Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical,Food, and Environmental Samples

Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.Botulinum neurotoxins (BoNTs) are the most toxic agents known, and as little as 30 ng neurotoxin is potentially lethal to humans (36). These toxins are responsible for botulism, a disease characterized by flaccid paralysis. Seven antigenically distinct BoNTs are known (types A to G), and BoNT types A, B, E, and F are the principal types associated with human botulism (37). Significant sequence diversity and antigenically variable subtypes have recently been reported for the type A, B, and E neurotoxin genes (14, 22, 23, 42).Apart from the species Clostridium botulinum, which itself consists of four phylogenetically distinct groups of organisms, some strains of other clostridia, namely Clostridium butyricum and Clostridium baratii, are also known to produce BoNTs (2, 4, 7, 13, 20, 26, 34, 44). Also, strains that produce two toxins and strains carrying silent toxin genes have been reported (8, 22, 24, 39). Due to the great physiological variation of the BoNT-producing clostridia, their isolation and identification cannot depend solely on biochemical characteristics (32). Indeed, the standard culture methods take into consideration only C. botulinum and not C. baratii and C. butyricum, and identification and confirmation require detection of BoNT by a standard mouse bioassay (SMB) (12). The SMB is highly sensitive and specific but also expensive, time-consuming, and undesirable because of the use of experimental animals. Detection of neurotoxin gene fragments by PCR is a rapid alternative method for detection and typing of BoNT-producing clostridia (3). Different PCR methods have been described for detecting neurotoxin type A-, B-, E-, and F-producing clostridia (9, 15-18, 21, 40, 41).A previously described multiplex PCR method able to simultaneously detect type A, B, E, and F neurotoxin genes is a useful tool for rapid detection of the BoNT-producing clostridia (31). While this method generally has a high level of inclusivity for detection of type B, E, and F neurotoxin genes, limitations for detection of the recently described subtype A2, A3, and A4 strains have been identified (6, 28). To increase the efficiency of this multiplex PCR method, new primers were designed to detect genes for all identified type A neurotoxin subtypes (19). Additionally, an internal amplification control (IAC) was added according to ISO 22174/2005. The specificity and selectivity of this multiplex PCR method were evaluated in comparison with an SMB (12) using target and nontarget strains, and the robustness was assessed using clinical, food, and environmental samples. Moreover, to evaluate the applicability of this multiplex PCR method, a survey with food and environmental samples was performed in a German food control laboratory.     

Comparative Genomic Hybridization Analysis of Two Predominant Nordic Group I (Proteolytic) Clostridium botulinum Type B Clusters

Comparative genomic hybridization analysis of 32 Nordic group I Clostridium botulinum type B strains isolated from various sources revealed two homogeneous clusters, clusters BI and BII. The type B strains differed from reference strain ATCC 3502 by 413 coding sequence (CDS) probes, sharing 88% of all the ATCC 3502 genes represented on the microarray. The two Nordic type B clusters differed from each other by their response to 145 CDS probes related mainly to transport and binding, adaptive mechanisms, fatty acid biosynthesis, the cell membranes, bacteriophages, and transposon-related elements. The most prominent differences between the two clusters were related to resistance to toxic compounds frequently found in the environment, such as arsenic and cadmium, reflecting different adaptive responses in the evolution of the two clusters. Other relatively variable CDS groups were related to surface structures and the gram-positive cell wall, suggesting that the two clusters possess different antigenic properties. All the type B strains carried CDSs putatively related to capsule formation, which may play a role in adaptation to different environmental and clinical niches. Sequencing showed that representative strains of the two type B clusters both carried subtype B2 neurotoxin genes. As many of the type B strains studied have been isolated from foods or associated with botulism, it is expected that the two group I C. botulinum type B clusters present a public health hazard in Nordic countries. Knowing the genetic and physiological markers of these clusters will assist in targeting control measures against these pathogens.Clostridium botulinum produces a potent neurotoxin during its growth. The toxin causes a potentially lethal paralytic disease, botulism, in humans and animals. The classical food-borne botulism follows the consumption of toxin-containing food or drink, while infant and adult intestinal botulism results from in vivo spore germination, outgrowth, and toxin production in the gut. Apart from attenuated intestinal microbial population, other factors affecting the colonization of C. botulinum in the intestinal forms of botulism are not known.Based on their physiology and genetic background, C. botulinum strains are divided into groups I to IV (13). Strains of groups I and II are associated with human disease. Group I strains produce neurotoxin serotypes A, B, and/or F, while the group II strains produce type B, E, or F toxin. Physiologically, groups I and II differ markedly from each other as well as from groups III and IV. Genomic analysis of group I and II C. botulinum strains by 16S rrn sequencing (13), ribotyping (10), and amplified fragment length polymorphism (11, 15, 16) is consistent with the divergent physiologies of the two groups (18).Nordic C. botulinum group I strains show a remarkable homogeneity (15, 20, 21, 23). In a large pulsed-field gel electrophoresis (PFGE) analysis, the majority of group I strains isolated from various sources from Finland, Norway, and Denmark formed type B neurotoxin and clustered into two large groups, with the members of each group sharing identical or nearly identical restriction patterns (20, 23). Many of these strains were recovered from honey for human consumption (23), and one strain was related to an infant botulism case (22). Apart from a recent study showing that strains of the two type B clusters, further referred to as clusters BI and BII, differ in their abilities to grow at extreme temperatures (12), the physiological, epidemiological, and genetic markers of the two clusters are not known. An understanding of such traits will assist in designing control measures against these potential food- and environment-borne pathogens.The availability of group I C. botulinum genome sequences has enabled the construction of whole-genome DNA microarrays and a comprehensive genomic analysis of C. botulinum strains (26, 27). In this paper, we describe a comparative genomic hybridization (CGH) analysis of 32 Nordic group I C. botulinum type B cluster BI or BII strains with a DNA microarray based on the protein-coding sequences (CDS) in the ATCC 3502 genome. Strains within each cluster showed no substantial variation. Furthermore, strains belonging to the two clusters differed by their responses to 145 CDS probes, suggesting differential resistance to toxic compounds and a relatively large antigenic variability. Sequencing of botB in a representative cluster BI strain and a representative cluster BII strain revealed subtype B2 neurotoxin genes in both strains.     

Expression of the Clostridium botulinum A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex

Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most potent toxins known in nature and are characterized as category A select agents since they are considered potential bioterrorism threats (3). BoNTs can be distinguished immunologically into seven serotypes by using homologous antitoxins, designated A to G. BoNT/A is of particular interest, since it is frequently implicated in cases of botulism and is a significant threat in bioterrorism (1, 10).BoNT is a 150-kDa protein composed of a heavy chain (100 kDa) and a light chain (50 kDa) linked by a disulfide bond and noncovalent molecular interactions (24). The heavy chain (H) has two functional domains, a transmembrane domain and a receptor binding domain. The light chain (L) is a zinc-dependent protease which specifically cleaves one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors, resulting in the blockage of evoked acetylcholine release at the skeletal neuromuscular junction (8).Previous studies have found that the bont genes of all strains of C. botulinum and neurotoxigenic strains of Clostridium butyricum and Clostridium baratii have a set of genes located upstream of the bont and ntnh genes that are organized as gene clusters (5, 7, 23). The two known primary types of clusters are (i) a hemagglutinin (ha) cluster and (ii) an orfX cluster with open reading frames (ORFs) of unknown functions. The ha cluster consists of genes encoding HA17, HA33, HA70, BotR, and NTNH. The orfX cluster consists of genes encoding ORFX3, ORFX2, ORFX1, P47, P21, and NTNH. Previous studies indicate that BoNT/A subtypes possess either a ha cluster or an orfX cluster associated with their expressed bont gene, depending on the subtype and strain (5, 11, 13-15, 33).It has been shown that the BoNT complex can form stable toxin complexes (TCs) of various sizes, including LL-TC (∼900 kDa), L-TC (∼500 kDa), and M-TC (∼300 kDa) composed of various combinations of HA proteins, NTNH, and BoNT (19, 21, 23, 29, 31, 34). M-TC contains BoNT and NTNH but has no HA proteins, whereas LL-TC and L-TC contain different ratios of the BoNT, NTNH, and HA proteins (21, 22, 29, 34). The biological and structural roles of the complex proteins are not completely characterized, although it has been proposed that they serve the role of protecting BoNT from harsh conditions, including pH, salt, temperature, and digestive enzymes, and that they assist BoNT translocation across the intestinal epithelial layer (2, 6, 17). A recent report indicated that the nontoxic proteins serve as adjuvants and contribute to the immunogenicity of BoNT/A (25).The production of botulinum TCs is known to vary with different serotypes and strains, medium composition, and culture conditions (21, 24, 31). The LL-TC has only been observed in proteolytic strains (group I). Serotype A to D strains produce M-TC and L-TC in their culture medium, while serotype E and F strains produce only M-TC (17, 18).In 1986, a Japanese group isolated four HA-negative C. botulinum strains from infant botulism cases that produced only M-TC (300 kDa). They assigned the strains to subtype A2 (14, 30). In 2004, our laboratory confirmed on a genomic level that the BoNT/A2 subtype contained the orfX cluster instead of the ha cluster (12). Since then, more arrangements and combinations of neurotoxin gene clusters were characterized along with more BoNT subtypes (13, 20, 33). However, the function of the orfX genes and the role of the presumptive protein products and their role in the TCs are still unknown, including whether ORFX proteins can form a TC with the expressed toxin analogous to the ha cluster proteins.In this study, the BoNT/A2 TC was purified from a native culture to determine if the orfX cluster proteins remain associated with BoNT/A2. To better understand the role of the orfX cluster genes, the orfX cluster proteins of C. botulinum A2 strains (ORFX1, ORFX3, P47, and the middle part of NTNH) was expressed using either an Escherichia coli or a C. botulinum expression system in this study. Antibodies against individual expressed orfX cluster proteins were then raised by immunizing a rabbit and mice. These antibodies were then used as probes to investigate the expression pattern of the orfX cluster genes in the native A2 culture. ORFX2, which could not be expressed, was detected by N-terminal protein sequencing.     

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10³ and 10⁵ CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.Botulinum neurotoxins (BoNTs), the causative agents of botulism, are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven serotypes, A to G. While the botulinum neurotoxins BoNT/A, BoNT/B, BoNT/E, and BoNT/F are known to cause botulism in humans, BoNT/C and BoNT/D are frequently associated with botulism in cattle and birds. Despite its toxicity, BoNT/G has not yet been linked to naturally occurring botulism (26).Botulism is a life-threatening illness caused by food contaminated with BoNT (food-borne botulism), by the uptake and growth of C. botulinum in wounds (wound botulism), or by colonization of the intestinal tract (infant botulism) (14). In addition, C. botulinum and the botulinum neurotoxins are regarded as potential biological warfare agents (8).The gold standard for the detection of BoNTs from food or clinical samples is still the mouse lethality assay, which is highly sensitive but rather time-consuming. In addition to various immunological assays for BoNT detection, several conventional and real-time PCR-based assays for the individual detection of bont genes have been reported (2, 9-12, 15, 20, 23, 27-30). A major improvement is the simultaneous detection of more than one serotype, which results in a reduction of effort and in the materials used. In recent years, both conventional and real-time PCR-based multiplex assays have been developed for the simultaneous detection of C. botulinum serotypes (1, 6, 22, 24). To date, however, no internally controlled multiplex real-time PCR assay for the simultaneous detection and differentiation of all four serotypes relevant for humans has been reported.We describe here a highly specific and sensitive multiplex real-time PCR assay based on the 5′-nuclease TaqMan chemistry (17) for the simultaneous detection of the C. botulinum types A, B, E, and F, including an internal amplification control (IAC). Furthermore, we developed six different singleplex assays based on the TaqMan chemistry for the detection of C. botulinum serotypes A to F. Assays were validated on 42 C. botulinum strains, 57 non-C. botulinum strains, on spiked food samples, and on real samples from cases of botulism in Germany.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Previously Unrecognized Amino Acid Substitutions in the Hemagglutinin and Fusion Proteins of Measles Virus Modulate Cell-Cell Fusion,Hemadsorption, Virus Growth,and Penetration Rate

Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.Measles virus (MV), which belongs to the genus Morbillivirus in the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. The MV genome encodes six structural proteins: the nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins. The P gene also encodes two other accessory proteins, the C and V proteins. The C protein is translated from an alternative translational initiation site leading a different reading frame, and the V protein is synthesized from an edited mRNA. MV has two envelope glycoproteins, the F and H proteins. The former is responsible for envelope fusion, and the latter is responsible for receptor binding (12).Wild-type MV strains isolated in B95a cells and laboratory-adapted MV strains have distinct phenotypes (18). Wild-type MV strains can grow in B95a cells but not in Vero cells, while laboratory-adapted MV strains can grow in both B95a and Vero cells. Wild-type MV strains do not cause hemadsorption (HAd) in African green monkey red blood cells (AGM-RBC), while most of laboratory-adapted MV strains cause HAd. Importantly, wild-type MV strains are pathogenic and induce clinical signs that resemble human measles in experimentally infected monkeys while laboratory-adapted MV strains do not.One approach to identify amino acid substitutions responsible for these phenotypic differences is the comparison of a wild-type MV strain with a standard laboratory-adapted MV strain such as the Edmonston strain. With regard to the H protein, amino acid substitutions important for HAd activity and cell-cell fusion in tissue culture cells were identified by expressing the H proteins in mammalian cells (15, 21). Recently, Tahara et al. revealed that the M, H, and L proteins are responsible for efficient growth in Vero cells by constructing a series of recombinant viruses in which part of the genome of the wild-type MV was replaced with the corresponding sequences of the Edmonston strain (45, 46, 47).Another approach is the comparison of wild-type MV strains with their Vero cell-adapted MV strains. It was reported that Vero cell-adapted MV strains could be obtained by successive blind passages of wild-type MV strains in Vero cells (18, 24, 30, 43). Interestingly, in vivo and in vitro phenotypes of Vero cell-adapted MV strains were similar to those of laboratory-adapted standard MV strains (18, 19, 24, 30, 43). Comparison of the complete nucleotide sequences of the genomes of wild-type MV strains with those of Vero cell-adapted wild-type MV strains revealed amino acid substitutions in the P, C, V, M, H, and L proteins (27, 42, 48, 53).At present, these phenotypic differences are explained mainly by the receptor usage of MV. Wild-type MV strains can use signaling lymphocyte activation molecule (SLAM; also called CD150) but not CD46 as a cellular receptor, whereas laboratory-adapted MV strains can use both SLAM and CD46 as cellular receptors (7, 10, 16, 29, 56, 60).However, receptor usage per se cannot explain all of the phenotypic differences (20, 25, 48, 53). For example, recombinant Edmonston strains expressing wild-type H proteins can grow in Vero cells to some extent (17, 54). Several reports suggested the presence of the third MV receptor on Vero cells (14, 44, 54, 60). Other reports indicated the contribution of the M protein on cell-cell fusion and growth of MV in Vero cells (4, 27, 47). Recently, the unidentified epithelial cell receptor for MV was predicted in primary culture of human cells (1, 55) and several epithelial cell lines (23, 51). However, the identity of the third receptor on Vero cells and the unidentified epithelial cell receptor is not clear yet. Thus, the mechanism of Vero cell adaptation of wild-type MV is not completely understood.In order to understand the molecular mechanism of these phenotypic changes of wild-type MV strains during adaptation in Vero cells, we determined the complete nucleotide sequences of the genomes of the wild-type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strains (43) and examined the effect of individual amino acid substitutions using a mammalian cell expression system and reverse genetics. We show here that previously unrecognized new amino acid substitutions in the H and F proteins are important for MV adaptation and HAd activity.     

Rapid Affinity Immunochromatography Column-Based Tests for Sensitive Detection of Clostridium botulinum Neurotoxins and Escherichia coli O157

Existing methods for detection of food-borne pathogens and their toxins are frequently time-consuming, require specialized equipment, and involve lengthy culture procedures and/or animal testing and are thus unsuitable for a rapid response to an emergency public health situation. A series of simple and rapid affinity immunochromatography column (AICC) assays were developed to detect Clostridium botulinum neurotoxin types A, B, E, and F and Escherichia coli O157 in food matrices. Specifically, for milk, grape juice with peach juice, and bottled water, the detection limit for the botulinum neurotoxin type A complex was 0.5 ng. Use of this method with a 10-ml sample would therefore result in a detection limit of 50 pg ml^−l. Thus, this assay is approximately 2 orders of magnitude more sensitive than a comparable lateral-flow assay. For botulinum neurotoxin complex types B, E, and F, the minimum detection limit was 5 ng to 50 ng. Sensitive detection of E. coli O157 was achieved, and the detection limit was 500 cells. The AICC test was also shown to be specific, rapid, and user friendly. This test takes only 15 to 30 min to complete without any specialized equipment and thus is suitable for use in the field. It has the potential to replace existing methods for presumptive detection of botulinum neurotoxin types A, B, E, and F and E. coli O157 in contaminated matrices without a requirement for preenrichment.The majority of conventional methods used for detection and identification of pathogenic microorganisms, viruses, and/or their toxins lack the speed and sensitivity necessary for use in the field (they typically are not completed in a single day) and also require specialized equipment (20). Rapid methods, including antibody-based and nucleic acid-based assays, have revolutionized the methodology for detection of microbial pathogens and their toxins in foods (16). However, while most antibody-based and nucleic acid-based assays are rapid, specialized equipment is often required, and specific enrichment is needed to achieve the necessary sensitivity. This means that the analysis time can still be several days (16). Lateral-flow assays (LFAs) and column flow assays are tests that have considerable merit in terms of rapidity and ease of use in the field without specialized equipment (4, 5, 8, 19, 34).Two contrasting agents were used as detection targets in this study: (i) a potent microbial toxin (Clostridium botulinum neurotoxin), including type A, B, E, and F neurotoxins; and (ii) an infectious pathogen, Escherichia coli O157. These two targets present different problems for detection; the first target is a protein toxin, and the second target is intact bacterial cells. The botulinum neurotoxin is the most potent toxin known, and as little as 30 to 100 ng has the potential to be fatal to humans (28). It is responsible for botulism, a severe neuroparalytic disease that affects humans and also animals and birds (28). There are seven antigenically distinct botulinum neurotoxins (types A to G), and a number of subtypes have also been described (9, 11, 15, 28, 36). Botulism in humans is associated principally with neurotoxin types A, B, E, and F (27, 29). Since the botulinum neurotoxins are the toxic agents and they can be produced by six physiologically distinct clostridia (28), considerable emphasis has been placed on detection of the neurotoxins rather than the bacteria. The “gold standard” method for detecting botulinum neurotoxins is the mouse bioassay due to its high levels of sensitivity and specificity. However, this technique is also problematic (33). It typically requires 24 to 48 h to yield results, is expensive, and is becoming less favored because of its use of animals (4). The alternative tests include enzyme-linked immunosorbent assays (ELISAs), lateral-flow assays (LFAs), a chemiluminescent slot blot immunoassay, surface plasmon resonance (SPR), the assay with a large immunosorbent surface area (ALISSA) test, and quantum dot immunoassays (4, 5, 7, 22, 43, 46). Lateral-flow assays are available and are convenient for toxin testing as they are easy to perform and rapid (<30 min) and no additional equipment is required. However, their poor sensitivity has limited their use (23).E. coli O157 produces a cytotoxin (verotoxin), and an E. coli O157 infection can lead to severe bloody diarrhea, kidney failure, brain damage, and death. Enumeration, identification, and control of this pathogen are challenging due to the low infectious dose necessary to cause disease, which is between 2 and 2,000 ingested cells (41). Sources of E. coli O157 infection include ground beef and unpasteurized milk and apple juice (1), raw milk (6), and spinach and lettuce (42). Isolation of E. coli O157:H7 from water, food, and environmental samples is laborious. Culture is difficult due to the large competing microflora that either overgrows or mimics the non-sorbitol-fermenting organism E. coli O157:H7 (12). According to Tokarskyy and Marshall (41), the largest group of rapid test kits commercially available for testing for the presence of E. coli O157 in food includes immunological methods, such as latex agglutination, reverse passive latex agglutination, immunodiffusion, ELISA, immunomagnetic separation (IMS), and immunoprecipitation. The other methods that have been developed include a dipstick test device (2), a lateral-flow immunoassay (8), real-time PCR (39), and an enzyme-linked immunomagnetic chemiluminescent assay (17). However, in many cases these tests require preenrichment or have limited sensitivity.The objective of the work described here was to develop a rapid sensitive diagnostic test for detection of botulinum neurotoxins A, B, E, and F and E. coli O157 that can be used without preenrichment.     

Horizontal Chromosome Transfer,a Mechanism for the Evolution and Differentiation of a Plant-Pathogenic Fungus

The tomato pathotype of Alternaria alternata produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene, ALT1, which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of ALT1 and another PKS gene, both of which reside on the CDC in the tomato pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five A. alternata tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the A. alternata strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among A. alternata strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.Fungi produce a huge variety of secondary metabolites. Some plant-pathogenic fungi, especially necrotrophic pathogens that kill plant cells during invasion, produce phytotoxic metabolites to impair host tissue functions (20, 30, 42, 47). Phytotoxins produced by fungal plant pathogens are generally low-molecular-weight secondary metabolites that exert toxic effects on host plants. Among these phytotoxins, host-specific toxins (HSTs) are critical determinants of pathogenicity or virulence in several plant-pathogen interactions (13, 30, 33, 40, 42, 47, 49).Recent advances in molecular biological techniques for fungi have led to the identification of fungal genes involved in pathogenesis, as exemplified by those used in the biosynthesis of toxic secondary metabolites, such as HSTs. Genes involved in the biosynthesis of secondary metabolites are typically clustered in filamentous fungi, including plant pathogens (20, 24, 44). The origins and evolutionary processes of these gene clusters, however, are largely unknown. Analysis of the arrangement and sequences of genes in the clusters would shed light on how the clusters themselves and their ability to produce toxic secondary metabolites evolved (20, 24, 44).The involvement of horizontal gene transfer (HGT) in the evolution of fungal secondary-metabolite gene clusters has been discussed (34, 44). HGT events are well known in prokaryotes (21, 29), and the genomic regions that have undergone HGT are referred to as pathogenicity or genomic islands (7). In prokaryotes, the mechanisms of HGT are also associated with conjugation, transformation, and transduction (21, 29). Although these transfer mechanisms are generally unknown in eukaryotes such as fungi, interspecific transfer of a virulence gene encoding the production of a critical toxin has been reported in Pyrenophora tritici-repentis (14). There is also clear evidence of recent lateral gene transfer of the ToxA gene from Stagonospora nodorum to P. tritici-repentis (14, 30).In Alternaria alternata plant pathogens (37), we have shown that all strains of the A. alternata pathotypes harbor small extra chromosomes of less than 1.7 Mb, whereas nonpathogenic isolates do not have these small chromosomes (5). A cyclic peptide synthetase gene, AMT, which is involved in host-specific AM toxin biosynthesis of the apple pathotype of A. alternata, was located on a small chromosome of 1.1 to 1.7 Mb, depending on the strain (22, 23). The AF toxin biosynthesis gene cluster was also present on a single small chromosome of 1.05 Mb in the strawberry pathotype of A. alternata (18). Based on biological and pathological observations, those small chromosomes were regarded as supernumerary chromosomes, or conditionally dispensable chromosomes (CDCs) (10, 18, 22). Fungal supernumerary chromosomes, which are not important for normal growth but confer advantages for colonizing an ecological niche, such as infecting host plants, are regarded as CDCs (21). The functions and pathological roles of CDCs have been studied in the pea pathogen Nectria haematococca (11, 17, 25, 32, 43, 46).The origin and evolution of CDCs have been intriguing issues in the study of plant-microbe interactions. The supernumerary chromosomes of certain strains of N. haematococca have been suggested to have a different evolutionary history than essential chromosomes (ECs) in the same genome, and they might have been introduced into the genome by horizontal transfer from another strain (10, 12, 36). In Colletotrichum gloeosporioides, the 2-Mb supernumerary chromosome was transferred from a biotype A strain to a vegetative incompatible biotype B strain (19, 31). Transfer of the chromosome, however, did not affect the pathogenicity of the recipient fungus, perhaps because it did not harbor pathogenicity genes (19, 31). These results suggest that supernumerary chromosomes of fungi might have the capacity for horizontal transfer across an incompatibility barrier between two distinct strains.AAL toxins are HSTs produced by the tomato pathotype of A. alternata (synonym A. alternata f. sp. lycopersici, synonym Alternaria arborescens), the causal agent of Alternaria stem canker disease in tomatoes, which causes severe necrosis of susceptible tomato cultivars (15, 26, 35). AAL toxins and fumonisins of the maize pathogen Gibberella moniliformis are structurally related to sphinganine and termed sphinganine-analogue mycotoxins. AAL toxins and fumonisins are sphinganine-analogue mycotoxins, which are toxic to some plant species and mammalian cells (16, 48). They cause apoptosis in susceptible tomato cells and mammalian cells by inhibiting ceramide biosynthesis (9, 41, 45). In the tomato pathotype of A. alternata-tomato interactions, a major factor in pathogenicity is the production of host-specific AAL toxins capable of inducing cell death only in susceptible cultivars (3, 9, 48).In this study, we describe evidence showing that the ability to produce the host-specific AAL toxin and to infect host tomato plants could potentially be distributed among a population of strains of the A. alternata tomato pathotype by horizontal transfer of an entire pathogenicity chromosome of the pathogen.     

Francisella tularensis Type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks Postinfection

Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown and to the organism''s potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype is caused by factor(s) secreted by amoebae and/or F. tularensis into the coculture medium. Further, our results indicate that in contrast to the live vaccine strain LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks postinfection and that the induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that interactions between F. tularensis strains and amoebae may play a role in the environmental persistence of F. tularensis.Francisella tularensis is the etiological agent of the zoonotic disease tularemia, also known as rabbit fever (35, 53). Strains belonging to F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, which are both prevalent in the Northern Hemisphere, cause the majority of reported cases of tularemia (36). Subspecies tularensis is highly contagious, with an infectious dose of 1 to 10 bacteria, and is associated with more severe disease (21). Though described more than a century ago as a disease common among hunters and trappers, tularemia has recently been reported in areas with no previous known risk (20, 25, 31, 42). F. tularensis infects a broad range of wildlife species (36), and a number of arthropods, such as ticks and flies, are known to be vectors (36, 49). Humans are usually infected either through an insect bite or by inhalation of aerosolized bacteria (49). Tularemia can be fatal in up to 30% of untreated cases (36, 49), with the mortality rate reaching 90% in pneumonic infections, as described in early studies conducted with vaccinated human volunteers (44-46, 49). Due to its highly infectious nature and its potential for use as a bioterrorism agent, F. tularensis has been classified as a class A biothreat pathogen by the Centers for Disease Control and Prevention (CDC), which has mandated that human tularemia be a reportable disease since 2000 (15, 37). In addition, the absence of a licensed vaccine for prophylaxis (36) makes understanding the virulence mechanisms used by this pathogen imperative for the development of efficacious measures to prevent or treat human disease.Though F. tularensis has been isolated from more than 250 wildlife species (21), the acute nature of the infections and the resultant high mortality rates in these hosts indicate that the bacterial reservoir(s) in nature have yet to be identified. Tularemia outbreaks involving F. tularensis subsp. holarctica have often been linked to water sources (6, 40), and a positive PCR field test was reported for Francisella during such an outbreak in Norway (5). Abd et al. reported that the F. tularensis live vaccine strain LVS is able to survive and replicate in the amoeba Acanthamoeba castellanii (1), suggesting a potential link between amoeba-Francisella interactions and environmental persistence. A. castellanii, a free-living environmental amoeba, is known to serve as a reservoir for a number of pathogenic microorganisms (24). However, to date, interactions of virulent F. tularensis subspecies tularensis strains with amoebae have not been documented. The ability of several human intracellular pathogens, including Legionella pneumophila and Mycobacterium avium, to infect and survive within amoebae has been well characterized (10, 12). In addition to playing a role in environmental survival and dissemination, growth in A. castellanii has been shown to enhance the ability of L. pneumophila and M. avium to survive and replicate in host macrophages (10, 12) and to enhance the virulence of both species in mice (7, 12). Since F. tularensis species are facultative intracellular pathogens that primarily survive in macrophages, probing the Francisella-amoeba interaction may provide insights into Francisella pathogenesis, as well as environmental survival. In this study, we investigated the ability of virulent type A strains of F. tularensis to survive in A. castellanii with a focus on understanding the role of Francisella-amoeba interactions in environmental persistence.