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Hill B.J.,National Institutes of Allergy and Infectious Diseases | Skerry J.C.,USAMRIID | Smith T.J.,USAMRIID | Arnon S.S.,Infant Botulism Treatment and Prevention Program | Douek D.C.,National Institutes of Allergy and Infectious Diseases
BMC Microbiology | Year: 2010

Background. Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin. Results. We assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies. Conclusions. While other studies have reported conventional or quantitative PCR-based assays for the detection of C. botulinum genes, our procedure's high-throughput capability and its portability allows most laboratories to quickly assess the possible presence of BoNTs either in food processing samples or in suspected cases of botulism. Thus, this assay provides rapid and specific detection of BoNT and toxin complex genes and would enable the targeting of appropriate therapeutics to infected individuals in a timely manner. © 2010 Hill et al; licensee BioMed Central Ltd. Source

Dabritz H.A.,Infant Botulism Treatment and Prevention Program | Conrad P.A.,University of California at Davis
Zoonoses and Public Health | Year: 2010

Summary Cats are popular as pets worldwide because they are easy to care for and provide companionship that enriches the lives of human beings. Little attention has been focused on their potential to contaminate the environment with zoonotic pathogens. One such pathogen, the protozoan parasite Toxoplasma gondii, rarely causes clinical manifestations in cats or immunocompetent humans; however, it can have serious adverse effects on human foetuses and immunocompromised patients. Many human infections are believed to be acquired from eating undercooked or raw meat, such as pork and lamb (Tenter et al. Int. J. Parasitol., 30, 2000, 1217; Dubey et al. J. Parasitol. 91, 2005, 1082). However, the prevalence of T. gondii infection in human populations that do not consume meat or eat it well-cooked suggests that the acquisition of infection from the environment, via oocysts in soil, water or on uncooked vegetables, is also important (Rawal. Trans. Royal Soc. Trop. Med. Hyg., 53, 1959, 61; Roghmann et al. Am. J. Trop. Med. Hyg., 60, 1999, 790; Chacin-Bonilla et al. Am. J. Trop. Med. Hyg., 65, 2001, 131). In the past 20 years, two changes occurred that significantly increased the size of the cat population in the USA. Pet cat ownership grew from 50 million to 90 million animals, and animal welfare activists created feeding stations for abandoned and free-roaming cats. As many cat owners allow their cats to deposit faeces outside and cats maintained in colonies always defecate outside, ample opportunity exists for T. gondii oocysts to enter the environment and be transmitted to humans. Prevention efforts should focus on educating cat owners about the importance of collecting cat faeces in litter boxes, spaying owned cats to reduce overpopulation, reducing the numbers of feral cats and promoting rigorous hand hygiene after gardening or soil contact. © 2009 Blackwell Verlag GmbH. Source

Abu-Madi M.A.,Qatar University | Behnke J.M.,University of Nottingham | Dabritz H.A.,Infant Botulism Treatment and Prevention Program
American Journal of Tropical Medicine and Hygiene | Year: 2010

Testing of patients who are deemed to be at high risk for TORCH pathogens, e.g., pregnant women, their fetuses, neonates, and acquired immunodeficiency syndrome (AIDS) patients, is important so that specific treatment can be initiated. This study included 1,857 such patients between 2005 and 2008. Logistic regression was used to evaluate factors associated with Toxoplasma gondii seropositivity. Among 823 women of childbearing age, 35.1% and 5.2% tested positive for T. gondii IgG and IgM, respectively. Three infants ≤ 6 months of age (0.8% of 353) were congenitally infected. Factors associated with T. gondii IgG seropositivity included older age, East Mediterranean or African nationality, positive cytomegalovirus (CMV) and herpes simplex virus (HSV)-1 serostatus, and negative rubella IgG results. The decreasing prevalence of IgM antibodies between 2005 and 2008 suggested that exposure to T. gondii from food or environmental sources declined over this period in Qatar. Population-based studies of newborns would be helpful to accurately estimate incidence of congenital toxoplasmosis. Copyright © 2010 by The American Society of Tropical Medicine and Hygiene. Source

Barash J.R.,Infant Botulism Treatment and Prevention Program | Arnon S.S.,Infant Botulism Treatment and Prevention Program
Journal of Infectious Diseases | Year: 2014

Background. Clostridium botulinum strain IBCA10-7060, isolated from a patient with infant botulism, produced botulinum neurotoxin type B (BoNT/B) and another BoNT that, by use of the standard mouse bioassay, could not be neutralized by any of the Centers for Disease Control and Prevention-provided monovalent polyclonal botulinum antitoxins raised against BoNT types A-G.Methods and Results. The combining of antitoxins to neutralize the toxicity of known bivalent C. botulinum strains Ab, Ba, Af, and Bf also failed to neutralize the second BoNT. Analysis of culture filtrate by double immunodiffusion yielded a single line of immunoprecipitate with anti-A, anti-B, and anti-F botulinum antitoxins but not with anti-E antitoxin. A heptavalent F(ab')2 botulinum antitoxin A-G obtained from the US Army also did not neutralize the second BoNT. An antitoxin raised against IBCA10-7060 toxoid protected mice against BoNT/B (Okra) and against the second BoNT but did not protect mice against BoNT/A (Hall) or BoNT/F (Langeland).Conclusion. The second BoNT thus fulfilled classic criteria for being designated BoNT/H. IBCA10-7060 is the first C. botulinum type Bh strain to be identified. BoNT/H is the first new botulinum toxin type to be recognized in >40 years, and its recognition could not have been accomplished without the availability of the mouse bioassay. © The Author 2013. Source

Dover N.,Infant Botulism Treatment and Prevention Program | Barash J.R.,Infant Botulism Treatment and Prevention Program | Hill K.K.,Los Alamos National Laboratory | Xie G.,Los Alamos National Laboratory | Arnon S.S.,Infant Botulism Treatment and Prevention Program
Journal of Infectious Diseases | Year: 2014

We sequenced the 2 botulinum toxin gene clusters of Clostridium botulinum strain IBCA10-7060 type Bh. The sequence of bont/H differed substantially from the sequences of the 7 known bont genes for toxin types A-G. The 5′ one-third terminus of bont/H that codes for the botulinum toxin light chain differed markedly from the light chain coding sequences of toxin types A-G. The 3′ two-thirds terminus of bont/H that codes for the botulinum toxin heavy chain contained a novel Hn translocation domain coding sequence and a nonneutralizing type A-like Hc binding domain coding sequence. bont/H was part of an orfX toxin gene cluster that was located at a unique chromosomal site distant from those used by other botulinum toxin gene clusters. The bont/B sequence was similar to that of subtype bont/B2 and was located within its ha toxin gene cluster at the oppA/brnQ site. Our findings further establish that C. botulinum IBCA10-7060 produces novel BoNT/H. © The Author 2013. Source

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