Effects of feeding plant-derived agents on the colonization of Campylobacter jejuni in broiler chickens.

The aim of this work was to test the potential use of plant-derived extracts and compounds to control Campylobacter jejuni in broiler chickens. Over a 7-wk feeding period, birds were fed a commercial diet with or without plant extracts (Acacia decurrens, Eremophila glabra), essential oil [lemon myrtle oil (LMO)], plant secondary compounds [terpinene-4-ol and α-tops (including α-terpineol, cineole, and terpinene-4-ol)], and the antibiotic virginiamycin. Traditional culture and real-time quantitative PCR techniques were used to enumerate the numbers of C. jejuni in chicken fecal and cecal samples. In addition, BW and feed intake were recorded weekly for the calculation of BW gain and feed conversion ratio. The mean log10 counts of C. jejuni were similar (P > 0.05) across treatments. However, significantly lower levels of fecal Campylobacter counts (P < 0.05) were recorded at d 41 for the α-tops treatment by culture methods. No differences (P > 0.05) in BW gain were obtained for dietary supplementation, except for the E. glabra extract, which had a negative impact (P < 0.001) on BW, resulting in sporadic death. Results from this study suggest that supplemental natural compounds used in the current study did not reduce the shedding of C. jejuni to desired levels.

Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25.

In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-ß and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation) were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis), but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia), implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT) (required to build cilia) is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.

Development of anti-infectives using phage display: biological agents against bacteria, viruses, and parasites.

The vast majority of anti-infective therapeutics on the market or in development are small molecules; however, there is now a nascent pipeline of biological agents in development. Until recently, phage display technologies were used mainly to produce monoclonal antibodies (MAbs) targeted against cancer or inflammatory disease targets. Patent disputes impeded broad use of these methods and contributed to the dearth of candidates in the clinic during the 1990s. Today, however, phage display is recognized as a powerful tool for selecting novel peptides and antibodies that can bind to a wide range of antigens, ranging from whole cells to proteins and lipid targets. In this review, we highlight research that exploits phage display technology as a means of discovering novel therapeutics against infectious diseases, with a focus on antimicrobial peptides and antibodies in clinical or preclinical development. We discuss the different strategies and methods used to derive, select, and develop anti-infectives from phage display libraries and then highlight case studies of drug candidates in the process of development and commercialization. Advances in screening, manufacturing, and humanization technologies now mean that phage display can make a significant contribution in the fight against clinically important pathogens.

Screening of Australian plants for antimicrobial activity against Campylobacter jejuni.

Campylobacter jejuni is the most common cause of acute enteritis in humans, with symptoms such as diarrhoea, fever and abdominal cramps. In this study, 115 extracts from 109 Australian plant species were investigated for their antimicrobial activities against two C. jejuni strains using an in vitro broth microdilution assay. Among the plants tested, 107 (93%) extracts showed activity at a concentration between 32 and 1024 µg/mL against at least one C. jejuni strain. Seventeen plant extracts were selected for further testing against another six C. jejuni strains, as well as Campylobacter coli, Escherichia coli, Salmonella typhimurium, Bacillus cereus, Proteus mirabilis and Enterococcus faecalis. The extract from Eucalyptus occidentalis demonstrated the highest antimicrobial activity, with an inhibitory concentration of 32 µg/mL against C. jejuni and B. cereus. This study has shown that extracts of selected Australian plants possess antimicrobial activity against C. jejuni and thus may have application in the control of this organism in live poultry and retail poultry products.

Antimicrobial activity of essential oils and five terpenoid compounds against Campylobacter jejuni in pure and mixed culture experiments.

The aim of this study was to examine the antimicrobial potential of three essential oils (EOs: tea tree oil, lemon myrtle oil and Leptospermum oil), five terpenoid compounds (α-bisabolol, α-terpinene, cineole, nerolidol and terpinen-4-ol) and polyphenol against two strains of Campylobacter jejuni (ACM 3393 and the poultry isolate C338), Campylobacter coli and other Gram negative and Gram positive bacteria. Different formulations of neem oil (Azadirachta indica) with these compounds were also tested for synergistic interaction against all organisms. Antimicrobial activity was determined by the use of disc diffusion and broth dilution assays. All EOs tested were found to have strong antimicrobial activity against Campylobacter spp. with inhibitory concentrations in the range 0.001-1% (v/v). Among the single compounds, terpinen-4-ol showed the highest activity against Campylobacter spp. and other reference strains. Based on the antimicrobial activity and potential commerciality of these agents, lemon myrtle oil, α-tops (α-terpineol+cineole+terpinen-4-ol) and terpinen-4-ol were also evaluated using an in vitro fermentation technique to test antimicrobial activity towards C. jejuni in the microbiota from the chicken-caecum. EO compounds (terpinen-4-ol and α-tops) were antimicrobial towards C. jejuni at high doses (0.05%) without altering the fermentation profile. EOs and terpenoid compounds can have strong anti-Campylobacter activity without adversely affecting the fermentation potential of the chicken-caeca microbiota. EOs and their active compounds may have the potential to control C. jejuni colonisation and abundance in poultry.

Phage-displayed peptides selected for binding to Campylobacter jejuni are antimicrobial.

In developed countries, Campylobacter jejuni is a leading cause of zoonotic bacterial gastroenteritis in humans with chicken meat implicated as a source of infection. Campylobacter jejuni colonises the lower gastrointestinal tract of poultry and during processing is spread from the gastrointestinal tract onto the surface of dressed carcasses. Controlling or eliminating C.jejuni on-farm is considered to be one of the best strategies for reducing human infection. Molecules on the cell surface of C.jejuni interact with the host to facilitate its colonisation and persistence in the gastrointestinal tract of poultry. We used a subtractive phage-display protocol to affinity select for peptides binding to the cell surface of a poultry isolate of C.jejuni with the aim of finding peptides that could be used to control this microorganism in chickens. In total, 27 phage peptides, representing 11 unique clones, were found to inhibit the growth of C.jejuni by up to 99.9% in vitro. One clone was bactericidal, reducing the viability of C.jejuni by 87% in vitro. The phage peptides were highly specific. They completely inhibited the growth of two of the four poultry isolates of C.jejuni tested with no activity detected towards other Gram-negative and Gram-positive bacteria.

The application of phage-displayed peptide libraries to ligand detection in eggs and larvae of Rhipicephalus (Boophilus) microplus.

The cattle tick, Rhipicephalus (Boophilus) microplus is an ectoparasite of cattle and is one of the major limiting factors in the use of Bos taurus cattle in tropical and subtropical countries. Current control relies heavily on chemotherapy with synthetic acaricides, which is threatened by the development of resistant tick populations. Novel approaches to target discovery in cattle ticks may provide alternative strategies for the control of these parasites. The value of phage-display technology in target discovery was assessed using late-stage (20 d) R. microplus eggs. Eight, 15-mer phage peptides were isolated which preferentially bound to the eggs, or to the larvae visible within. Western blot analyses indicated that the phage clones all bound to the same 16 kDa tick antigen. The results indicate the potential utility of phage-display in detecting 'unknown' cell surface targets on R. microplus, or indeed a range of other parasite species, which may be suitable targets for chemotherapy or vaccination strategies.

Peptides selected for binding to a virulent strain of Haemophilus influenzae by phage display are bactericidal.

Nontypeable Haemophilus influenzae (NTHi) is an obligate parasite of the oropharynx of humans, in whom it commonly causes mucosal infections such as otitis media, sinusitis, and bronchitis. We used a subtractive phage display approach to affinity select for peptides binding to the cell surface of a novel invasive NTHi strain R2866 (also called Int1). Over half of the selected phage peptides tested were bactericidal toward R2866 in a dose-dependent manner. Five of the clones encoded the same peptide sequence (KQRTSIRATEGCLPS; clone hi3/17), while the remaining four clones encoded unique peptides. All of the bactericidal phage peptides but one were cationic and had similar physical-chemical properties. Clone hi3/17 possessed a similar level of activity toward a panel of clinical NTHi isolates and H. influenzae type b strains but lacked bactericidal activity toward gram-positive (Enterococcus faecalis, Staphylococcus aureus) and gram-negative (Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella enterica) bacteria. These data indicate that peptides binding to bacterial surface structures isolated by phage display may prove of value in developing new antibiotics.

Phage-displayed peptides as developmental agonists for Phytophthora capsici zoospores.

As part of its pathogenic life cycle, Phytophthora capsici disperses to plants through a motile zoospore stage. Molecules on the zoospore surface are involved in reception of environmental signals that direct preinfection behavior. We developed a phage display protocol to identify peptides that bind to the surface molecules of P. capsici zoospores in vitro. The selected phage-displayed peptides contained an abundance of polar amino acids and proline but were otherwise not conserved. About half of the selected phage that were tested concomitantly induced zoospore encystment in the absence of other signaling agents. A display phage was shown to bind to the zoospore but not to the cyst form of P. capsici. Two free peptides corresponding to active phage were similarly able to induce encystment of zoospores, indicating that their ability to serve as signaling ligands did not depend on their exact molecular context. Isolation and subsequent expression of peptides that act on pathogens could allow the identification of receptor molecules on the zoospore surface, in addition to forming the basis for a novel plant disease resistance strategy.