PathOrganic – Risks and Recommendations Regarding Human Pathogens in Organic Vegetable Production Chains

PathOrganic assesses risks associated with the consumption of fresh and minimally processed vegetables due to the prevalence of bacterial human pathogens in plant produce. The project evaluates whether organic production poses a risk on food safety, taking into consideration sources of pathogen transmission (e.g. animal manure). The project also explores whether organic versus conventional production practices may reduce the risk of pathogen manifestation. In Europe, vegetable-linked outbreaks are not well investigated. A conceptual model together with novel sampling strategies and specifically adjusted methods provides the basis for large-scale surveys of organically grown plant produce in five European countries. Critical control points are determined and evaluated and factors contributing to a food safety problem are analyzed in greenhouse and field experiments. The project aims at developing a quantitative risk assessment model and at formulating recommendations for improving food safety in organic vegetable production

A Deletion in the N-Myc Downstream Regulated Gene 1 (NDRG1) Gene in Greyhounds with Polyneuropathy

The polyneuropathy of juvenile Greyhound show dogs shows clinical similarities to the genetically heterogeneous Charcot-Marie-Tooth (CMT) disease in humans. The pedigrees containing affected dogs suggest monogenic autosomal recessive inheritance and all affected dogs trace back to a single male. Here, we studied the neuropathology of this disease and identified a candidate causative mutation. Peripheral nerve biopsies from affected dogs were examined using semi-thin histology, nerve fibre teasing and electron microscopy. A severe chronic progressive mixed polyneuropathy was observed. Seven affected and 17 related control dogs were genotyped on the 50k canine SNP chip. This allowed us to localize the causative mutation to a 19.5 Mb interval on chromosome 13 by homozygosity mapping. The NDRG1 gene is located within this interval and NDRG1 mutations have been shown to cause hereditary motor and sensory neuropathy-Lom in humans (CMT4D). Therefore, we considered NDRG1 a positional and functional candidate gene and performed mutation analysis in affected and control Greyhounds. A 10 bp deletion in canine NDRG1 exon 15 (c.1080_1089delTCGCCTGGAC) was perfectly associated with the polyneuropathy phenotype of Greyhound show dogs. The deletion causes a frame shift (p.Arg361SerfsX60) which alters several amino acids before a stop codon is encountered. A reduced level of NDRG1 transcript could be detected by RT-PCR. Western blot analysis demonstrated an absence of NDRG1 protein in peripheral nerve biopsy of an affected Greyhound. We thus have identified a candidate causative mutation for polyneuropathy in Greyhounds and identified the first genetically characterized canine CMT model which offers an opportunity to gain further insights into the pathobiology and therapy of human NDRG1 associated CMT disease. Selection against this mutation can now be used to eliminate polyneuropathy from Greyhound show dogs

Persistence of Two Campylobacter jejuni Strains in Soil and on Spinach Plants

There are indications that the more frequent use of untreated organic residues for fertilization results in increased risk of contamination with human pathogens. Here, we evaluate the ability of two different strains of Campylobacter jejuni to persist in manure and soil as well as spread to spinach plants. It was revealed that different strategies for inoculation of C. jejuni contribute to the persistence of the bacterium in soil, roots, and shoots. Upon inoculation of the bacteria into manure prior to soil application,the amount of C. jejuni subsequently recovered in soil was higher than that from treatments involving the addition of C. jejuni cells to the soil after plant emergence. Irrespective of the bacterial inoculation dose and strategy employed, the C. jejuni content in soil remained relatively constant, whereas the majority of C. jejuni cells applied to spinach leaves could be recovered during the whole evaluation period of 21 days

Surveying Pathogenic Bacteria in Vegetables

Bacterial pathogens have increasingly been identified as disease causing agents in vegetable-linked outbreaks. Consumers nowadays show higher demands for fresh or minimally processed fruits and vegetables, and at the same time potential sources of pathogen infestation are increasing due to the more frequent use of animal manures as fertilizers in organic than in conventional agriculture. On the other hand, a higher antagonistic potential against invading pathogens is implicated from the more diverse microbiota in organic soils. Vegetable-associated outbreaks in Europe are not well documented, and guidelines are missing for reducing risks of pathogen infestation. Thus, a survey on organically grown vegetables has been carried out by the “PathOrganic” consortium in five countries. In total, 4620 plants of lettuce, 1900 plants of spinach and 500 carrots from 14 fields were collected and by using PCR based detection methods were analyzed for the prevalence of pathogenic E. coli, Salmonella sp., Staphylococcus aureus, Listeria sp. and Campylobacter spp. Common protocols were used regarding harvest and sample preparation; and molecular analyses were shared among labs according to expertise. Preceding the vegetable screenings, animal manures of various origin used for fertilizing the fields were analyzed for the presence of the same pathogens, aimed at pin-pointing potential sources of contamination. Results of the vegetable and manure surveys are presented and the concept of microbiological risk assessment followed in the project is discussed. Besides yielding a large dataset concerning pathogen infestation of vegetables and manures, the screenings provided a collection of vegetable-derived pathogen isolates available for further analysis. Together with information obtained in experimental assays, the survey results may enlarge our understanding of vegetable-associated human pathogenic bacteria and their interrelations with common microbial colonizers of plants and soils