Bovine tuberculosis in Europe from the perspective of an officially tuberculosis free country: trade, surveillance and diagnostics.

Switzerland has been officially free of bovine tuberculosis (OTF) since 1960. Since 1980 the control of bovine tuberculosis (bTB) has been reduced to passive abattoir surveillance. Isolated cases of bTB, partly due to reactivation of human Mycobacterium bovis infections with subsequent transmission to cattle, have been noticed in the last years. In Europe, the overall prevalence of bTB is slightly increasing. Both OTF and non-OTF countries report increases in the proportion of bTB positive cattle herds. Current bTB eradication and control programs in Europe are facing a range of challenges. Whole herd depopulation is becoming a less attractive option for economic reasons and due to animal welfare concerns. Live animal trade is increasing both at national and international levels. Regarding these tendencies and taking into account the chronicity of bTB infection, pre-movement testing is becoming increasingly important as a central tool for eradication and for protection against re-introduction of bTB. Pre-movement testing, however specifically focuses on the infection status in individuals, requiring a high level of diagnostic accuracy to correctly diagnose infected animals. Current screening tests for bTB, however, have been designed to meet demands as herd tests. This illustrates that the modification of existing and/or the development of new diagnostics for bTB might be needed. The tuberculin skin test (TST), the primary screening test for bTB may in certain situations have low sensitivity. The interferon gamma (IFN-γ) assay is accepted to be more sensitive compared to TST. Reduced specificity, however, especially in areas of low bTB prevalence raises concerns. New antigen combinations including Rv3615c, OmpATb and others have been shown to complement ESAT-6 and CFP-10 in the whole blood IFN-γ assay and resulted in improved sensitivity (compared to ESAT-6 and CFP-10) and specificity (compared to tuberculins). Lesion detection after slaughter represents a cost-effective procedure for passive surveillance of bTB, especially in areas of low prevalence or in regions free of bTB; however, its sensitivity is very low. This illustrates that trade is linked with a certain risk to re-introduce bTB in OTF regions or countries and that there may be delays in detecting a re-introduction of bTB. In conclusion, regarding the fact that some parameters linked with bTB programs are changing, the development of improved diagnostic tests with a high reliability for use as individual animal tests will be important for future eradication of bTB, in line with international commitment to high standard animal health programs.

Bovine tuberculosis: effect of the tuberculin skin test on in vitro interferon gamma responses.

Bovine tuberculosis (bTB) is a disease of zoonotic and economic importance. In many countries, control is based on test and slaughter policies and/or abattoir surveillance. For testing, cell mediated immune- (CMI-) based assays (i.e., tuberculin skin test (TST) supplemented by the interferon gamma (IFN-gamma) assay) are the primary surveillance and disease control tests for bTB. The combined use of the in vivo and in vitro CMI assays to increase overall sensitivity has raised the question of whether the IFN-gamma response is influenced by injection of purified protein derivatives (PPDs) for TST. Published data on the influence of the TST, applied as the caudal fold test (CFT) or the comparative cervical test (CCT), on the IFN-gamma assay are contradictory. Reviewing published data and including additional data, the following conclusions can be drawn: (1) in naturally infected cattle, PPD administration for the single or repeated short-interval CCT neither boosts nor depresses PPD-specific IFN-gamma production. Disparate results have been concluded from some studies using experimental infections, emphasizing the importance of confirming initial experimental-based findings with studies using cattle naturally infected with Mycobacterium bovis. (2) In cattle experimentally infected with M. bovis, PPD administration for CFT boosts PPD-specific IFN-gamma production for up to 7 days without any effect on test interpretation. Importantly, in naturally infected cattle, CFT-related boosting selectively increases the in vitroM. bovis PPD (PPD-B) response 3 days after CFT, resulting in an increased PPD-B response relative to the response to Mycobacterium avium PPD (PPD-A). In non-infected cattle, it cannot be excluded that the CFT induces a mild boost of the PPD-specific response, particularly in animals sensitized to environmental, non-tuberculous mycobacteria, thus decreasing the specificity of the IFN-gamma assay. (3) In general, there is a lack of data clearly characterizing the effect of TSTs on the IFN-gamma assay. Further studies are required to clearly describe the effects of both CFT and CCT in non-infected animals and in naturally infected cattle, especially in low reacting infected cattle.

Bayesian receiver operating characteristic estimation of multiple tests for diagnosis of bovine tuberculosis in Chadian cattle.

BACKGROUND: Bovine tuberculosis (BTB) today primarily affects developing countries. In Africa, the disease is present essentially on the whole continent; however, little accurate information on its distribution and prevalence is available. Also, attempts to evaluate diagnostic tests for BTB in naturally infected cattle are scarce and mostly complicated by the absence of knowledge of the true disease status of the tested animals. However, diagnostic test evaluation in a given setting is a prerequisite for the implementation of local surveillance schemes and control measures. METHODOLOGY/PRINCIPAL FINDINGS: We subjected a slaughterhouse population of 954 Chadian cattle to single intra-dermal comparative cervical tuberculin (SICCT) testing and two recently developed fluorescence polarization assays (FPA). Using a Bayesian modeling approach we computed the receiver operating characteristic (ROC) curve of each diagnostic test, the true disease prevalence in the sampled population and the disease status of all sampled animals in the absence of knowledge of the true disease status of the sampled animals. In our Chadian setting, SICCT performed better if the cut-off for positive test interpretation was lowered from >4 mm (OIE standard cut-off) to >2 mm. Using this cut-off, SICCT showed a sensitivity and specificity of 66% and 89%, respectively. Both FPA tests showed sensitivities below 50% but specificities above 90%. The true disease prevalence was estimated at 8%. Altogether, 11% of the sampled animals showed gross visible tuberculous lesions. However, modeling of the BTB disease status of the sampled animals indicated that 72% of the suspected tuberculosis lesions detected during standard meat inspections were due to other pathogens than Mycobacterium bovis. CONCLUSIONS/SIGNIFICANCE: Our results have important implications for BTB diagnosis in a high incidence sub-Saharan African setting and demonstrate the practicability of our Bayesian approach for diagnostic test evaluation.

Optimization of a whole-blood gamma interferon assay for detection of Mycobacterium bovis-infected cattle.

Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermal injection in cattle. In vitro, such antigens stimulate the production of gamma interferon (IFN-gamma) by bovine T cells in whole-blood culture (IFN-gamma assay). We have analyzed various parameters of the in vitro IFN-gamma assay, ranging from blood sampling to execution of the IFN-gamma test, in view of potential simplifications of the assay. Here, we show that IFN-gamma responses may be reduced under certain animal handling/holding conditions and that a delayed time from blood collection to culture may lead to a reduced in vitro IFN-gamma response. Delayed initiation of culture in a purified-protein-derivative-based assay (24 h compared to 8 h after blood collection), however, resulted in a significant improvement of specificity (97% compared to 85%), whereas there was only a modest reduction of sensitivity (from 96% to 90%), which was statistically not significant. Furthermore, we show that the stimulation temperature needs to be 33 degrees C or higher; that carbon dioxide is not required for stimulation; and that various plate formats, ranging from 24 to 96 wells per plate, can be utilized. The produced IFN-gamma is stable at 4 degrees C for 28 days as well as after repeated freeze-thaw cycles. Thus, stimulation of samples may be initiated in the field without the need for a carbon dioxide source, and bovine IFN-gamma is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-gamma test platform and flexibilities in test application.

Assessment of Mycobacterium tuberculosis OmpATb as a novel antigen for the diagnosis of bovine tuberculosis.

In the search for better tools to control bovine tuberculosis, the development of diagnostic tests with improved specificity and sensitivity has a high priority. We chose to search for novel immunodiagnostic reagents. In this study, Rv0899 (outer membrane protein A of Mycobacterium tuberculosis [OmpATb]) was evaluated as a stimulation antigen in a gamma interferon (IFN-gamma) release assay to diagnose bovine tuberculosis. OmpATb induced IFN-gamma responses in cattle experimentally infected with M. bovis as early and as persistently as ESAT-6 and CFP-10, the current lead diagnostic antigens. In naturally infected cattle, OmpATb stimulated IFN-gamma production in 22 of 26 animals (85%). Importantly, OmpATb detected a portion of M. bovis-infected cattle which did not respond to ESAT-6 and CFP-10 (five of six cattle). The combined diagnostic sensitivity of OmpATb, ESAT-6, and CFP-10 for a preselected group consisting of naturally infected cattle with an overrepresentation of ESAT-6/CFP-10 nonresponders was 96% (25 of 26 animals). The specificity of OmpATb for uninfected cattle was 100% (27 cattle were tested; 12 of them gave false-positive results with tuberculins). In summary, our results indicate that OmpATb has the potential to enhance the sensitivity of previously described diagnostic tests based on ESAT-6 and CFP-10 and that the combined use of OmpATb, ESAT-6, CFP-10, and other proteins may achieve at least equal sensitivity to that obtained with purified protein derivative, but at a higher specificity. Further studies evaluating the diagnostic performance of OmpATb in combination with other proteins are ongoing.

Comparative assessment of fluorescence polarization and tuberculin skin testing for the diagnosis of bovine tuberculosis in Chadian cattle.

Effective surveillance of bovine tuberculosis (BTB) in developing countries where reliable data on disease prevalence is scarce or absent is a precondition for considering potential control options. We conducted a slaughterhouse survey to assess for the first time the burden of BTB in Southern Chad. Altogether, 954 slaughter animals were consecutively sampled and tested using the single intra-dermal comparative cervical tuberculin (SICCT) test, a recently developed fluorescence polarization assay (FPA) and routine abattoir meat inspection after slaughter. Gross visible lesions were detected in 11.3% (CI: 9.4-13.5%) of the animals examined and they were mostly located in the lymph nodes and the lung. Significantly more Mbororo zebus (15.0%) were affected by lesions than Arab zebus (9.9%; OR=2.20, CI: 1.41-3.41%; p<0.001). Of all animals tested, 7.7% (CI: 6.2-9.6%) reacted positively to SICCT if OIE guidelines were applied. However, receiver operating characteristic (ROC) analysis using Mycobacterium tuberculosis complex (MTBC) infected animals as the positive population and lesion negative animals as the negative population, revealed a better SICCT performance if the cut-off value was decreased to >2mm. SICCT reactor prevalence rose to 15.5% (CI: 13.3-18.0%) and FPA did not perform better than SICCT, when this setting adapted cut-off was applied.

The prion protein is neuroprotective against retinal degeneration in vivo.

A common feature of neurodegenerative disorders is acute or progressive loss of neurons due to apoptosis. The pathological isoform of the prion protein is associated with retinal apoptosis and the cellular isoform (PrPc) has been shown to mediate protection from apoptosis in cell culture and in neonatal retinal explants. Using a model of light-induced photoreceptor apoptosis, we show in vivo that the levels of PrPc expression in the retina inversely correlate with the susceptibility of photoreceptors to light damage. Dissection of apoptotic signalling cascades suggests that PrPc acts neuroprotectively downstream of AP-1 induction. Our results reveal PrP as a neuroprotective/anti-apoptotic factor in vivo and suggest that PrPc may function as a guardian of neuronal integrity.

Comparison of immunohistochemistry and two rapid tests for detection of abnormal prion protein in different brain regions of sheep with typical scrapie.

One of the "gold standard" techniques for postmortem confirmation of scrapie diagnosis in sheep and goats is immunohistochemical examination of brain tissue. Active surveillance for scrapie is mainly performed by rapid diagnostic tests on the basis of postmortem immunochemical detection of prion protein (PrP) in the obex tissue. The aim of this study was to determine the performance of 2 rapid tests, Prionics-Check LIA (a chemiluminescence sandwich enzyme-linked immunosorbent assay) and Prionics-Check Western blot for scrapie diagnosis when applied to brain areas other than the obex, in comparison with the recognized immunohistochemistry. Prion protein was detected in the obex, cervical spinal cord, and thalamus from all the scrapie-positive sheep by the 3 tests. Western blot and LIA were negative in other areas of the brain, although weak immunohistochemical staining was detected. The results show that the 2 rapid tests studied may detect PrP in brain areas other than the obex, although with a lower sensitivity than immunohistochemistry when there is minimal PrP deposition.

Immunodetection of disease-associated mutant PrP, which accelerates disease in GSS transgenic mice.

The absence of infectivity-associated, protease-resistant prion protein (PrP(Sc)) in the brains of spontaneously sick transgenic (Tg) mice overexpressing PrP linked to Gerstmann-Sträussler Scheinker syndrome, and the failure of gene-targeted mice expressing such PrP to develop disease spontaneously, challenged the concept that mutant PrP expression led to spontaneous prion production. Here, we demonstrate that disease in overexpressor Tg mice is associated with accumulation of protease-sensitive aggregates of mutant PrP that can be immunoprecipitated by the PrP(Sc)-specific monoclonal antibody designated 15B3. Whereas Tg mice expressing multiple transgenes exhibited accelerated disease when inoculated with disease-associated mutant PrP, Tg mice expressing mutant PrP at low levels failed to develop disease either spontaneously or following inoculation. These studies indicate that inoculated mutant PrP from diseased mice promotes the aggregation and accumulation of pre-existing pathological forms of mutant PrP produced as a result of transgene overexpression. Thus, while pathological mutant PrP possesses a subset of PrP(Sc) characteristics, we now show that the attribute of prion transmission suggested by previous studies is more accurately characterized as disease acceleration.

Aggravation of ischemic brain injury by prion protein deficiency: role of ERK-1/-2 and STAT-1.

The cellular isoform of prion protein, PrPc, may confer neuroprotection in the brain, according to recent studies. To elucidate the role of PrPc in stroke pathology, we subjected PrPc-knockout (Prnp(0/0)), wild-type and PrPc-transgenic (tga20) mice to 30 min of intraluminal middle cerebral artery occlusion, followed by 3, 24 or 72 h reperfusion, and examined how PrPc levels influence brain injury and cell signaling. In immunohistochemical experiments and Western blots, we show that PrPc expression is absent in the brains of Prnp(0/0) mice, detectable in wild-type controls and approximately 4.0-fold elevated in tga20 mice. We provide evidence that PrPc deficiency increases infarct size by approximately 200%, while transgenic PrPc restores tissue viability, albeit not above levels in wild-type animals. To elucidate the mechanisms underlying Prnp(0/0)-induced injury, we performed Western blots, which revealed increased activities of ERK-1/-2, STAT-1 and caspase-3 in ischemic brains of Prnp(0/0)mice. Our data suggest a role of cytosolic signaling pathways in Prnp(0/0)-induced cell death.

Diagnosis of prion diseases.

Prion diseases are usually diagnosed clinically and confirmed by post-mortem histopathological examination of brain tissue. The only reliable molecular marker for prion diseases is PrP(Sc), the pathological conformer of the prion protein that accumulates in the central nervous system and, to a lesser extent, in lymphoreticular tissues. For BSE, several commercial diagnostic kits based on the post-mortem immunochemical detection of PrP(Sc) in brain tissue are now available. These rapid screening tests have been used in active surveillance of BSE and have greatly improved the detection of infected cattle before their entry into the human food chain. At present, no diagnostic test exists for the detection of prion diseases in live animals or humans. New diagnostic techniques aimed at increasing sensitivity and specificity of PrP(Sc) detection in body fluids and at identifying novel surrogate markers are under development. In this report, we review the classical diagnostic methods as well as present and future tools for the diagnosis of prion diseases.

PrP(CWD) lymphoid cell targets in early and advanced chronic wasting disease of mule deer.

Up to 15% of free-ranging mule deer in northeastern Colorado and southeastern Wyoming, USA, are afflicted with a prion disease, or transmissible spongiform encephalopathy (TSE), known as chronic wasting disease (CWD). CWD is similar to a subset of TSEs including scrapie and variant Creutzfeldt-Jakob disease in which the abnormal prion protein isoform, PrP(CWD), accumulates in lymphoid tissue. Experimental scrapie studies have indicated that this early lymphoid phase is an important constituent of prion replication interposed between mucosal entry and central nervous system accumulation. To identify the lymphoid target cells associated with PrP(CWD), we used triple-label immunofluorescence and high-resolution confocal microscopy on tonsils from naturally infected deer in advanced disease. We detected PrP(CWD) primarily extracellularly in association with follicular dendritic and B cell membranes as determined by frequent co-localization with antibodies against membrane bound immunoglobulin and CD21. There was minimal co-localization with cytoplasmic labels for follicular dendritic cells (FDC). This finding could indicate FDC capture of PrP(CWD), potentially in association with immunoglobulin or complement, or PrP(C) conversion on FDC. In addition, scattered tingible body macrophages in the germinal centre contained coarse intracytoplasmic aggregates of PrP(CWD), reflecting either phagocytosis of PrP(CWD) on FDC processes, apoptotic FDC or B cells, or actual PrP(CWD) replication within tingible body macrophages. To compare lymphoid cell targets in early and advanced disease, we also examined: (i) PrP(CWD) distribution in lymphoid cells of fawns within 3 months of oral CWD exposure and (ii) tonsil biopsies from preclinical deer with naturally acquired CWD. These studies revealed that the early lymphoid cellular distribution of PrP(CWD) was similar to that in advanced disease, i.e. in a pattern suggesting FDC association. We conclude that in deer, PrP(CWD) accumulates primarily extracellularly and associated with FDCs and possibly B cells - a finding which raises questions as to the cells responsible for pathological prion production.

Prion protein interaction with glycosaminoglycan occurs with the formation of oligomeric complexes stabilized by Cu(II) bridges.

Several lines of evidence have shown glycosaminoglycans (GAGs) to be physiological ligands of the prion protein (PrP), but the molecular and regulatory aspects of the interaction remain unknown. Using full-length recombinant prion protein and low molecular mass heparin and heparan sulfate as glycosaminoglycans, we have found that the interaction occurs with the formation of oligomeric complexes. Within the protein-glycosaminoglycan complexes, PrP exhibited an enhanced fluorescence emission and a reduced solvent exposure. The pH and ionic strength-dependence of the interaction reveals His residues as the main binding sites at acid pH. A synthetic peptide consisting of four octarepeats is able to reproduce the His-dependent binding of the protein, thus demonstrating the role of the octarepeats in the GAG interaction. Alternatively, PrP can bind GAGs through His-bound Cu(II). These Cu(II) bridges promote a tighter interaction, as shown by the increased resistance to ionic strength, to protease action, and to pH-induced cation release. Inspection of other cations shows that Zn(II) but not Ni(II) shares the interaction trend. Taken together, our data suggest that the octarepeat region constitutes a novel GAG-binding sequence and that His-bound Cu(II) may act as a cofactor for intermolecular recognition reactions, allowing the formation of PrP-Cu(II)-glycosaminoglycan assemblies that may be crucial entities in the PrP metabolism.

Validation of a luminescence immunoassay for the detection of PrP(Sc) in brain homogenate.

A luminescence immunoassay (LIA) was developed for the diagnosis of bovine spongiform encephalopathy (BSE) in brain tissue using two different monoclonal antibodies for capture and detection of the protease-resistant fragment of the pathological prion protein (PrP27-30). PrP27-30 currently represents the most reliable marker for the infectious particle (denominated prion) causing transmissible spongiform encephalopathies (TSEs). Internal and official validation studies of this assay are described using brain homogenates from ascertained BSE positive and negative cows. Using more than 300 positive and 1400 negative bovine or ovine samples, an excellent sensitivity and specificity of 100% were demonstrated. More than 1000-fold dilutions of a BSE positive homogenate still resulted in a clear positive signal. In combination with a simple homogenisation procedure for the preparation of the samples, this assay lends itself for large scale screening of cattle and sheep for TSEs using complete automation of the process.

PrP(CWD) in the myenteric plexus, vagosympathetic trunk and endocrine glands of deer with chronic wasting disease.

Accumulated evidence in experimental and natural prion disease systems supports a neural route of infectious prion spread from peripheral sites of entry to the central nervous system. However, little is known about prion trafficking routes in cervids with a naturally occurring prion disease known as chronic wasting disease (CWD). In the brain, the pathogenic isoform of the prion protein (PrP(CWD)) accumulates initially in the dorsal motor nucleus of the vagus nerve. To assess whether alimentary-associated neural pathways may play a role in prion trafficking, neural and endocrine tissues from mule deer naturally infected with CWD (n=6) were examined by immunohistochemistry. PrP(CWD) was detected in the myenteric plexus, vagosympathetic trunk, nodose ganglion, pituitary, adrenal medulla and pancreatic islets. No to scant PrP(CWD) staining was detected in other nerves or ganglia (brachial plexus, sciatic nerve, gasserian ganglion, coeliac ganglion, cranial cervical ganglion, spinal nerve roots) of CWD-positive deer and no PrP(CWD) was detected in nerves or endocrine tissues from 11 control deer. These findings suggest that: (i) transit of PrP(CWD) in nerves, either centrifugally or centripetally, is one route of prion trafficking and organ invasion and (ii) endocrine organs may also be targets for cervid pathogenic prion accumulation.