Constraint Handling Guided by Landscape Analysis in Combinatorial and Continuous Search Spaces.

The notion and characterisation of fitness landscapes has helped us understand the performance of heuristic algorithms on complex optimisation problems. Many practical problems, however, are constrained, and when significant areas of the search space are infeasible, researchers have intuitively resorted to a variety of constraint-handling techniques intended to help the algorithm manoeuvre through infeasible areas and toward feasible regions of better fitness. It is clear that providing constraint-related feedback to the algorithm to influence its choice of solutions overlays the violation landscape with the fitness landscape in unpredictable ways whose effects on the algorithm cannot be directly measured. In this work, we apply metrics of violation landscapes to continuous and combinatorial problems to characterise them. We relate this information to the relative performance of six well-known constraint-handling techniques to demonstrate how some properties of constrained landscapes favour particular constraint-handling approaches. For the problems with sampled feasible solutions, a bi-objective approach was the best performing approach overall, but other techniques performed better on problems with the most disjoint feasible areas. For the problems with no measurable feasibility, a feasibility ranking approach was the best performing approach overall, but other techniques performed better when the correlation between fitness values and the level of constraint violation was high.

Bronchoalveolar lavage for diagnosis of tuberculosis infection in elephants.

Tuberculosis (TB) has been known to affect elephants for thousands of years. It was put into spotlight when few circus elephants were diagnosed carrying Mycobacterium (M.) tuberculosis. Because of the zoonotic risk and high susceptibility to M. tuberculosis, periodic testing was enacted since, in captive breeding programmes. Presently, trunk wash is the recommended diagnostic procedure for TB. Trunk wash, however, puts the operator at risk, has low sensitivity, and is prone to contamination. Here, bronchoalveolar lavage is described for the first time for TB diagnosis in elephants. Bronchial, trunk and mouth fluids were investigated using bacterial culture, M. tuberculosis complex (MTC)-specific real-time quantitative PCR (qPCR) and mycobacterial genus-specific qPCR for overall presence of mycobacteria or mycobacterial DNA including bacteria or DNA of closely related genera, respectively, in 14 elephants. Neither bacteria of the MTC nor their DNA were identified in any of the elephants. Yet, 25% of the cultures grew non-tuberculous mycobacteria (NTM) or closely related bacterial species. Furthermore, 85% of the samples contained DNA of NTM or closely related bacterial genera. This finding might explain continued false-positive results from various serological tests. From a zoonotic point of view, bronchoalveolar lavage is safer for the testing personal, has higher probability of capturing MTC and, through PCR, identifies DNA NTM in elephants. Yet, necessary endoscopic equipment, animal sedation and access to a TB reference laboratory might pose challenging requirements in remote conditions in some elephant range countries.

Identifying Features of Fitness Landscapes and Relating Them to Problem Difficulty.

Complex combinatorial problems are most often optimised with heuristic solvers, which usually deliver acceptable results without any indication of the quality obtained. Recently, predictive diagnostic optimisation was proposed as a means of characterising the fitness landscape while optimising a combinatorial problem. The scalars produced by predictive diagnostic optimisation appear to describe the difficulty of the problem with relative reliability. In this study, we record more scalars that may be helpful in determining problem difficulty during the optimisation process and analyse these in combination with other well-known landscape descriptors by using exploratory factor analysis on four landscapes that arise from different search operators, applied to a varied set of quadratic assignment problem instances. Factors are designed to capture properties by combining the collinear variances of several variables. The extracted factors can be interpreted as the features of landscapes detected by the variables, but disappoint in their weak correlations with the result quality achieved by the optimiser, which we regard as the most reliable indicator of difficulty available. It appears that only the prediction error of predictive diagnostic optimisation has a strong correlation with the quality of the results produced, followed by a medium correlation of the fitness distance correlation of the local optima.

Mycobacterium tuberculosis exposure of livestock in a German dairy farm: implications for intra vitam diagnosis of bovine tuberculosis in an officially tuberculosis-free country.

Germany has been an officially bovine tuberculosis (bTB)-free (OTF) country since 1996. Gradually rising numbers of bTB herd incidents due to Mycobacterium bovis and M. caprae in North-Western and Southern Germany during the last few years prompted the competent authorities to conduct a nationwide bTB survey in 2013/2014. This led to the detection of a dairy herd in which as many as 55 cattle reacted positively to consecutive intra vitam testing. Test-positive animals lacked visible lesions indicative of bTB at necropsy. Extensive mycobacterial culturing as well as molecular testing of samples from 11 tissues for members of the M. tuberculosis complex (MTC) yielded negative results throughout. However, caseous lymphadenitis of Ln. mandibularis accessorius was observed during meat inspection of a fattening pig from the same farm at regular slaughter at that time. Respective tissue samples tested MTC positive by polymerase chain reaction, and M. tuberculosis T1 family were identified by spoligotyping. Four human reactors within the farmer's family were also found to be immunoreactive. As exposure of livestock to M. tuberculosis is not generally considered, its impact may result in regulatory and practical difficulties when using protocols designed to detect classical bTB, particularly in OTF countries.

[Tuberculosis infection control - recommendations of the DZK]. / Infektionsprävention bei Tuberkulose - Empfehlungen des DZK.

The epidemiological situation of tuberculosis (TB) in Germany has improved considerably during the past few years. However, those in unprotected contact with infectious tuberculosis patients frequently and/or over longer periods of time and/or intensively continue to have a higher risk for TB infection. Rapid diagnosis, prompt initiation of effective treatment, and adequate infection control measures are of particular importance to prevent infection. The present recommendations depict the essentials of infection control as well as specific measures in the hospital (isolation, criteria for its duration and technical requirements, types of respiratory protection, disinfection measures, waste disposal). The specific requirements for outpatients (medical practice), at home, for ambulance services, and in congregate settings, including prisons, are also addressed. Compared with the previous recommendations the pattern of respiratory protection measures has been simplified. As a rule, hospital staff and those visiting infectious tuberculosis patients are advised to wear respiratory protection that satisfies the criteria of FFP2-masks (DIN EN 149), while patients should wear mouth-nose protectors (surgical masks) in the presence of others and outside the isolation room. A detailed depiction of criteria for isolation and its duration in smear positive and only culturally confirmed pulmonary tuberculosis has been added.

[Tuberculosis infection control--recommendations of the DZK]. / Infektionsprävention bei Tuberkulose--Empfehlungen des DZK.

The epidemiological situation of tuberculosis (TB) in Germany has improved considerably during the past few years. However, those in unprotected contact with infectious tuberculosis patients frequently and/or over longer periods of time and/or intensively continue to have a higher risk for TB infection. Rapid diagnosis, prompt initiation of effective treatment, and adequate infection control measures are of particular importance to prevent infection. The present recommendations depict the essentials of infection control as well as specific measures in the hospital (isolation, criteria for its duration and technical requirements, types of respiratory protection, disinfection measures, waste disposal). The specific requirements for outpatients (medical practice), at home, for ambulance services, and in congregate settings, including prisons, are also addressed. Compared with the previous recommendations the pattern of respiratory protection measures has been simplified. As a rule, hospital staff and those visiting infectious tuberculosis patients are advised to wear respiratory protection that satisfies the criteria of FFP2-masks (DIN EN 149), while patients should wear mouth-nose protectors (surgical masks) in the presence of others and outside the isolation room. A detailed depiction of criteria for isolation and its duration in smear positive and only culturally confirmed pulmonary tuberculosis has been added.

Bovine tuberculosis: making a case for effective surveillance.

In 2008, a cow with marked gross lesions suspicious for bovine tuberculosis (bTB) was identified by meat inspection at home slaughtering in north-western Germany. Epidemiological investigations led to the identification of another 11 affected farms with a total of 135 animals which reacted positive to the skin test. Eight affected farms had been in trade contact with the putative index farm. While the source for the initial introduction remained unknown, it was shown that all isolates tested shared the same molecular characteristics suggesting a common source of infection. The findings demonstrate that bTB can easily be transmitted via animal trade and may remain undetected for years in herds in the absence of tuberculin testing. Hence, we believe that bTB surveillance should not rely only on meat inspection, but on a combination of both meat inspection and intradermal tuberculin testing.

The sources and dynamics of fine-grained sediment degrading the Freshwater Pearl Mussel (Margaritifera margaritifera) beds of the River Torridge, Devon, UK.

The Freshwater Pearl Mussel (Margaritifera margaritifera) is an endangered organism across its entire range. It has a complex life cycle and stringent habitat requirements and is therefore an indicator species for the general ecosystem health of host rivers. Whereas historical intensive pearl fishing contributed to population declines, excess nutrient and sediment loss associated with current land use pressures in host river catchments, including modern intensive farming practices, are now highlighted as primary contributory factors. Accordingly, this study investigated the sources and dynamics of fine-grained sediment sampled in the mussel beds of the River Torridge, SW England. Sediment source fingerprinting using a combination of colorimetric and radiometric tracers to construct different composite signatures revealed the importance of roads both as a sediment source and delivery pathway for fine-grained sediment mobilised from fields predominantly supporting lowland livestock farming. Grassland fields with evidence of soil poaching were highlighted as important sediment sources, but equally, riparian woodland was also identified as important, especially during the latter stages of consecutive runoff events when its rainfall buffering capacity was exceeded. Bed sediment storage levels (median up to 393 g m-2) were found to be low (41st percentile) compared to typical values reported by a recent strategic scale survey across England and Wales, whereas elevated turbidity peaks were shown to be long duration (days) in conjunction with consecutive days of rainfall and corresponding runoff events. Hysteresis patterns varied but were generally clockwise during the largest runoff events associated with consecutive rain days; again, suggesting mobilisation of sediment from proximal woodland sources following exceedance of rainfall buffering capacity. In combination, the data assembled by this study provides a basis for planning sediment control measures for protecting the Freshwater Pearl Mussel (FPM) beds from excessive fine-grained sediment inputs associated with the intensive use of primarily grazing land.

Mycobacterium pinnipedii: transmission from South American sea lion (Otaria byronia) to Bactrian camel (Camelus bactrianus bactrianus) and Malayan tapirs (Tapirus indicus).

Tuberculosis infections caused by Mycobacterium (M.) pinnipedii in a South American sea lion, Bactrian camel, and Malayan tapirs kept in two zoological gardens spanning a time period of 5 years are reported. The zoos were linked by the transfer of one tapir. Conventional bacteriological and molecular methods were applied to detect the pathogen. Spoligotyping and MIRU/VNTR-typing performed to assess the genetic similarity revealed identical molecular characteristics of the isolates from all animals involved. Anti-tuberculosis antibodies were detected using ELISA and a recently developed serological rapid test. The study shows that: (i) using molecular methods, the assessment of the genetic relationship of infectious agents helps to confirm the routes of infection, and that (ii) immunological tests may help to detect tuberculosis infections ante mortem more reliably and early. This would prevent the transfer of tuberculosis by asymptomatic animals.

Multiple granulomas in three squirrel monkeys (Saimiri sciureus) caused by Mycobacterium microti.

Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, causes tuberculosis in small rodents and occasionally in other mammals including man. Three adult male squirrel monkeys, two with a history of lethargy, weakness and stridor and one with paralysis of the hind legs, were presented for necropsy. One of the two lethargic animals showed multiple granulomas in the mesentery, mesenteric lymph nodes, lung, liver, kidneys and spleen, while the other showed granulomas only in the lung. The animal with paralysis of the legs had an abscess-like lesion in the skeletal muscle of the neck, granulomas in the mesenteric and mediastinal lymph nodes, and a fracture of the thirteenth thoracic vertebra with severe lesions of the spinal cord. Histologically the granulomas showed typical features of tuberculous granulomas, i.e., central necrosis surrounded by epithelioid cells, multinucleated giant cells, inflammatory cells and a border of connective tissue. Ziehl-Neelsen stain demonstrated sporadic acid-fast bacilli within the granulomas, these organisms being identified as M. microti by microbiological and molecular methods.

Outbreak of Mycobacterium bovis infection in a wild animal park.

An outbreak of tuberculosis due to Mycobacterium bovis occurred in a wild animal park. Three pot-bellied pigs (Sus scrofa vittatus), one red deer (Cervus elaphus), one buffalo (Bison bonasus) and two European lynxes (Lynx lynx) were affected and showed clinical signs including weight loss, enlarged lymph nodes and paralysis of the hindlimbs. Postmortem examinations revealed multifocal granulomatous lesions in various organs, including the lymph nodes, lungs, intestines, kidneys and the central nervous system. Acid-fast organisms were demonstrated in various organs histologically and bacteriologically. Spoligotyping of 17 isolates from various organs of the affected animals confirmed an infection by M bovis and revealed an identical pattern indicating a common origin. The spoligotype was different from the pattern of M bovis recorded in the cattle population in Germany between 2000 and 2006. Investigations of sentinel animals such as an aged silver fox (Vulpes vulpes), a badger (Meles meles), a ferret (Mustela putorius) and rodents, and tuberculin skin tests of the animal attendants and randomly collected faecal samples from the enclosures were all negative for M bovis.

Nationwide Cross-Sectional Study on Bovine Tuberculosis by Intra Vitam Testing in Germany, 2013-2014.

Germany was declared officially free from bovine tuberculosis (bTB) effective from 1 July 1996. After the occurrence of several Mycobacterium (M.) bovis outbreaks in north-western Germany in recent years with high intraherd prevalence at the time of detection, the reliability of abattoir surveillance as the principal component of the national bTB control programme was debated by veterinary public health officials. Rising numbers of wildlife-associated outbreaks caused by M. caprae in southern Germany eventually prompted a nationwide cross-sectional study on bTB. A total of 51 999 cattle, that is 0.41% of the national herd kept on 1.73% of German cattle farms, were tested. Despite 4 positive and 152 inconclusive single intradermal comparative cervical test results, none of the animals was confirmed as bTB-positive by a subsequent interferon-release assay or by post-mortem PCR testing. The estimated prevalence of bTB in Germany was thus calculated as 0.0% (CI 0.0000-0.0064%) affirming that Germany still qualifies as an officially tuberculosis-free (OTF) country. Occasional randomized nationwide testing can be an appropriate tool to reassure the OTF status and may also help to maintain an appropriate training level for the diagnostic procedures and for supporting sustained disease awareness among stakeholders.

Comparative macrorestriction and RFLP analysis of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis isolates from man, pig, and cattle.

In Germany, tuberculous lesions in slaughtered pigs due to infection with members of the Mycobacterium avium complex are increasingly reported. Contaminated food originating from pig or other livestock is discussed as potential source of human infection. M. avium isolates from man (n=45), pig (n=29), and cattle (n=13) were characterised by restriction fragment length polymorphism (RFLP) with respect to insertion sequences IS1245 and IS901 as well as by XbaI-based pulsed-field gel electrophoresis (PFGE) and the results were compared by computer cluster correlation analysis, to determine potential sources of infection in man. By PCR, 55% of animal isolates was identified as M. avium subsp. avium, and 45% as M. a. hominissuis. All human isolates belonged to M. a. hominissuis. IS1245-RFLP and PFGE resulted in two distinct main groupings reflecting the two subspecies, and dividing the isolates into several subgroups. Animal isolates of M. a. hominissuis were widely distributed within the subgroups of human isolates. M. a. avium isolates, further discriminated by IS901-RFLP, formed host-associated subgroups for animals. Comparison of RFLP patterns with those of PFGE resulted in different subgroups as well as different pairs of isolates with high similarities. Only two isolates exhibited identical patterns by both methods. In general, results of both methods support the possibility that M. a. hominissuis isolates from livestock represent a source of infection for man, probably by common environmental reservoirs. There was no evidence of human infections caused by M. a. avium in Germany.

Bovine Tuberculosis and Brucellosis in Cattle and African Buffalo in the Limpopo National Park, Mozambique.

Bovine tuberculosis (BTB) and brucellosis are prevalent in buffaloes of the Kruger National Park (KNP, South Africa). Both diseases were considered to have no or a very low prevalence in wildlife and livestock in and around the Limpopo National Park (LNP, Mozambique). The same applies for tuberculosis in Gonarezhou National Park (GNP, Zimbabwe), but just recently, BTB was detected in buffaloes in the GNP and fears arose that the disease might also spread to the LNP as a result of the partial removal of the fences between the three parks to form the Great Limpopo Transfrontier Park. To assess the status of both diseases in and around LNP, 62 buffaloes were tested for bovine tuberculosis (BTB) and bovine brucellosis. The percentage of positive BTB reactors in buffalo was 8.06% using BovidTB Stat-Pak® and 0% with BOVIGAM® IFN-γ test and IDEXX ELISA. The brucellosis seroprevalence in buffalo was found to be 17.72% and 27.42% using Rose Bengal Test (RBT) and ELISA, respectively. In addition, 2445 cattle in and around the LNP were examined for BTB using the single intradermal cervical comparative tuberculin test (SICCT), and an apparent prevalence of 0.98% was found with no significant difference inside (0.5%) and outside (1.3%) the park. This is the first published report on the presence of positive reactors to BTB and bovine brucellosis in buffalo and cattle in and outside the LNP. Monitoring the wildlife-livestock-human interface of zoonotic high-impact diseases such as BTB and brucellosis is of outmost importance for the successful implementation and management of any transfrontier park that aims to improve the livelihoods of the local communities.

Primary structure analysis and adhesion studies on the major outer membrane protein of Campylobacter jejuni.

The major outer membrane protein of Campylobacter jejuni (MOMP, 43 kDa), supposed to be one of the structures responsible for adhesion to INT 407 cells, was isolated from the crude outer membrane preparation by treatment with n-octyl-beta-D-glucopyranoside followed by preparative SDS-polyacrylamide gel electrophoresis. By cleavage of the isolated protein with cyanogen bromide and proteolytic enzymes, peptides were generated, separated by reverse phase high pressure liquid chromatography, and sequenced by automatic Edman degradation. The protein was aligned by identification of overlapping peptides. Treatment of bacteria with proteinase K prior to preparation of the outer membrane yielded a truncated MOMP with an apparent molecular mass of 25 kDa consisting of the C-terminal part of the protein. The isolated MOMP was functionally characterized by significant binding activity towards INT 407 cell membranes when isolated by preparative native gel electrophoresis, however, no binding activity was detected when the protein was isolated in the presence of SDS.

Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes.

Campylobacter jejuni is one of the major causes of human diarrhea throughout the world. Attachment to host cells and extracellular matrix proteins is considered to be an essential primary event in the pathogenesis of enteritis. Outer membrane proteins of three C. jejuni strains, one of which was aflagellate, were investigated for their contribution to the process of adhesion to INT 407 cell membranes and the extracellular matrix protein fibronectin. Using a ligand-binding immunoblotting assay the flagellin, the major outer membrane protein and a 59-kDa protein were detected to be involved in adhesion to both substrates. The MOMP was able to inhibit the attachment of the bacteria to INT 407 cell membranes partly, when the protein was isolated under native conditions. However, it was totally lost when the protein was isolated in the presence of SDS. The 59-kDa protein of one strain was identified by N-terminal sequencing, and regarding the first 14 amino acids it was found to be identical to the 37-kDa CadF protein just recently described as fibronectin-binding protein of C. jejuni. Especially for the aflagellate strain this protein may be of special importance for adhesion of the bacteria to different substrates.

BioMEMS device with integrated microdialysis probe and biosensor array.

The fabrication of a microdevice for continuous sampling and on-line monitoring of glucose is described. The device comprised a microdialysis sampling system integrated on the flow through channel of a microfabricated enzyme sensor. The sensor was produced by thin film technology and was assembled to a printed circuit board (PCB) that provided the means for both electrical and fluidic connections. A polyacrilonitrile fibre, with a cut-off of 50 kDa, was used in the fabrication of the sampling probe. The performance of the device was evaluated in-vitro. High sampling efficiency of the microdialysis probe was achieved by appropriate selection of the perfusion fluid flow rate. Response times varying from 1.5 to 3.0 min were determined for flow rates ranging between 1 and 0.2 micro l/min. The linear response range was up to 30 mM glucose and interference from other electroactive substances was almost negligible. The device showed excellent stability under continuous operation for at least 5 days and sensitivity variation less than 3% over a period of 15 days.

Microdevice with integrated dialysis probe and biosensor array for continuous multi-analyte monitoring.

The simultaneous on-line determination of glucose and lactate using a microdevice that consisted of a dialysis sampling system incorporated to the flow-through cell of a microfabricated biosensor array is presented. The fluidic connections between the different device's components were realized by subsequent processing of stacked dry resist layers on a plastic support that provided also the means for electric connections. The performance of the device was evaluated in vitro. The cross-talk effect on the downstream sensor was investigated and found to be negligible. Recoveries of over 95% for both analytes were achieved when flow rates of the perfusion fluid

Continuous measurement of subcutaneous lactate concentration during exercise by combining open-flow microperfusion and thin-film lactate sensors.

The present study was carried out to investigate in vivo in healthy humans the method of open-flow microperfusion for monitoring of the subcutaneous (s.c.) lactate concentration during rest and cycle ergometer exercise. Using open-flow microperfusion, a perforated double lumen catheter with an inflow and an outflow connection is inserted into the s.c. adipose tissue and perfused with a sterile, isotonic, ionfree fluid. Due to the low flow rate, the fluid partially equilibrates with the surrounding tissue. The equilibrated perfusate passes a sensor flow chamber where the substance of interest and the rate of recovery (i.e. the ratio of sampled concentration to interstitial concentration) are continuously monitored. Within this study, the method was evaluated in four healthy volunteers during cycle ergometer exercise. The relative increase of the lactate concentration was approximately a third in the s.c. tissue compared to the capillary blood and the peak time was delayed on average by 10 min. The correlation coefficient between blood and s.c. tissue lactate concentration ranged from r = 0.41 to r = 0.90 (n = 29) in the individual experiments. The combination of open-flow microperfusion and lactate and conductivity sensors enables on-line monitoring of the s.c. lactate concentration without in vivo calibration during steady-state and cycle ergometer exercise.

Miniaturized thin film glutamate and glutamine biosensors.

Integrated thin film biosensors were developed for the simultaneous measurement of L-glutamine and L-glutamate in a mu-flow cell. Due to a novel glutaminase with an activity optimum in the neutral pH range, direct monitoring of glutamine in a mammalian cell culture medium could be performed. The glutamine bienzyme sensor was prepared by co-immobilization of glutaminase with glutamate oxidase within a photopatterned poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel membrane. The sensor response was linear in the concentration range of 50 mumol to 10 mmol glutamine/l. Additionally, a glutamate biosensor was integrated on the sensor chip for difference measurement of possible glutamate interferences. The sensor-chip could be used for at least 300 measurements without any alteration in the performance of its sensors. A new sensor-chip with an integrated flow cell provided the possibility of simultaneous measurement of four different parameters at a cell volume of 1 microliter. In order to complete the microsystem, and in order to obtain a "lab on chip", a battery operated surface mounted device (SMD) potentiostat was developed.