The objectives of the Global Health Security Agenda (GHSA) will require not only a “One Health” approach to counter natural disease threats against humans, animals, and the environment, but also a security focus to counter deliberate threats to human, animal, and agricultural health and to nations' economies. We have termed this merged approach “One Health Security.” It will require the integration of professionals with expertise in security, law enforcement, and intelligence to join the veterinary, agricultural, environmental, and human health experts essential to One Health and the GHSA. Working across such different professions, which occasionally have conflicting aims and different professional cultures, poses multiple challenges, but a multidisciplinary and multisectoral approach is necessary to prevent disease threats; detect them as early as possible (when responses are likely to be most effective); and, in the case of deliberate threats, find who may be responsible. This article describes 2 project areas that exemplify One Health Security that were presented at a workshop in January 2014: the US government and private industry efforts to reduce vulnerabilities to foreign animal diseases, especially foot-and-mouth disease; and AniBioThreat, an EU project to counter deliberate threats to agriculture by raising awareness and implementing prevention and response policies and practices.
This thesis deals with the methodological advances of diagnostic PCR including reliability of PCR and pre-PCR processing of food and feed samples. Diagnostic PCR has been greatly improved by the introduction of the second generation of PCR, so-called real-time PCR. Automated closed-tube quantitative real-time PCR favours the analysis of food-borne pathogenic bacteria. However, in common with conventional PCR, the real-time PCR technology requires pre-PCR processing of the food/feed sample (i) to remove PCR-inhibitory substances, (ii) facilitate detection of low concentrations of target bacteria, (iii) to convert a heterogeneous bulk sample to a more homogeneous PCR sample, and (iv) to restrict competitive background flora. The aim of the research presented in this thesis was to adapt microbiological food sampling to PCR by designing pre-PCR processing strategies for future routine analysis of Yersinia enterocolitica and Salmonella in complex samples from the food-chain. To enable the detection of low concentrations of Y. enterocolitica and Salmonella in PCR-inhibitory samples, such as pork and animal feed, enrichment PCR procedures have been employed. The reliability of PCR detection of pathogenic Y. enterocolitica using a developed multiplex PCR assay was studied using a logistic regression model for determination of the detection probability. The probability of detecting 1´104 CFU/ml Y. enterocolitica was estimated to be 85.4%. A Yersinia-PCR-compatible enrichment (YPCE) medium was developed to remove the necessity for sample preparation prior to PCR detection of Y. enterocolitica. The pre-PCR processing strategy allows detection of low concentrations (101 CFU/ml) in the presence of background flora in concentrations up to three orders of magnitude higher than Y. enterocolitica. The YPCE medium can, form part of integrated and automated pre-PCR processing protocol, especially for swab samples. To complement the Yersinia assay a RAPD protocol was developed for inter-laboratory use. The stringent RAPD protocol was evaluated on 70 Yersinia strains and allowed discrimination at serotype level, and the sub-clusters of Y. enterocolitica correlated with the geographic origin of isolates, where especially O:3 strains from Scandinavia formed a homogeneous sub-cluster. Enrichment PCR of Salmonella enterica was studied using real-time PCR. A model was developed to describe the 5' nuclease real-time PCR performance in the presence buffered peptone water (BPW) and brain heart infusion. Using the model it was found that the rTth DNA polymerase mixture was more resistant to the presence of BPW than AmpliTaq Gold. Accurate detection of 1 CFU/ml S. Enteritidis inoculated in BPW required 8.4 hours’ enrichment using the rTth DNA polymerase mixture, while AmpliTaq Gold required 11.6 h. Using an alternative DNA polymerase, Tth instead of Taq, facilitated the PCR detection of Salmonella in animal feed. The PCR protocol was more sensitive than the traditional culture-based standard method (NMKL-71), since out of 155 feed samples, 8% were positive for PCR detection of Salmonella in comparison with 3% with the NMKL-71 method. (Less)
Preparedness for bioterrorism is based on communication between people in organizations who are educated and trained in several disciplines, including law enforcement, health, and science. Various backgrounds, cultures, and vocabularies generate difficulties in understanding and interpretating terms and concepts, which may impair communication. This is especially true in emergency situations, in which the need for clarity and consistency is vital. The EU project AniBioThreat initiated methods and made a rough estimate of the terms and concepts that are crucial for an incident, and a pilot database with key terms and definitions has been constructed. Analysis of collected terms and sources has shown that many of the participating organizations use various international standards in their area of expertise. The same term often represents different concepts in the standards from different sectors, or, alternatively, different terms were used to represent the same or similar concepts. The use of conflicting terminology can be problematic for decision makers and communicators in planning and prevention or when handling an incident. Since the CBRN area has roots in multiple disciplines, each with its own evolving terminology, it may not be realistic to achieve unequivocal communication through a standardized vocabulary and joint definitions for words from common language. We suggest that a communication strategy should include awareness of alternative definitions and ontologies and the ability to talk and write without relying on the implicit knowledge underlying specialized jargon. Consequently, cross-disciplinary communication skills should be part of training of personnel in the CBRN field. In addition, a searchable repository of terms and definitions from relevant organizations and authorities would be a valuable addition to existing glossaries for improving awareness concerning bioterrorism prevention planning.
Detection of avian influenza virus (AIV) in poultry meat is hampered by the lack of an efficient analytical method able to extract and concentrate viral RNA prior to PCR. In this study we developed a method for extracting and detecting AIV from poultry meat by a previously standardized 1-step real-time reverse transcriptase PCR (RRT-PCR) assay. In addition, a new process control, represented by feline calicivirus (FCV), was included in the original protocol, to evaluate all analytical steps from sample preparation to the detection phase. The detection limit was below 1×10−1 TCID50 of AIV per sample, and the quantification limit corresponded to 1×101 TCID50 of AIV per sample. Moreover, the addition of 1×102 TCID50/sample of FCV did not affect the quantification and detection limit of the reaction. These results show that the developed assay is suitable for detecting small amounts of AIV in poultry meat. In addition, the developed biopreparedness protocol can be applied to detect AIV in legal or illegal imported broiler chicken meat. The availability of a rapid and sensitive diagnostic method based on molecular identification of AIV in poultry meat provides an important tool in the prevention of AIV circulation.
Botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishes. The disease in animals is mainly caused by toxins produced by Clostridium botulinum strains belonging to group III, although outbreaks due to toxins produced by group I and II organisms have been recognized. Group III strains are capable of producing botulinum toxins of type C, D, and C/D and D/C mosaic variants. Definitive diagnosis of animal botulism is made by combining clinical findings with laboratory investigations. Detection of toxins in clinical specimens and feed is the gold standard for laboratory diagnosis. Since toxins may be degraded by organisms contained in the gastrointestinal tract or may be present at levels below the detection limit, the recovery of C. botulinum from sick animal specimens is consistent for laboratory confirmation. In this article we report the development and in-house validation of a new multiplex real-time PCR for detecting and typing the neurotoxin genes found in C. botulinum group III organisms. Validation procedures have been carried out according to ISO 16140, using strains and samples recovered from cases of animal botulism in Italy and France.
IntroductionVarious biological agents such as bacteria, parasites, viruses and toxins may be deliberately released and spread through feed, food, water and air to cause harm and panic (Rotz et al., 2004).These biological agents can infect humans and animals but also crops (Gullino, 2008).Bioterrorism is probably the most inter-sectoral and international challenges among Chemical, Biological, Radiological, and Nuclear (CBRN) threats.To improve the interactions between these sectors there are some key issues involving R&D, training, event exercises, early warning and effective communication strategies that need to be addressed to rapidly share event information related to detection and identification.In this perspective, diagnostic capabilities are critical components to enhance the preparedness against bioterrorism (Morse, 2004).Covert and overt incidents will lead to various alarm chains.In a covert incident, which is characterized by an unannounced release, the early response and detection will be driven by public health organizations.However, an overt incident is characterized by the fact that the perpetrator announces responsibility and the response will therefore be driven by law enforcement.A diagnostic response strategy must be able to address both types of incidents.This requires a multidisciplinary network composed of diagnostic capabilities both in law enforcement agencies and public health organizations such as environmental, agricultural, veterinary, and food.As a result, laboratory response networks have been developed in different countries.
It is expected that the increasing genetic information on Bacillus cereus in genome databases will pave the way for investigating its evolution, ecology, and virulence, and, finally, will contribute to the development of new strategies to control and prevent foodborne diseases caused by B. cereus. This chapter talks about toxins and population structure of the B.cereus group. Many bacterial genome sequences, including B. cereus strains, are available in draft versions only and are missing a few percent of the sequence. The chapter deals with only complete genome sequences, and illustrates that species definition in the B. cereus group remains an open question and it may well be that the discussion will gain momentum with daily growing genome information, especially of "borderline strains". It focuses on pan genome, core genome, accessory genome, and mobilome of the B. cereus group. B. cereus geomics suggest that the mobilome of this species group is important not only to model its evolution, but also to differentiate and detect the different pathotypes. It is clearly visible that the high number of B. anthracis and B. cereus strains already known is improving research tools to study pathogenicity, ecology, and host and environmental adaptation.
