Surveillance of bovine tuberculosis and risk estimation of a future reservoir formation in wildlife in Switzerland and Liechtenstein.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis or M. caprae has recently (re-) emerged in livestock and wildlife in all countries bordering Switzerland (CH) and the Principality of Liechtenstein (FL). Comprehensive data for Swiss and Liechtenstein wildlife are not available so far, although two native species, wild boar (Sus scrofa) and red deer (Cervus elaphus elaphus), act as bTB reservoirs elsewhere in continental Europe. Our aims were (1) to assess the occurrence of bTB in these wild ungulates in CH/FL and to reinforce scanning surveillance in all wild mammals; (2) to evaluate the risk of a future bTB reservoir formation in wild boar and red deer in CH/FL. Tissue samples collected from 2009 to 2011 from 434 hunted red deer and wild boar and from eight diseased ungulates with tuberculosis-like lesions were tested by direct real-time PCR and culture to detect mycobacteria of the Mycobacterium tuberculosis complex (MTBC). Identification of suspicious colonies was attempted by real-time PCR, genotyping and spoligotyping. Information on risk factors for bTB maintenance within wildlife populations was retrieved from the literature and the situation regarding identified factors was assessed for our study areas. Mycobacteria of the MTBC were detected in six out of 165 wild boar (3.6%; 95% CI: 1.4-7.8) but none of the 269 red deer (0%; 0-1.4). M. microti was identified in two MTBC-positive wild boar, while species identification remained unsuccessful in four cases. Main risk factors for bTB maintenance worldwide, including different causes of aggregation often resulting from intensive wildlife management, are largely absent in CH and FL. In conclusion, M. bovis and M. caprae were not detected but we report for the first time MTBC mycobacteria in Swiss wild boar. Present conditions seem unfavorable for a reservoir emergence, nevertheless increasing population numbers of wild ungulates and offal consumption may represent a risk.

Pulmonary Disease due to Mycobacterium tuberculosis in a Horse: Zoonotic Concerns and Limitations of Antemortem Testing.

A case of pulmonary tuberculosis caused by Mycobacterium tuberculosis was diagnosed in a horse. Clinical evaluation performed prior to euthanasia did not suggest tuberculosis, but postmortem examination provided pathological and bacteriological evidence of mycobacteriosis. In the lungs, multiple tuberculoid granulomas communicating with the bronchiolar lumen, pleural effusion, and a granulomatous lymphadenitis involving mediastinal and tracheobronchial lymph nodes were found. Serologic response to M. tuberculosis antigens was detected in the infected horse, but not in the group of 42 potentially exposed animals (18 horses, 14 alpacas, 6 donkeys, and 4 dogs) which showed no signs of disease. Diagnosis of tuberculosis in live horses remains extremely difficult. Four of 20 animal handlers at the farm were positive for tuberculous infection upon follow-up testing by interferon-gamma release assay, indicating a possibility of interspecies transmission of M. tuberculosis.

Comparative study of IFNγ and antibody tests for feline tuberculosis.

This study describes the comparison of the cell-based interferon-gamma (IFNγ) test with serological rapid antibody tests (STAT-PAK and DPP VetTB) for the ante mortem testing of tuberculosis in domestic cats. The antibody specificities of rapid antibody test-positive cats were further discerned using multi-antigen print immunoassay. A total of 62 cats with culture-confirmed Mycobacterium bovis, Mycobacterium microti, Mycobacterium avium and Mycobacterium malmoense, as well as negative controls and dangerous-contact cats were tested. Tests were also applied longitudinally to one further cat undergoing TB chemotherapy for suspected M. bovis infection. Our data from this small study show excellent test specificity (100% for all tests) and encouraging levels of test sensitivity for M. bovis and TB Complex infections (IFNγ 70-100% depending upon test interpretation criteria; rapid tests both 90% for M. bovis infection and up to 46.2% for M. microti infection). The differential diagnosis of very pathogenic TB Complex (M. bovis, Mycobacterium tuberculosis), as opposed to less-pathogenic TB Complex (M. microti) was possible where positive responses to the protein cocktail ESAT6/CFP10 were observed (80% of M. bovis-infected cats in this study showed positive IFNγ responses to ESAT6/CFP10, while 20% had antibody responses to ESAT6/CFP10 using MAPIA). Finally, preliminary data from a longitudinal study of one M. bovis-exposed cat with a positive IFNγ test pre-treatment suggest that a decrease in bacterial burden may be reflected in the IFNγ response, and thus the IFNγ test may provide a monitor for TB chemotherapy.

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.

Experimental enteric infection of gnotobiotic piglets with Chlamydia suis strain S45.

Enteric chlamydial infections of pigs with Chlamydia (C.) suis are frequent and often subclinical. The enteric pathogenicity of C. suis strain S45 was investigated in gnotobiotic piglets. Piglets from three litters (n=31) were inoculated with egg-grown chlamydiae at 2-3 days of age (n=17) or used as controls (n=14). They were observed for clinical signs, killed and necropsied sequentially at 2-13 days postinoculation (DPI). Feces were collected daily and investigated with an ELISA for chlamydial antigen. At necropsy, specimens were collected for histopathology and for immunohistochemical, PCR-based, and serological (complement fixation test, ELISA) detection of chlamydiae. Chlamydial replication and associated symptoms and lesions were observed from 2 to 13 DPI and were particularly pronounced within the first week PI. Clinical symptoms consisted of moderate-to-severe diarrhea, slight and transient anorexia, weakness and body weight loss. Immunohistochemistry and ELISA revealed that chlamydial replication was particularly marked at 2-4 DPI and primarily located in the small intestinal villus enterocytes. Further sites of replication included large intestinal enterocytes, the lamina propria and Tunica submucosa, and the mesenteric lymphnodes. Histopathological changes included moderate-to-severe villus atrophy with flattened enterocytes and focal villus tip erosions, and moderate mucosal inflammatory cell infiltrates and lymphangitis in the small intestine. PCR of spleen tissue and blood was mostly negative for chlamydiae, indicating that they did not substantially disseminate into the host up to 13 DPI. All sera were negative for anti-chlamydial antibodies. In conclusion, C. suis strain S45 elicited significant enteric disease and lesions in gnotobiotic piglets indicating its pathogenic potential for swine.

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.

Mixed infections in vitro with different Chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus.

Assuming a synergistic or additive effect of Chlamydiaceae in coexistence with other enteropathogenic agents, the viral/bacterial interaction between a cell culture adapted porcine epidemic diarrhea virus (ca-PEDV) and different Chlamydiaceae strains was studied in vitro. Vero cells were dually infected with ca-PEDV and one of the three chlamydial strains Chlamydia trachomatis S45, Chlamydophila abortus S26/3 or Chlamydophila pecorum 1710S. Three experimental protocols were designed varying the inoculation sequence. Cell layers were first inoculated with Chlamydiaceae and 20 h later with ca-PEDV in protocol one. In protocol two, both agents were administered concurrently, whereas in protocol three, ca-PEDV was applied 20 h in advance of the Chlamydiaceae. Immunofluorescence techniques, immunohistochemical (IH) staining and electron microscopy were subsequently employed to investigate the cell layers. Using indirect immunofluorescence (IF) labeling, all mixed infections revealed dually infected cells, however, only incidentally and in low numbers. Characteristically, ca-PEDV syncytia with one or more chlamydial inclusions were detected but dually infected single cells were absent. Some syncytial cells contained enlarged C. abortus or C. pecorum inclusions with abnormally large developmental forms. In comparison with simultaneously conducted monoinfections, larger chlamydial inclusions were observed in dually infected cell layers. Experiments with C. trachomatis showed significantly increased numbers of chlamydial inclusions in dually infected cell layers compared to monoinfected ones. These findings indicate an influence of ca-PEDV on the chlamydial developmental cycle and in the case of C. trachomatis, a positive effect on chlamydial colonization in mixed infections.

Growth characteristics of porcine chlamydial strains in different cell culture systems and comparison with ovine and avian chlamydial strains.

Porcine Chlamydiaceae were cultivated under various culture conditions and we compared their growth characteristics with those of ruminant and avian strains. The combination of centrifugation assisted cell culture infection and cycloheximide treatment of Vero cell coverslip cultures provided the highest inclusion numbers with all chlamydial strains. Interestingly, the use of Iscove's modified Dulbecco's medium instead of Eagle's minimal essential medium significantly increased Chlamydia suis inclusion counts. C. suis and Chlamydophila pecorum inclusion numbers were markedly increased in CaCo cells, compared with Vero cells. This accelerated growth of porcine Chlamydiaceae under certain cultivation conditions may be helpful for the propagation of low chlamydial numbers or for their isolation from field samples. The intracellular distribution of porcine Chlamydiaceae in polarised CaCo cells clearly demonstrated differences between the chlamydial strains: C. pecorum 1710S inclusions were predominantly localised in the apical cytoplasm, C. suis S45 inclusions, however, were mostly situated in lower cytoplasmatic compartments. These findings might reflect biological differences in vivo.

Molecular characterisation of chlamydial isolates from birds.

Fifty-one chlamydial isolates from birds collected in Switzerland were classified by amplification and restriction analysis of the 16S-23S rRNA intergenic spacer region as Chlamydophila psittaci. The aim was to characterise a broad panel of chlamydial strains from birds and to apply and verify the methods of classification and differentiation described for chlamydial organisms. Two of the six known avian chlamydial serovars (A and B) were found by serotyping with monoclonal antibodies. One isolate was not typable. Digestion of ompA-PCR amplicons by AluI generated four distinct restriction patterns (genotypes A, B, F and G). Genotypes A and B correlated in most cases to serovars A and B, respectively. One serovar A isolate was verified as genotype B instead of A and one serovar B isolate belonged to genotype A. The non-serotypable isolate was of genotype F and one serovar A generated genotype G. OmpA sequences of one strain of each genotype were determined and compared to data bank entries. Amino acid sequences of genotype A and B strains corresponded well, showing more than 98.0% homology. The homologies of genotypes F and G sequences to genotype A strain were 82.0 and 83.0% respectively.

Establishment of proliferative cell nuclear antigen gene as an internal reference gene for polymerase chain reaction of a wide range of archival and fresh mammalian tissues.

Polymerase chain reaction (PCR) from paraffin-embedded tissues provides a powerful tool to amplify DNA from a variety of recent and archival material. Because DNA from paraffin-embedded samples is more degraded than from fresh material, the amplification of reference genes is essential to exclude false-negative results. This study describes the use of the proliferative cell nuclear antigen (PCNA) gene as a reference gene in a range of animal species and in humans. The PCNA-PCR to amplify a fragment extending from exon 5 through exon 6 and including the intervening intron 6 gave a reproducible pattern, with a 280-base pair (bp) band from canine, equine, bovine, ovine, and caprine samples showing high sequence homology. Porcine, guinea pig, tiger, and lion samples, however, gave an additional fragment of approximately 197 bp. The whole intron 6 from these fragments is missing, possibly representing a pseudogene. In feline samples only the 197-bp fragment could be detected. This study shows that the PCNA gene is highly conserved across a broad range of animal species and is well suited as an internal control for PCR analysis in veterinary medicine.