The Development of a Novel qPCR Assay-Set for Identifying Fecal Contamination Originating from Domestic Fowls and Waterfowl in Israel.

Yechezkel Kashi,Jaime Prilusky,Vered Chalifa-Caspi,Shoshanit Ohad,Shifra Ben-Dor,Efrat Rorman,Bareket Dassa,Valeria Kravitz

doi:10.3389/fmicb.2016.00145

Abstract

The emerging microbial source tracking (MST) methodologies aim to identify fecal contamination originating from domestic and wild animals, and from humans. Avian MST is especially challenging, primarily because the Aves class includes both domesticated and wild species with highly diverse habitats and dietary characteristics. The quest for specific fecal bacterial MST markers can be difficult with respect to attaining sufficient assay sensitivity and specificity. The present study utilizes high throughput sequencing (HTS) to screen bacterial 16S rRNA genes from fecal samples collected from both domestic and wild avian species. Operational taxonomic unit (OTU) analysis was then performed, from which sequences were retained for downstream quantitative polymerase chain reaction (qPCR) marker development. Identification of unique avian host DNA sequences, absent in non-avian hosts, was then carried out using a dedicated database of bacterial 16S rRNA gene taken from the Ribosomal Database Project. Six qPCR assays were developed targeting the 16S rRNA gene of Lactobacillus, Gallibacterium, Firmicutes, Fusobacteriaceae, and other bacteria. Two assays (Av4143 and Av163) identified most of the avian fecal samples and demonstrated sensitivity values of 91 and 70%, respectively. The Av43 assay only identified droppings from battery hens and poultry, whereas each of the other three assays (Av24, Av13, and Av216) identified waterfowl species with lower sensitivities values. The development of an MST assay-panel, which includes both domestic and wild avian species, expands the currently known MST analysis capabilities for decoding fecal contamination.

Highlights

Microbial source tracking (MST) methodologies (Stoeckel and Harwood, 2007; Gourmelon et al, 2010; Marti et al, 2013) improve microbial monitoring resolution of surface and underground water influenced by human and animal fecal contamination
The second, compared the16S rRNA gene sequences of the selected Operational taxonomic unit (OTU) to database of bacteria originating from other host fecal samples, in order to identify unique avian DNA sequences
These analyses were the basis for planning and design of quantitative polymerase chain reaction (qPCR) assays, which were evaluated to establish a battery of Avian MST assays

Summary

Introduction

Microbial source tracking (MST) methodologies (Stoeckel and Harwood, 2007; Gourmelon et al, 2010; Marti et al, 2013) improve microbial monitoring resolution of surface and underground water influenced by human and animal fecal contamination. The Aves class (Zhu et al, 2002; Scupham et al, 2008; Lu et al, 2009; Xenoulis et al, 2010), comprising both domestic and wild birds, demonstrates highly diverse habitats and dietary characteristics which challenge MST marker design and evaluation These factors have a direct influence on assay sensitivity and specificity values, which are crucial for assay assessment, because they reflect true positive and true negative detection rates. Testing pigeon droppings using the Gull quantitative polymerase chain reaction (qPCR) assays yielded amplification levels comparable to those found in gull droppings, and in selected cases, even higher This finding emphasizes other aspects of the complexity of avian MST design as different birds share the same habitats/environmental niches and possess similar bacterial communities. They can fly and change locations carrying bacteria to different geographic locations

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