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Acoustic techniques remain the bedrock of pipeline leak detection, particularly for the water industry. The correlation technique, in which leak noise measurements are made at accessible locations on the pipe, either side of the leak, is used world-wide. Unfortunately, especially in the case of plastic pipes, access points are often not spaced closely enough for effective leak detection to take place. An alternative to sensing on the pipe is to measure directly on the ground surface, using discrete sensors such as geophones or accelerometers. However, to do this, the vibrational field on the ground, produced by the leak, needs to be fully understood. The present author, alongside colleagues, has developed an analytical model to show how axisymmetric elastic waves propagating within the pipe radiate to the ground surface. The model, only valid directly above the pipe, shows that, dependent on the soil properties, both a conical shear wave and a conical compressional wave may radiate into the soil, and thence propagate to the ground surface. Moreover, the axial dependence of the ground surface response mirrors the axial dependence of the waves propagating within the pipe. Here, a simplified analytical model of the conical pipe-radiated waves, which encapsulates the essential phase-related features of the more complex development described previously, is presented. This then allows a relatively simple extension to predict the off-axis ground surface as well as that directly above the pipe. Numerical simulations and experimental investigations are also carried out to demonstrate the potentialities of the proposed model to reveal the underlying physics through a simple way.

One way to locate a buried plastic water pipe is to measure the surface vibration due to a leak in the region above the pipe, and to process the data to infer the pipe location. This paper investigates the physical mechanisms that propagate leak noise through the pipe and the surrounding soil to the ground surface. An analysis is carried out of the relative phase between vertical ground vibration measurements at points in a grid above the pipe. The study involves experimental measurements from a site in the UK with a more realistic leak mechanism compared to recent research, a simplified analytical model to gain insight into the underlying physics, and a numerical model to validate some of the assumptions made in the derivation of the analytical model. Three waves are principally involved in propagating leak noise to the ground surface from the pipe, namely the predominantly fluid-borne wave in the pipe, and the shear and compressional waves in the soil radiating from the pipe. Their influence on the ground surface vibration is investigated through measured and simulated phase contours over a rectangular grid of surface velocity measurements. It is shown how shear and compressional waves combine to affect the shape of the lines of constant phase on the ground. The results demonstrate the potential of the proposed analytical and numerical models to investigate wave radiation from buried water pipes, and possible pipe location strategies using phase data from surface vibration measurements.

Buried pipes are used worldwide to transport water. Although they are convenient, a large amount of water is wasted during the transportation. To minimize such a problem, water companies apply different technologies to locate leaks in their pipe networks, where the buried pipe is usually located first. Electromagnetic techniques can be used to locate buried pipes, but their performance is limited by the moisture content of the surrounding soil. Active vibro-acoustic techniques have also been investigated to locate buried pipes, in which vibro-acoustic energy is introduced into the soil by an excitation source. Although they are promising, their practical application can be expensive and complex due to the setup of an excitation mechanism. The aim of this paper is to present a passive vibro-acoustic localization technique for buried water pipes, in which a leak is the source of excitation. The localization technique is based on the calculation of an approximate slope for the unwrapped phase between a pair of sensors placed on the ground surface. The performance of the two-sensor technique is tested with two datasets, one extracted from a numerical model of the buried pipe system and the other extracted from an experiment carried out on a test rig. The results highlight the potential of the two-sensor technique in locating buried water pipes.

© 2018 Elsevier Ltd In many countries, leaks are located in water distribution pipes by using the cross-correlation of pipe vibration measured either side of a suspected leak. However, in modern plastic pipes this can be problematic due to strong coupling between the water, the pipe and the soil, affecting the propagation of leak noise within the pipe. This paper concerns an analytical, numerical and experimental investigation into the way in which soil properties influence leak noise propagation in buried plastic water pipes. The analytical model allows a detailed investigation into the physical effects of the soil on leak noise (wave) propagation in the pipe, in particular on the wave-speed and wave attenuation. Results highlight that, in addition to the pipe hoop stiffness, the shear stiffness of the soil can have a significant effect on the wave-speed in the pipe. Experimental measurements were conducted at two different sites - one in the UK and the other in Brazil. In the UK system, both dilatational and shear waves in the soil propagate away from the pipe, resulting in large wave attenuation in the pipe. However, in the Brazilian system, only shear waves propagate resulting in smaller wave attenuation in the pipe.

A new and improved vaccine against tuberculosis (TB) would provide a powerful tool to conquer one of the most insidious infectious diseases of mankind. Protection afforded by bacillus Calmette-Guérin (BCG) has been shown to be limited and inconsistent, especially in adults that are known to transmit TB disease. In the last two decades, several new vaccines have been developed and tested with the aim to elicit robust and long-lived T-cell responses against Mycobacterium tuberculosis antigens. Although much progress has been made in the TB vaccine field, there is an urgent need to address critical research questions about TB immunity with a special focus on designing vaccines aimed at preventing infection and transmission of TB. Here, we discuss the rationale behind the current immunization strategies being implemented for TB vaccines and provide some suggestions for hypothesis driven research to encourage the development of novel TB vaccines.

Among the few well characterized virulence factors of Mycobacterium tuberculosis (Mtb) is the heparin-binding hemagglutinin (HBHA). HBHA is a 21-kDa protein that localizes to the mycobacterial surface where it can interact with host components. Interaction with epithelial cells and components of the extracellular matrix is mediated by the methylated lysine-rich C-terminal domain of the protein. The N-terminal end of HBHA contains a coiled coil motif which is involved in protein oligomerization and bacterial-bacterial aggregation. In this report, we will focus our attention on what is known about the structure of the HBHA protein and the protein function and role in TB pathogenesis.

Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence and the infection of host cells by mycobacteria. In the studies presented here, we have expressed the PE_PGRS 33 gene in a non-pathogenic fast-growing Mycobacterium smegmatis strain and demonstrated that it survives better in macrophage cultures, in vitro as well as in mice after intraperitoneal administration, than the parental strain containing the vector only or a strain expressing only the PE domain of PE_PGRS 33. In macrophages, enhanced colonization by the M. smegmatis expressing PE_PGRS 33 was associated with macrophage aggregation and clearance of macrophage monolayers, visible cell necrosis and significantly greater levels of TNF (TNF-alpha) in the cultures compared with controls. The presence of macrophage cell necrosis was confirmed by measurement of significantly greater levels of lactate dehydrogenase and nucleosomes in the supernatants of the macrophage cultures infected with M. smegmatis expressing PE_PGRS 33. Antibodies directed against TNF partially reduced cytolysis, suggesting that this cytokine is critical but not sufficient for the observed macrophage necrosis and enhanced mycobacterial survival. These results extend earlier observations, which suggested that PE_PGRS proteins may have a role in the pathogenesis of mycobacterial disease and that there may be a specific role for these proteins in influencing host cell responses to infection.

Evaluation of expression of 16 PE_PGRS genes present in Mycobacterium tuberculosis under various growth conditions demonstrated constitutive expression of 7 genes, variable expression of 7 genes, and no expression of 2 genes. An inverse expression profile for genes PE_PGRS16 and PE_PGRS26 was observed to occur in macrophages and in mice infected with M. tuberculosis. Variable expression of PE_PGRS proteins could have implications for their role in the immunopathogenesis of tuberculosis.

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guérin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

The heparin-binding hemagglutinin (HBHA) of Mycobacterium tuberculosis is a surface-expressed adhesin that can affect binding to host cells via a unique, methylated, carboxyl-terminal, lysine-, alanine-, and proline-rich repeat region. It has been implicated in extrapulmonary dissemination of M. tuberculosis from the lung following the initial infection of the host. To assess the vaccine potential of this protein, purified preparations of HBHA were emulsified in a dimethyldioctadecylammonium bromide-monophosphoryl lipid A adjuvant and tested for the ability to reduce M. tuberculosis infection in the mouse aerosol challenge model for tuberculosis. The HBHA-containing vaccine gave a approximately 0.7-log reduction in CFU in both mouse lungs and spleens compared to adjuvant controls 28 days following challenge. Although a notable level of serum antibody to HBHA was elicited after three immunizations and the antibodies were able to bind to the surface of M. tuberculosis, passive immunization with monoclonal antibodies directed against HBHA did not protect in the challenge model. Compared to adjuvant controls, an elevated gamma interferon response was generated by splenic and lymph node-derived T cells from immunized mice in the presence of macrophages pulsed with purified HBHA or infected with live M. tuberculosis, suggesting that the effective immunity may be cell mediated. Efforts to construct effective recombinant HBHA vaccines in fast-growing Mycobacterium smegmatis have been unsuccessful so far, which indicates that distinctive posttranslational modifications present in the HBHA protein expressed by M. tuberculosis are critical for generating effective host immune responses. The vaccine studies described here demonstrate that HBHA is a promising new vaccine candidate for tuberculosis.

The Heparin-Binding Haemagglutinin (HBHA) is a mycobacterial adhesin involved in the dissemination of Mycobacterium tuberculosis from the site of primary infection and a potential candidate for the development of a new vaccine against tuberculosis. Methylation of HBHA is a novel post-translational event that imparts important immunological properties to the protein. Since recombinant HBHA expressed in Escherichia coli is not methylated, we investigated the possibility of producing recombinant methylated HBHA in fast growing mycobacteria for use in immunological and biochemical studies. The complete coding sequence of HBHA was cloned in the plasmid pMV206, under the control of a strong promoter (hsp60) or its own promoter. The constructs generated were electroporated into Mycobacterium smegmatis and the recombinant strains obtained were analyzed for the presence of the HBHA protein using the anti-HBHA monoclonal antibodies D2 and E4. Our results indicate that expression of high amounts of intact protein can be toxic for the mycobacteria, that methylated HBHA can be obtained in M. smegmatis only when using a promoter sequence weaker than hsp60 and that the expression of the complete structural gene is required in order to obtain methylated HBHA. We constructed a recombinant M. smegmatis strain (pMV3-38) that expresses a histidine-tagged methylated HBHA that can be easily purified. The use of fast-growing strains of M. smegmatis to obtain significant amounts of purified HBHA protein within a short timeframe, should be an effective strategy for the evaluation of a new HBHA-based vaccine candidate for tuberculosis.

The PE multigene family of Mycobacterium tuberculosis is remarkable in that it is composed of approximately 100 highly homologous genes that are found only in mycobacteria. Early evidence suggests that proteins encoded by certain members of this gene family could be present in the mycobacterial cell wall, impact antigen-presentation pathways and the ensuing host immune responses, and also provide a mechanism for generating antigenic diversity in mycobacteria.

Sequencing of the entire genome of Mycobacterium tuberculosis identified a novel multigene family composed of two closely related subfamilies designated PE and PE_PGRS. The major difference between these two families is the presence of a domain containing numerous Gly-Ala repeats extending to the C terminus of the PE_PGRS genes. We have used a representative PE_PGRS gene from M. tuberculosis, Rv1818c (1818PE_PGRS), and its amino-terminal PE region (1818PE), to investigate the immunological response to these proteins during experimental tuberculosis and following immunization with DNA constructs. During infection of mice with M. tuberculosis, a significant humoral immune response was observed against recombinant 1818PE_PGRS but not toward the 1818PE protein. Similarly, immunization with a 1818PE_PGRS DNA construct induced antibodies directed against 1818PE_PGRS but not against 1818PE proteins, and no humoral response was induced by 1818PE DNA. These results suggest that certain PE_PGRS genes are expressed during infection of the host with M. tuberculosis and that an antibody response is directed solely against the Gly-Ala-rich PGRS domain. Conversely, splenocytes from 1818PE-vaccinated mice but not mice immunized with 1818PE_PGRS secreted gamma interferon following in vitro restimulation and demonstrated protection in the mouse tuberculosis challenge model. These results suggest that the PE vaccine can elicit an effective cellular immune response and that immune recognition of the PE antigen is influenced by the Gly-Ala-rich PGRS domain.

The elucidation of the genomic sequence of Mycobacterium tuberculosis revealed the presence of a novel multigene family designated PE/PE_PGRS that encodes numerous, highly related proteins of unknown function. In this study, we demonstrate that a transposon insertion in a PE_PGRS gene (1818(PE_PGRS)) found in Mycobacterium bovis BCG Pasteur, which is the BCG homologue of the M. tuberculosis H37Rv gene Rv1818c, introduces new phenotypic properties to this BCG strain. These properties include dispersed growth in liquid medium and reduced infection of macrophages. Complementation of the 1818(PE_PGRS)::Tn5367 mutant with the wild-type gene restores both aggregative growth (clumping) in liquid medium and reestablishes infectivity of macrophages to levels equivalent to those for the parent BCG strain. Western blot analysis using antisera raised against the 1818(PE_PGRS) protein shows that PE_PGRS proteins are found in cell lysates of BCG and M. tuberculosis H37Ra and in the cell wall fraction of M. tuberculosis H37Rv. Moreover, immunofluorescent labeling of mycobacteria indicates that certain PE_PGRS proteins are localized at the cell surface of BCG and M. tuberculosis. Together these results suggest that certain PE_PGRS proteins may be found at the surface of mycobacteria and influence both cell surface interactions among mycobacteria as well as the interactions of mycobacteria with macrophages.

Tuberculosis remains the world's leading cause of death due to a single infectious agent, Mycobacterium tuberculosis, with 3 million deaths and 10 million new cases per year. The infection initiates in the lungs and can then spread rapidly to other tissues. The availability of the entire M. tuberculosis genome sequence and advances in gene disruption technologies have led to the identification of several mycobacterial determinants involved in virulence. However, no virulence factor specifically involved in the extrapulmonary dissemination of M. tuberculosis has been identified to date. Here we show that the disruption of the M. tuberculosis or Mycobacterium bovis Bacille Calmette-Guérin (BCG) hbhA gene encoding the heparin-binding haemagglutinin adhesin (HBHA) markedly affects mycobacterial interactions with epithelial cells, but not with macrophage-like cells. When nasally administered to mice, the mutant strains were severely impaired in spleen colonization, but not in lung colonization. Coating wild-type mycobacteria with anti-HBHA antibodies also impaired dissemination after intranasal infection. These results provide evidence that adhesins such as HBHA are required for extrapulmonary dissemination, and that interactions with non-phagocytic cells have an important role in the pathogenesis of tuberculosis. They also suggest that antibody responses to HBHA may add to immune protection against tuberculosis.

Identification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines. Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation in vitro. In this study, further molecular and biochemical analysis of HBHA demonstrates that it has three functional domains: a transmembrane domain of 18 amino acids residing near the N terminus, a large domain of 81 amino acids consistent with an alpha-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domain that mediates binding to proteoglycans. Using His-tagged recombinant HBHA proteins and nickel chromatography we demonstrate that HBHA polypeptides which contain the coiled-coil region form multimers. This tendency to oligomerize may be responsible for the induction of mycobacterial aggregation since a truncated N-terminal HBHA fragment containing the coiled-coil domain promotes mycobacterial aggregation. Conversely, a truncated C-terminal HBHA fragment which contains Lys-Pro-Ala-rich repeats binds to the proteoglycan decorin. These results indicate that HBHA contains at least three distinct domains which facilitate intercalation into surface membranes, promote bacterium-bacterium interactions, and mediate the attachment to sulfated glycoconjugates found in host tissues.

A 69-kDa protein has been identified on the surface of the Gram-negative pathogen Bordetella pertussis that can elicit a protective immune response in animal models. This protein is associated with virulent strains of B. pertussis but its function has remained unclear. In this report we demonstrate that purified preparations of the 69-kDa outer membrane protein can promote the attachment of Chinese hamster ovary (CHO) cells. The interaction between the mammalian cells and this protein can be specifically inhibited by an Arg-Gly-Asp (RGD)-containing synthetic peptide that is homologous with a region found in the 69-kDa protein sequence. These studies indicate that a specific cell binding site containing an Arg-Gly-Asp sequence may be involved in the interaction of this bacterial protein with mammalian cell surfaces. To further investigate the role of this protein as a bacterial adhesin, a mutant of B. pertussis W28 that does not express the 69-kDa protein was constructed using the plasmid vector pRTP1. This mutant was 30-40% less efficient at adhering to CHO cells and to human HeLa cells than was the parent strain. These data support a role for this 69-kDa outer membrane protein in the attachment of B. pertussis to mammalian cells. We propose the name "pertactin" for this protein.