Biological control of weeds in the 22 Pacific island countries and territories: current status and future prospects

Biological control of introduced weeds in the 22 Pacific island countries and territories (PICTs) began in 1911, with the lantana seed-feeding fly introduced into Fiji and New Caledonia from Hawaii. To date, a total of 62 agents have been deliberately introduced into the PICTs to control 21 weed species in 17 countries. A further two agents have spread naturally into the region. The general impact of the 36 biocontrol agents now established in the PICTs ranges from none to complete control of their target weed(s). Fiji has been most active in weed biocontrol, releasing 30 agents against 11 weed species. Papua New Guinea, Guam, and the Federated States of Micronesia have also been very active in weed biocontrol. For some weeds such as Lantana camara, agents have been released widely, and can now be found in 15 of the 21 PICTs in which the weed occurs. However, agents for other commonly found weeds, such as Sida acuta, have been released in only a few countries in which the weed is present. There are many safe and effective biocontrol agents already in the Pacific that could be utilised more widely, and highly effective agents that have been released elsewhere in the world that could be introduced following some additional host specificity testing. This paper discusses the current status of biological control efforts against introduced weeds in the 22 PICTs and reviews options that could be considered by countries wishing to initiate weed biological control programmes.

Zmienność genetyczna, specyficzność żywicielska i zdolność do przenoszenia wirusa mozaiki smugowatej pszenicy (WSMV) w obrębie kompleksu gatunków Aceria tosichella

Wydział Biologii: Instytut Biologii Środowiska

Carriage of Staphylococcus aureus by free-living wild animals in Spain

The presence of methicillin-susceptible Staphylococcus aureus (MSSA) was analyzed in different free-living wild animals to assess the genetic diversity and predominant genotypes on each animal species. Samples were taken from the skin and/or nares, and isolates were characterized by spa typing, multilocus sequence typing (MLST) and antimicrobial susceptibility testing. The proportion of MSSA carriers were 5.00, 22.93, 19.78, and 17.67% in Eurasian griffon vulture, Iberian ibex, red deer, and wild boar, respectively (P = 0.057). A higher proportion of isolates (P = 0.000) were recovered from nasal samples (78.51%) than skin samples (21.49%), but the 9.26% of red deer and 18.25% of wild boar would have been undetected if only nasal samples had been tested. Sixty-three different spa types were identified, including 25 new spa types. The most common were t528 (43.59%) in Iberian ibex, t548 and t11212 (15.79% and 14.04%) in red deer, and t3750 (36.11%) in wild boar. By MLST, 27 STs were detected, of which 12 had not been described previously. The most frequent were ST581 for Iberian ibex (48.72%), ST425 for red deer (29.82%), and ST2328 for wild boar (42.36%). Isolates from Eurasian griffon vulture belong to ST133. Host specificity has been observed for the most frequent spa types and STs (P = 0.000). The highest resistance percentage was found against benzylpenicillin (average, 22.2%), although most of the S. aureus isolates were susceptible to all antimicrobial tested. Basically, MSSA isolates were different from those MRSA isolates previously detected in the same animal species.

Vaccination of koalas with a recombinant Chlamydia pecorum major outer membrane protein induces antibodies of different specificity compared to those following a natural live infection

Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity, infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas (particularly blindness and infertility in females) would provide an important management tool to prevent further population decline of this species. An important step towards developing a vaccine in koalas is to understand the host immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cell epitopes across the Major Outer Membrane Protein (MOMP) of four C. pecorum strains/genotypes that are recognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasma antibodies from the koalas naturally infected with a C. pecorum G genotype strain recognised the epitopes located in the variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from an animal infected with a C. pecorum F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not from other genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein they produced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infected koalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserved domains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer wide cross-protection against the variety of chlamydial infections circulating in wild koala populations.

The host range of three biotypes of Dactylopius tomentosus (Lamarck) (Hemiptera: Dactylopiidae) and their potential as biological control agents of Cylindropuntia spp. (Cactaceae) in Australia

The host range of two newly imported biotypes of Dactylopius tomentosus and their potential as biological control agents of Cylindropuntia spp. were investigated. A third biotype (imbricata) of D. tomentosus previously released in Australia to control C. imbricata was also screened to determine if it will feed on other species of Cylindropuntia occurring in Australia. Efficacy trials were conducted to evaluate the ability of the biotypes to retard the growth or kill those plant species supporting development of four or more individuals in the host test trials. The host range of the three biotypes of D. tomentosus was restricted to the genus Cylindropuntia. However, the biotypes showed varying degrees of specificity within this genus. The imbricata biotype was the only biotype to develop on Australian C. rosea provenances, albeit with a range of developmental success on all C. rosea provenances tested. The Spanish provenance supported the highest development success followed by Grawin (NSW), Lorne Station (NSW) while the least preferred was the Mexican provenance. The rosea and cholla biotypes were unsuitable candidates to control C. rosea in Australia. However, the efficacy trials showed that the cholla biotype had a high impact on four of the eight naturalised Cylindropuntia species in Australia. This biotype established rapidly and the sustained feeding of one fecund female and her progeny killed potted plants of C. imbricata and C. fulgida at week 18. This biotype has the potential to be an effective agent against C. fulgida, C. imbricata, C. kleiniae and C. tunicata and, as a consequence, an application seeking its release in Australia has been lodged.

The host range of four new biotypes of Dactylopius tomentosus (Hemiptera: Dactylopiidae) from southern USA and their potential as biological control agents of Cylindropuntiaspp.(Cactaceae) in Australia: Part II.

Eight Cylindropuntia species have naturalised in Australia and pose serious economic, environmental and social impacts. Two biotypes of Dactylopius tomentosus have been used as bio-control agents to control different Cylindropuntia species. The host range of four additional biotypes of Dactylopius tomentosus from southern USA was investigated. Feeding and development were restricted to the genus Cylindropuntia. However, they showed differences in specificity within this genus and some biotypes discriminated between the provenances of C. rosea and C. tunicata. Efficacy trials were conducted to determine whether populations of each biotype could be sustained on the naturalised Cylindropuntia species and if these populations could retard the growth or kill these plants. The acanthocarpa biotype offers potential control of C. rosea (Lorne Station), while the cylindropuntia sp. biotype shows great potential to control C. rosea (Grawin). The cylindropuntia sp. biotype also had a high impact on C. kleiniae and C. imbricata, and a moderate impact on C. leptocaulis and C. prolifera. The acanthocarpa X echinocarpa biotype had its greatest impact on C. tunicata (Grawin), killing this plant in 18 weeks. A fourth biotype, leptocaulis, was damaging to some species, but was less effective than the other biotypes. Cylindropuntia spinosior is the only naturalised species in Australia where no effective biocontrol agent has been found.

Specific Sequence of a Beta Turn in Human La Protein May Contribute to Species Specificity of Hepatitis C Virus

Human La protein is known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with the HCV internal ribosomal entry site (IRES) around the initiator AUG within stem-loop IV form a beta-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that the HCV RNA-interacting beta-turn is conserved only in humans and chimpanzees, the species primarily known to be infected by HCV. A 7-mer peptide corresponding to the HCV RNA-interacting region of human La inhibits HCV translation, whereas another peptide corresponding to the mouse La sequence was unable to do so. Furthermore, IRES-mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed enhanced replication with HCV subgenomic and full-length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring a human La beta-turn sequence in mouse cells. Taken together, our results raise the possibility of creating an immunocompetent HCV mouse model using human-specific cell entry factors and a humanized form of La protein.

Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N-6-adenine DNA methyltransferases of Neisseria meningitidis

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N-6-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N-6-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5'-CGY(m6)AG-3'), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5'-AC(m6)ACC-3') and ModD1 (exemplified by M.Nme579I) (5'-CC(m6)AGC-3'). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5'-CGY(m6)AG-3'. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5'-GCGC(m6)AGG-3' sites, to 100% at 5'-ACGT(m6)AGG-3' sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.

A host transcriptional signature for presymptomatic detection of infection in humans exposed to influenza H1N1 or H3N2.

There is great potential for host-based gene expression analysis to impact the early diagnosis of infectious diseases. In particular, the influenza pandemic of 2009 highlighted the challenges and limitations of traditional pathogen-based testing for suspected upper respiratory viral infection. We inoculated human volunteers with either influenza A (A/Brisbane/59/2007 (H1N1) or A/Wisconsin/67/2005 (H3N2)), and assayed the peripheral blood transcriptome every 8 hours for 7 days. Of 41 inoculated volunteers, 18 (44%) developed symptomatic infection. Using unbiased sparse latent factor regression analysis, we generated a gene signature (or factor) for symptomatic influenza capable of detecting 94% of infected cases. This gene signature is detectable as early as 29 hours post-exposure and achieves maximal accuracy on average 43 hours (p = 0.003, H1N1) and 38 hours (p-value = 0.005, H3N2) before peak clinical symptoms. In order to test the relevance of these findings in naturally acquired disease, a composite influenza A signature built from these challenge studies was applied to Emergency Department patients where it discriminates between swine-origin influenza A/H1N1 (2009) infected and non-infected individuals with 92% accuracy. The host genomic response to Influenza infection is robust and may provide the means for detection before typical clinical symptoms are apparent.

Specificity enhancement of polyclonal antisera by the induction of tolerance to unwanted cross-reacting determinants

Steroids form a structurally closely related group. As a result, antibodies produced for use in immunoassays regularly show unwanted cross-reactivities, These may be reduced by altering hapten-protein coupling procedures, thereby reducing the exposure of the determinants giving rise to the undesirable cross-reaction. However, these procedures carl prove to be complex, expensive and nor totally predictable in outcome. Exploitation of the clonal selection theory is an attractive alternative approach. The host is primed with the interfering cross-reactant coupled to a non-immunogenic amino acid copolymer to inactivate the B-lymphocyte clones specific for this steroid, producing a specific immunotolerance. Then, 3 days Inter, the host is immunized with the steroid against which nn antibody is required. The clones producing antibody to this immunogen are unaffected and the cross-reactivity is significantly reduced or deleted The technique has been applied to the reduction of endogenous sex steroid cross-reactivity from antibodies prepared against synthetic and semi-synthetic androgens (17 alpha-methyltestosterone, 19-nor-beta-testosterone) and the progestogen medroxyprogesterone. Antibodies prepared against the synthetic oestrogen zeranol using this technique have significantly reduced its undesirable cross-reactivity with the fungal metabolite 7 alpha-zearalenol. Highly specific antisera have been generated in all cases, the only adverse effect being a reduction in the titres achieved in comparison with rabbits receiving the conventional immunizing regime.

Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues

The metalloproteases ZapA of Proteus mirabilis and LasB of Pseudomonas aeruginosa are known to be virulence factors their respective opportunistic bacterial pathogens, and are members of the structurally related serralysin and thermolysin families of bacterial metalloproteases respectively. Secreted at the site of infection, these proteases play a key role in the infection process, contributing to tissue destruction and processing of components of the host immune system. Inhibition of these virulence factors may therefore represent an antimicrobial strategy, attenuating the virulence of the infecting pathogen. Previously we have screened a library of N-alpha mercaptoamide dipeptide inhibitors against both ZapA and LasB, with the aim of mapping the S1' binding site of the enzymes, revealing both striking similarities and important differences in their binding preferences. Here we report the design, synthesis, and screening of several inhibitor analogues, based on two parent inhibitors from the original library. The results have allowed for further characterization of the ZapA and LasB active site binding pockets, and have highlighted the possibility for development of broad-spectrum bacterial protease inhibitors, effective against enzymes of the thermolysin and serralysin metalloprotease families.

Burkholderia cenocepacia BC2L-C Is a Super Lectin with Dual Specificity and Proinflammatory Activity

Lectins and adhesins are involved in bacterial adhesion to host tissues and mucus during early steps of infection. We report the characterization of BC2L-C, a soluble lectin from the opportunistic pathogen Burkholderia cenocepacia, which has two distinct domains with unique specificities and biological activities. The N-terminal domain is a novel TNF-alpha-like fucose-binding lectin, while the C-terminal part is similar to a superfamily of calcium-dependent bacterial lectins. The C-terminal domain displays specificity for mannose and L-glycero-D-manno-heptose. BC2L-C is therefore a superlectin that binds independently to mannose/heptose glycoconjugates and fucosylated human histo-blood group epitopes. The apo form of the C-terminal domain crystallized as a dimer, and calcium and mannose could be docked in the binding site. The whole lectin is hexameric and the overall structure, determined by electron microscopy and small angle X-ray scattering, reveals a flexible arrangement of three mannose/heptose-specific dimers flanked by two fucose-specific TNF-alpha-like trimers. We propose that BC2L-C binds to the bacterial surface in a mannose/heptose-dependent manner via the C-terminal domain. The TNF-alpha-like domain triggers IL-8 production in cultured airway epithelial cells in a carbohydrate-independent manner, and is therefore proposed to play a role in the dysregulated proinflammatory response observed in B. cenocepacia lung infections. The unique architecture of this newly recognized superlectin correlates with multiple functions including bacterial cell cross-linking, adhesion to human epithelia, and stimulation of inflammation.

Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia

Background: Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare.

Objectives: We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. 

Methods: A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >10⁴ colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1β), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. 

Results: Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1β was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1β and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). 

Conclusions: Low BALF IL-1β in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship.

Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

We describe a protocol for the generation and validation of bacteria microarrays and their application to the study of specific features of the pathogen's surface and interactions with host receptors. Bacteria were directly printed on nitrocellulose-coated glass slides, using either manual or robotic arrayers, and printing quality, immobilization efficiency and stability of the arrays were rigorously controlled by incorporating a fluorescent dye into the bacteria. A panel of wild type and mutant strains of the human pathogen Klebsiella pneumoniae, responsible for nosocomial and community-acquired infections, was selected as model bacteria, and SYTO-13 was used as dye. Fluorescence signals of the printed bacteria were found to exhibit a linear concentration-dependence in the range of 1 x 10(8) to 1 x 10(9) bacteria per ml. Similar results were obtained with Pseudomonas aeruginosa and Acinetobacter baumannii, two other human pathogens. Successful validation of the quality and applicability of the established microarrays was accomplished by testing the capacity of the bacteria array to detect recognition by anti-Klebsiella antibodies and by the complement subcomponent C1q, which binds K. pneumoniae in an antibody-independent manner. The biotin/AlexaFluor-647-streptavidin system was used for monitoring binding, yielding strain-and dose-dependent signals, distinctive for each protein. Furthermore, the potential of the bacteria microarray for investigating specific features, e.g. glycosylation patterns, of the cell surface was confirmed by examining the binding behaviour of a panel of plant lectins with diverse carbohydrate-binding specificities. This and other possible applications of the newly developed arrays, as e.g. screening/evaluation of compounds to identify inhibitors of host-pathogen interactions, make bacteria microarrays a useful and sensitive tool for both basic and applied research in microbiology, biomedicine and biotechnology.

Geographic and host factors shape the evolution of a newly recognized subgroup within the U genogroup of the fish rhabdovirus IHNV

Thesis (Master's)--University of Washington, 2015