Comparison of immunoassay and LC-tandem mass spectrometry analyses of ractopamine in hog oral fluid.

The accurate detection of ractopamine in food animals is crucial for marketing since some entities require animals or animal carcasses to be free of ractopamine residues. Field-based ractopamine screening tests that are rapid, sensitive, and capable of high-throughput are highly desirable to ensure that inadvertent exposure to ractopamine did not occur in animals marketed as animals that have not been fed ractopamine. An immunochemically based lateral flow assay was used to analyze oral fluids from hogs never exposed to ractopamine and from hogs that were presumed positives and results were confirmed using an enhanced sensitivity LC-MSMS method. We found that an immunochemically based lateral flow system having a working range of 2.5 to 15 ng mL-1 worked well as a screening assay with 1.7% false positive results in freshly collected hog oral fluids. Using ractopamine glucuronide standards and LC-MSMS, we determined that the false positive results were not due to the presence of ractopamine glucuronide metabolites in oral fluids.

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi.

Perylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.