slaughtered SICTT/mQFT-positive animals. The mQFT shows promise as a practical test for M. bovis infection in buffaloes and shows a sensitivity and specificity at least similar to that of the TST.

Agreement between assays of cell-mediated immunity utilizing Mycobacterium bovis-specific antigens for the diagnosis of tuberculosis in African buffaloes (Syncerus caffer).

We assessed the use of Mycobacterium bovis-specific peptides for the diagnosis of tuberculosis in African buffaloes (Syncerus caffer) by evaluating the agreement between the single intradermal comparative tuberculin test (SICTT), the Bovigam(®) EC (BEC) assay, the Bovigam(®) HP (BHP) assay and two assays utilizing the QuantiFERON(®) TB-Gold (in tube) system employing 20 h (mQFT20 assay) and 30 h (mQFT30 assay) whole blood incubation periods. Of 84 buffaloes, 45% were SICTT-positive, 48% were BEC-positive, 50% were BHP-positive, 37% were mQFT20-positive and 43% were mQFT30-positive. Agreement between the BEC and BHP Bovigam(®) assays was high (κ=0.86, 95% CI 0.75-0.97) and these detected the most test-positive animals suggesting that they were the most sensitive assays. Interferon-gamma release was significantly greater in buffaloes that were test-positive for all tests than in animals with discordant but positive Bovigam(®) results. Agreement between the mQFT assays was equally high (κ=0.88, 95% CI 0.77-0.98); however, all buffaloes with discordant mQFT results (n=6) were mQFT30-positive/mQFT20-negative, including three confirmed M. bovis-infected animals, suggesting that the mQFT30 assay is the more sensitive of the two. Agreements between the two Bovigam(®) and two mQFT assays were moderate, suggesting that in its current format the mQFT assay is less sensitive than either the BEC or the BHP assays.

IP-10 Is a Sensitive Biomarker of Antigen Recognition in Whole-Blood Stimulation Assays Used for the Diagnosis of Mycobacterium bovis Infection in African Buffaloes (Syncerus caffer)

ABSTRACT African buffaloes (Syncerus caffer) are maintenance hosts of Mycobacterium bovis, the causative agent of bovine tuberculosis. They act as reservoirs of this infection for a wide range of wildlife and domestic species, and the detection of infected animals is important to control the geographic spread and transmission of the disease. Interferon gamma (IFN-γ) release assays (IGRAs) utilizing pathogen-derived peptide antigens are highly specific tests of M. bovis infection; however, the diagnostic sensitivities of these assays are suboptimal. We evaluated the diagnostic utility of measuring antigen-dependent interferon gamma-induced protein 10 (IP-10) release as an alternative to measuring IFN-γ levels. M. bovis-exposed buffaloes were tested using the Bovigam PC-EC and Bovigam PC-HP assays and a modified QuantiFERON TB-Gold (mQFT) assay. IP-10 was measured in the harvested plasma and was produced in significantly greater abundance in response to M. bovis antigens in Bovigam-positive than in Bovigam-negative animals. For each assay, using the Bovigam results as a reference, receiver operating characteristic curve analysis was done to determine diagnostically relevant cutoff values for IP-10. Thereafter, mQFT test results derived from measurement of IP-10 and IFN-γ were compared and a larger number of Bovigam-positive animals were detected using IP-10 as a diagnostic marker. Moreover, using IP-10, agreement between the mQFT assay and the Bovigam assays was increased, while the excellent agreement between the Bovigam assays was retained. We conclude that IP-10 is a sensitive marker of antigen recognition and that measurement of this cytokine in antigen-stimulated whole blood might increase the sensitivity of conventional IGRAs in African buffaloes.

Detection of Mycobacterium kansasii infection in a rhesus macaque (Macaca mulatta) using a modified QuantiFERON-TB Gold assay

A modification of a highly practical human interferon-gamma release assay for the diagnosis of tuberculosis (TB), the QuantiFERON((R))-TB Gold (In-Tube Method) (QFG-IT) assay, was evaluated for diagnosing natural mycobacterial infection in rhesus macaques (Macaca mulatta). All animals in a captive colony were tested using the QFG-IT and tuberculin skin test (TST). Animals testing positive to these tests were euthanised and necropsied. Selected tissues were processed for histopathology and mycobacterial culture, and positive cultures were speciated by Mycobacterium tuberculosis complex polymerase chain reaction (PCR) and 16S rRNA gene PCR sequencing techniques. M. tuberculosis was cultured from a TST-positive/QFG-IT-positive animal which showed gross pulmonary pathology typical of TB. Additionally, Mycobacterium kansasii was cultured from a TST-negative/QFG-IT-positive animal which had no pathological or histopathological signs of mycobacterial infection. The detection of M. kansasii infection in a QFG-IT-positive animal which showed no evidence of disease indicates that this test might be a highly sensitive tool for the diagnosis of mycobacterial infection in rhesus macaques. However, these findings highlight the limitations of the QFG-IT to specifically detect infection by the pathogens M. tuberculosis and Mycobacterium bovis.