Food Safety: at the center of a One Health approach for combating zoonoses.

Food Safety is at the center of One Health. Many, if not most, of all important zoonoses relate in some way to animals in the food production chain. Therefore, the food becomes an important vehicle for many, but not all, of these zoonotic pathogens. One of the major issues in food safety over the latest decennia has been the lack of cross-sectoral collaboration across the food production chain. Major food safety events have been significantly affected by the lack of collaboration between the animal health, the food control, and the human health sector. Examples range from BSE and E. coli outbreaks over dioxin crises to intentional melamine contamination. One Health formulates clearly both the need for and the benefit of cross-sectoral collaboration. In this chapter, we will focus on the human health risk related to zoonotic microorganisms present both in food animals and food from these animals, and typically transmitted to humans through food. We focus on these issues because they are very important in relation to the human disease burden, but also because this is the area where some experience of cross-sectoral collaboration already exist. Food related zoonoses can be separated in three major classes: parasites, bacteria, and viruses. While parasites often relate to very specific animal hosts and contribute significantly to the human disease burden, virus have often been related to major, well-published global outbreaks, e.g. SARS and avian- and swine-influenza. The bacterial zoonoses on the other hand often result in sporadic, but very wide-spread disease cases, resulting in a major disease burden in all countries, e.g. Salmonella and Campylobacter. Next to these traditional zoonotic problems, the use of antimicrobials in (food) animals has also caused the emergence of antimicrobial resistant (AMR) zoonotic bacteria. It is important to realize the difference in the nature of disease epidemiology, as well as, in society's reaction to these diseases in different socio-economic settings. Some diseases have global epidemic-or pandemic-potential, resulting in dramatic action from international organizations and national agricultural-and health authorities in most countries, for instance as was the case with avian influenza. Other diseases relate to the industrialized food production chain and have been-in some settings-dealt with efficiently through farm-to-fork preventive action in the animal sector, e.g. Salmonella. Finally, an important group of zoonotic diseases are 'neglected diseases' in poor settings, while they have been basically eradicated in affluent economies through vaccination and culling policies in the animal sector, e.g. Brucella.

Evaluation of DNA extraction methods for Bacillus anthracis spores spiked to food and feed matrices at biosafety level 3 conditions.

The DNA extraction efficiency from milk, whey, soy, corn gluten meal, wheat powders and heat-treated corn grain that were spiked with Bacillus anthracis and Bacillus thuringiensis spores was determined. Two steps were critical: lysis of the spores and binding of the free DNA to the DNA binding magnetic beads in the presence of the interfering powders. For the guanidine-thiocyanate based Nuclisens lysis buffer from Biomerieux we found that between 15 and 30% of the spores survived the lysis step. As most lysis buffers in DNA/RNA extraction kits are guanidine based it is likely that other lysis buffers will show a similar partial lysis of the Bacillus spores. Our results show that soybean flour and wheat flour inhibited the DNA extraction process strongest, leading to unreliable DNA extractions when using too much of the matrix. For corn gluten meal, heat-treated corn grain and milk powders, DNA extraction efficiencies in the presence of 100mg and 10mg of powder resulted in 70%-95% reduced DNA recoveries. The inhibition was, however, reliable and intermediate compared to the inhibition by soy and wheat. Whey powder had the lowest inhibitory effect on DNA-extraction efficiency and recoveries of 70-100% could be reached when using 10mg of powder. The results show that reducing the amount of matrix leads to better DNA-extraction efficiencies, particularly for strongly inhibiting powders such as soy and wheat. Based on these results, a standard protocol to directly isolate DNA from micro-organisms present in complex matrixes such as food and feed powders was designed.

Towards an international standard for detection and typing botulinum neurotoxin-producing Clostridia types A, B, E and F in food, feed and environmental samples: a European ring trial study to evaluate a real-time PCR assay.

A real-time PCR method for detection and typing of BoNT-producing Clostridia types A, B, E, and F was developed on the framework of the European Research Project "Biotracer". A primary evaluation was carried out using 104 strains and 17 clinical and food samples linked to botulism cases. Results showed 100% relative accuracy, 100% relative sensitivity, 100% relative specificity, and 100% selectivity (inclusivity on 73 strains and exclusivity on 31 strains) of the real-time PCR against the reference cultural method combined with the standard mouse bioassay. Furthermore, a ring trial study performed at four different European laboratories in Italy, France, the Netherlands, and Sweden was carried out using 47 strains, and 30 clinical and food samples linked to botulism cases. Results showed a concordance of 95.7% among the four laboratories. The reproducibility generated a relative standard deviation in the range of 2.18% to 13.61%. Considering the high level of agreement achieved between the laboratories, this real-time PCR is a suitable method for rapid detection and typing of BoNT-producing Clostridia in clinical, food and environmental samples and thus support the use of it as an international standard method.

A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types.

Bacillus anthracis is closely related to the endospore forming bacteria Bacillus cereus and Bacillus thuringiensis. For accurate detection of the life threatening pathogen B. anthracis, it is essential to distinguish between these three species. Here we present a novel multiplex real-time PCR for simultaneous specific identification of B. anthracis and discrimination of different B. anthracis virulence types. Specific B. anthracis markers were selected by whole genome comparison and different sets of primers and probes with optimal characteristic for multiplex detection of the B. anthracis chromosome, the B. anthracis pXO1 and pXO2 plasmids and an internal control (IC) were designed. The primer sets were evaluated using a panel of B. anthracis strains and exclusivity was tested using genetically closely related B. cereus strains. The robustness of final primer design was evaluated by laboratories in three different countries using five different real-time PCR thermocyclers. Testing of a panel of more than 20 anthrax strains originating from different locations around the globe, including the recent Swedish anthrax outbreak strain, showed that all strains were detected correctly.

Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry flea-borne Rickettsia species.

BACKGROUND: Hard ticks have been identified as important vectors of rickettsiae causing the spotted fever syndrome. Tick-borne rickettsiae are considered to be emerging, but only limited data are available about their presence in Western Europe, their natural life cycle and their reservoir hosts. Ixodes ricinus, the most prevalent tick species, were collected and tested from different vegetation types and from potential reservoir hosts. In one biotope area, the annual and seasonal variability of rickettsiae infections of the different tick stages were determined for 9 years. RESULTS: The DNA of the human pathogen R. conorii as well as R. helvetica, R. sp. IRS and R. bellii-like were found. Unexpectedly, the DNA of the highly pathogenic R. typhi and R. prowazekii and 4 other uncharacterized Rickettsia spp. related to the typhus group were also detected in I. ricinus. The presence of R. helvetica in fleas isolated from small rodents supported our hypothesis that cross-infection can occur under natural conditions, since R. typhi/prowazekii and R. helvetica as well as their vectors share rodents as reservoir hosts. In one biotope, the infection rate with R. helvetica was ~66% for 9 years, and was comparable between larvae, nymphs, and adults. Larvae caught by flagging generally have not yet taken a blood meal from a vertebrate host. The simplest explanation for the comparable prevalence of R. helvetica between the defined tick stages is, that R. helvetica is vertically transmitted through the next generation with high efficiency. The DNA of R. helvetica was also present in whole blood from mice, deer and wild boar. CONCLUSION: Besides R. helvetica, unexpected rickettsiae are found in I. ricinus ticks. We propose that I. ricinus is a major reservoir host for R. helvetica, and that vertebrate hosts play important roles in the further geographical dispersion of rickettsiae.

Persistent detection of Babesia EU1 and Babesia microti in Ixodes ricinus in the Netherlands during a 5-year surveillance: 2003-2007.

We report the finding of Babesia EU1 and Babesia microti in Ixodes ricinus ticks in the Netherlands. During 5 years of surveillance between 2003 and 2007, 1488 ticks were collected in a dune forest area near the North Sea and were screened for Babesia infections. In 17 ticks, DNA of the protozoan parasite genus Babesia was detected using a Babesia-specific 18S rRNA polymerase chain reaction. Further, reverse line blot analysis and DNA sequence analysis showed that 13 of these ticks carried Babesia EU1, two ticks carried B. microti, and one tick carried B. divergens. This study shows that the human pathogenic species Babesia EU1 and B. microti can complete their life cycle in the Netherlands.

Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands.

The protozoan parasite Cryptosporidium is found world-wide and can cause disease in both humans and animals. To study the zoonotic potential of Cryptosporidium in The Netherlands we isolated this parasite from the faeces of infected humans and cattle and genotyped those isolates for several different markers. The overall genotyping results showed: for humans isolates, 70% Cryptosporidium hominis, 19% Cryptosporidium parvum, 10% a combination of C. hominis and C. parvum, and 1% Cryptosporidium felis; and for cattle isolates 100% C. parvum. Analysis of the genetic variants detected for the HSP70, ML1 and GP60 markers showed: for human isolates, one C. hominis and two C. parvum variants (C. parvum and C. parvum NL) for HSP70, one C. hominis and five C. parvum variants (C1, C2, C3, and C2 NL1 and C2 NL2) for ML1, four C. hominis (mainly IbA10G2) and four C. parvum variants (mainly IIaA15G2R1) for GP60; and the cattle isolates only C. parvum (not C. parvum NL1) for HSP70, C1 and C2 for ML1, and 17 different IIa sub-types (mainly IIaA15G2R1) for GP60. Molecular epidemiological analysis of the human data showed a C. hominis peak in autumn. The majority (80%) of the human cases were children aged between 0 and 9 years and >70% of these were caused by C. hominis. Patients >25 years of age were infected mainly with C. parvum. We conclude that C. hominis IbA10G2 is found at high frequencies in autumn in humans and not in cattle. The high prevalence of C. parvum IIaA15G2R1 in both humans and cattle indicates that cattle may be a reservoir for this sub-type in The Netherlands.

cGMP transport by vesicles from human and mouse erythrocytes.

cGMP secretion from cells can be mediated by ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCC11. Indirect evidence suggests that ABCC4 and ABCC5 contribute to cGMP transport by erythrocytes. We have re-investigated the issue using erythrocytes from wild-type and transporter knockout mice. Murine wild-type erythrocyte vesicles transported cGMP with an apparent Km that was 100-fold higher than their human counterparts, the apparent Vmax being similar. Whereas cGMP transport into human vesicles was efficiently inhibited by the ABCC4-specific substrate prostaglandin E1, cGMP transport into mouse vesicles was inhibited equally by Abcg2 and Abcc4 inhibitors/substrates. Similarly, cGMP transport into vesicles from Abcc4-/- and Abcg2-/- mice was 42% and 51% of that into wild-type mouse vesicles, respectively, whereas cGMP transport into vesicles from Abcc4(-/-)/Abcg2(-/-) mice was near background. The knockout mice were used to show that Abcg2-mediated cGMP transport occurred with lower affinity but higher Vmax than Abcc4-mediated transport. Involvement of Abcg2 in cGMP transport by Abcc4-/- erythrocyte vesicles was supported by higher transport at pH 5.5 than at pH 7.4, a characteristic of Abcg2-mediated transport. The relative contribution of ABCC4/Abcc4 and ABCG2/Abcg2 in cGMP transport was confirmed with a new inhibitor of ABCC4 transport, the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride.

Longitudinal analysis of tick densities and Borrelia, Anaplasma, and Ehrlichia infections of Ixodes ricinus ticks in different habitat areas in The Netherlands.

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m2). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m2. The highest tick density was found in the dune area (139 to 551/100 m2). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.

Mice lacking Mrp3 (Abcc3) have normal bile salt transport, but altered hepatic transport of endogenous glucuronides.

BACKGROUND/AIM: Multidrug Resistance Protein 3 (MRP3) transports bile salts and glucuronide conjugates in vitro and is postulated to protect the liver in cholestasis. Whether the absence of Mrp3 affects these processes in vivo is tested. METHODS: Mrp3-deficient mice were generated and the contribution of Mrp3 to bile salt and glucuronide conjugate transport was tested in (1): an Ussing-chamber set-up with ileal explants (2), the liver during bile-duct ligation (3), liver perfusion experiments, and (4) in vitro vesicular uptake experiments. RESULTS: The Mrp3((-/-)) mice show no overt phenotype. No differences between WT and Mrp3-deficient mice were found in the trans-ileal transport of taurocholate. After bile-duct ligation, there were no differences in histological liver damage and serum bile salt levels between Mrp3((-/-)) and WT mice, but Mrp3-deficient mice had lower serum bilirubin glucuronide concentrations. Glucuronide conjugates of hyocholate and hyodeoxycholate are substrates of MRP3 in vitro and in livers that lack Mrp3, there is reduced sinusoidal secretion of hyodeoxycholate-glucuronide after perfusion with hyodeoxycholate. CONCLUSIONS: Mrp3 does not have a major role in bile salt physiology, but is involved in the transport of glucuronidated compounds, which could include glucuronidated bile salts in humans.

Mice lacking multidrug resistance protein 3 show altered morphine pharmacokinetics and morphine-6-glucuronide antinociception.

Glucuronidation is a major detoxification pathway for endogenous and exogenous compounds in mammals that results in the intracellular formation of polar metabolites, requiring specialized transporters to cross biological membranes. By using morphine as a model aglycone, we demonstrate that multidrug resistance protein 3 (MRP3/ABCC3), a protein present in the basolateral membrane of polarized cells, transports morphine-3-glucuronide (M3G) and morphine-6-glucuronide in vitro. Mrp3(-/-) mice are unable to excrete M3G from the liver into the bloodstream, the major hepatic elimination route for this drug. This results in increased levels of M3G in liver and bile, a 50-fold reduction in the plasma levels of M3G, and in a major shift in the main disposition route for morphine and M3G, predominantly via the urine in WT mice but via the feces in Mrp3(-/-) mice. The pharamacokinetics of injected morphine-glucuronides are altered as well in the absence of Mrp3, and this results in a decreased antinociceptive potency of injected morphine-6-glucuronide.

Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells.

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.