Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4,182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 µM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 µM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide and pyridazinone. Exploitation of analogues of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

Rapid and efficient screening of a Representational Difference Analysis library using reverse Southern hybridisation: Identification of genetic differences between Haemophilus parasuis isolates

Representational Difference Analysis (RDA) is an established technique used for isolation of specific genetic differences between or within bacterial species. This method was used to investigate the genetic basis of serovar-specificity and the relationship between serovar and virulence in Haemophilus parasuis. An RDA clone library of 96 isolates was constructed using H. parasuis strains H425(P) (serovar 12) and HS1967 (serovar 4). To screen such a large clone library to determine which clones are strain-specific would typically involved separately labelling each clone for use in Southern hybridisation against genomic DNA from each of the strains. In this study, a novel application of reverse Southern hybridisation was used to screen the RDA library: genomic DNA from each strain was labelled and used to probe the library to identify strain-specific clones. This novel approach represents a significant improvement in methodology that is rapid and efficient.

Current and emerging screening methods to identify post-head-emergence frost adaptation in wheat and barley

Cereal crops can suffer substantial damage if frosts occur at heading. Identification of post-head-emergence frost (PHEF) resistance in cereals poses a number of unique and difficult challenges. Many decades of research have failed to identify genotypes with PHEF resistance that could offer economically significant benefit to growers. Research and breeding gains have been limited by the available screening systems. Using traditional frost screening systems, genotypes that escape frost injury in trials due to spatial temperature differences and/or small differences in phenology can be misidentified as resistant. We believe that by improving techniques to minimize frost escapes, such ofalse-positive' results can be confidently identified and eliminated. Artificial freezing chambers or manipulated natural frost treatments offer many potential advantages but are not yet at the stage where they can be reliably used for frost screening in breeding programmes. Here we describe the development of a novel photoperiod gradient method (PGM) that facilitates screening of genotypes of different phenology under natural field frosts at matched developmental stages. By identifying frost escapes and increasing the efficiency of field screening, the PGM ensures that research effort can be focused on finding genotypes with improved PHEF resistance. To maximize the likelihood of identifying PHEF resistance, we propose that the PGM form part of an integrated strategy to (i) source germplasm;(ii) facilitate high throughput screening; and (iii) permit detailed validation. PGM may also be useful in other studies where either a range of developmental stages and/or synchronized development are desired.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5–6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30 mg semi-engorged ticks fed more reliably, ticks as small as 15 mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10 mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.