Campylobacters are emerging as one of the most significant causes of human infections worldwide, and the role that waterfowl and the aquatic environment have in the spread of disease is beginning to be elucidated. On a world scale campylobacters are possibly the major cause of gastrointestinal infections. Campylobacters are common commensals in the intestinal tract of many species of wild birds, including waterfowl. They are also widely distributed in aquatic environments where their origins may include waterfowl as well as sewage effluents and agricultural runoff. Campylobacters have marked seasonal trends. In temperate aquatic environments they peak during winter, whereas spring-summer is the peak period for human infection. Campylobacter species may survive, and remain potentially pathogenic, for long periods in aquatic environments. The utility of bacterial fecal indicators in predicting the presence of campylobacters in natural waters is questionable. Viable but nonculturable Campylobacter cells may occur, but whether they have any role in the generation of outbreaks of campylobacteriosis is unclear. The routine detection of Campylobacter spp. in avian feces and environmental waters largely relies on conventional culture methods, while the recognition of a particular species or strain is based on serotyping and increasingly on molecular methods. Thus, PCR combined with selective enrichment enhances the detection of campylobacters in water and feces, while DNA sequencing facilitates recognition of particular species and strains.

In this study, 14 isolates of Acanthamoeba from both clinical and environmental sources belonging to seven different species were assayed for tolerance of high osmotic pressure, temperature tolerance, extracellular proteases, and cytopathic effects (CPE) on immortalized rabbit corneal epithelial cells. On the basis of the results, amoeba isolates were divided into pathogenic and nonpathogenic groups. Ribosomal DNA sequencing was performed on these isolates. Phylogenetic relationships revealed that all the pathogenic strains tested clustered together as one group, while nonpathogenic strains clustered into other groups. Sequence comparisons with previously published sequences determined that among the six new pathogenic isolates used in this study, five belong to T4 genotype and one to T11. This is the first report of a T11 genotype being found in Acanthamoeba keratitis.

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 μg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 μg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts.

Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genusAcanthamoeba. Although not allAcanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenicAcanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenicAcanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoebaisolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed withAcanthamoeba-conditioned medium as well as withAcanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenicAcanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.

Acanthamoeba are opportunistic pathogens with invasive and noninvasive species. For clinical purposes it is important to differentiate potentially pathogenic from nonpathogenic isolates. For the rapid and sensitive identification of Acanthamoeba at the genus level, we used a polymerase chain reaction (PCR)-based method which detected as few as five cells. Further, we tested nine isolates of Acanthamoeba for their ability to produce cytopathic effects (CPE) on corneal epithelial cells. On the basis of the results, Acanthamoeba were divided into pathogenic or nonpathogenic groups. However, because CPE assays are not available to every diagnostic laboratory, we developed a simple plating assay based on osmotolerance which correlated well with the CPE assays. Pathogenic Acanthamoeba showed growth on higher osmolarity (agar plates containing one molar mannitol), while growth of nonpathogens was inhibited on these plates. In conclusion, we have developed methods for the rapid identification and differentiation of Acanthamoeba.