Nova Scotia health inspectors apologize to breastfeeding farmers’ market vendor

From the no-risk-assessment-in-sight files, the Nova Scotia Department of Agriculture has apologized to Tanessa Holt, owner of Food Noise, after citing her for food safety violations for breastfeeding while selling food.

According to media reports and her Facebook page Holt’s risk management strategy wasn’t good enough for the initial inspector.tanessa-holt

Holt writes:

I recently received a call from a food safety representative informing me that I cannot nurse my child at my booth as it is a violation of the food safety regulations.

I explained that there is a handwashing station behind my booth, I have gloves for handling food and that I am always more than covered when nursing. Further, my food is served in closed containers.

Despite this I was informed that I will have to have someone else sit with me the entire day to handle food if I nurse my child even once during the day while at the market.

CTV News reports in a follow-up that Holt received an email from higher-ups apologizing for the misunderstanding.

“I am happy to receive an apology, because I thought it was outrageous to begin with,” she told CTVNews.ca.

Holt has been selling dry food items, including granola and oatmeal, at several markets in the Halifax area since last August.

Holt explained that her food is served in containers, that she uses gloves and a nearby hand washing station, and that she covers up while nursing. Still, she was told that she would have to get another person to handle the food if she is breastfeeding her son.

As that is not a feasible option for her, she had decided to pull out of the farmers’ markets altogether.

In the apology, the department said that the message from the food safety inspector was a “misunderstanding,” and Holt was welcome to breastfeed her son at the market so long as she followed proper hand-washing procedures.

 

Cross-contamination at Jade Dragon Wok-Out leads to poor inspection

A couple of years I saw cross-contamination in the wild while sitting at a bar that faced an open kitchen. The was grilling and used the same tongs for raw and cooked (but not temped) burgers.

And placed the cooked burgers back on the same tray he used for raw patties.Screen Shot 2015-01-14 at 12.27.20 PM

I chatted with the manager and was assured that the burgers wouldn’t make it service.

Our food safety infosheet evaluation work showed that cross contamination was pretty prevalent (on average, one cross-contamination event per food handler, per hour).

And the Jade Dragon Wok-Out in Lawrenceville, GA, according to the Atlanta Journal-Constitution has an issue with cross-contamination.

During a recent inspection at Jade Dragon Wok-Out in Lawrenceville, an employee dipped a ladle that was contaminated by raw meat into a container of spices.

The spices were discarded, and points were deducted from the health score. Points were also taken off because raw shrimp were prepped next to broccoli without any separation.

Employees were performing multiple tasks using single-use gloves instead of changing the gloves and washing their hands, the inspector said. One employee touched raw meat then touched a ladle. Another touched raw egg then touched a utensil and a to-go container.

Norovirus outbreak, including a death, linked to Kansas Meals on Wheels; handwashing issues cited

When I was a kid I used to visit my grandparents in Campbellford,Ontario (that’s in Canada) a bunch. My grandparents lived most of their lives in Toronto (that’s also in Canada) but had retired to this town about 2hrs outside of the city.

My grandparents were into community stuff: volunteering for the hospital auxiliary, organizing charity curling bonspiels and golf tournaments and driving some of their more elderly neighbors to doctor’s appointments.Unknown-1

And my grandfather drove around some food for Meals on Wheels.

I remember being about 8 and going on his route one spring break morning. He grabbed some already-food-filled covered trays and then took a bunch of apples and oranges in bulk. This was before I knew anything about food safety; I didn’t really notice anything about bare hand contact or handwashing.

According to chanute.com, a norovirus outbreak, tragically including a death, has been linked to a Meals on Wheels service in Kansas.

It has been confirmed by the Kansas Department of Health and Environment that the source of the norovirus outbreak in the Neosho County area last week was Meals on Wheels in southeast Kansas.

KDHE Health Programs Public Information Officer Aimee Rosenow said the numbers of confirmed ill have not changed since Friday when there were 29 confirmed ill. 

Rosenow said epidemiology staff are still working on the case to narrow down the source of infection.

A Jan. 8 food safety inspection of the Chanute Senior Services of SEK facility found problems with hand washing and properly arranging food in coolers to avoid cross-contamination. Samples were also taken of all food from the batch delivered Jan. 6.

KDHE Public Health Public Information Officer Aimee Rosenow said KDHE does not have an autopsy report confirming the cause of death as Norovirus infection and cannot release the name of the deceased. 

“We do know that the patient was ill and has been served by this program,” she said.

Over the past year UNC-Chapel Hill food policy student (and frequent barfblog contributor) Ashley Chaifetz has been researching food handling practices and infrastructure at food pantries in North Carolina (as part of a USDA CAP grant on STECs). Volunteers provide a particular challenge for food safety as they may be transient and have varying food safety values. 

 

 

 

CEO draws link between food safety, worker treatment

There’s a strong link between food safety and how a company’s employees are treated, according to the CEO of a major baked goods company.

food.safety.workerWorkers who play a critical role in handling food products often aren’t paid much or given opportunities for advancement, said Paula Marshall, CEO of the Bama Companies.

“There’s a problem, in my mind, with that model,” said Marshall, who delivered a keynote address this week at the Northwest Food Processors Association’s annual conference in Portland.

Farmers and food manufacturers face a threat from employees who simply don’t care about the final product or even hold a grudge against the company, she said.

To get workers committed to food safety, companies must show them respect — not only with better pay, but with opportunities for training and education that allow them to rise in the corporation, Marshall said.

India midday meal horror: 23 kids get food poisoning, admitted to Sion hospital

In yet another case of food poisoning due to midday meals, 23 schoolchildren, all 13-year-olds were admitted to Sion hospital on Tuesday. The incident occurred at 3.40pm in Dayanand Balak Vidyalaya, Matunga East.

pulaoThe children were admitted for observation after at least six started vomitting and got loose motions. Others complained of pain in the abdomen, said Dr Avinash Supe, dean, Sion hospital. “We have administered intravenous drugs and other antibiotics to the children. Fortunately, all are stable and under observation,” he added. 

‘I don’t walk around with baby puke on me’: Breastfeeding mom forced to quit selling at Halifax market

Tanessa Holt opened her first business in the fall, selling dry foods at local farmers’ markets. She said she has been forced to stop selling containers of soup mix, homemade protein bars and energy balls, and prepackaged oatmeal and granola.

tanessa-holtHolt was bringing her 7½-month-old son to the markets, since he has never liked taking bottles and is still dependent on breast milk.

But that was a problem, a food safety inspector told Holt on Monday.

“I have no problem with you breastfeeding at the booth, as long as there is another person that is at the booth with you, who can serve food to the customers,” wrote the inspector in an email provided to CBC News.

Holt said she has no other option but to shut down her stall. As a fledgling business owner, she can’t afford to hire someone.

Holt will soon open a store in Dartmouth, but right now farmers’ markets — in Halifax, Dartmouth and Beaver Bank — make up half her business.

In a followup conversation with the inspector, Holt learned breastfeeding even once at the booth disqualifies her from handling food, no matter how clean she stays or what precautions she takes.

“If I set up there in the morning and I nurse him, at say, 10 o’clock in the morning, then for the remainder of the day, someone else has to handle the food,” she said.

Vomit, feces from baby are concerns, says inspector

tanessa-holtIn the email, the inspector wrote: “The food safety concern is contamination of food through possible throw up and/or feces coming from the baby. This would include you burping the baby after nursing or you having to change the infant’s diaper between serving customers.”

Holt has been food safety certified and said this kind of contamination isn’t happening.

There’s a bathroom nearby to change the baby and wash up. Her clothes are covered with a receiving blanket when she nurses, and, like most mothers, she has extra layers to wear in case the blanket doesn’t do the job.

At the market or at home, “I don’t want to walk around with baby puke on me,” she said.

Too risky: Petco stops selling pet treats from China

Petco says it has cleared its store shelves of all dog and cat treats made in China, in response to consumers’ concerns about contamination.

sadie.dog.powellThe announcement includes more than 1,300 stores nationwide, including 42 in Ohio, as well as Unleashed by Petco stores and online sales at Petco.com

Petco says that makes it the first national pet retailer to stop selling China-made pet treats. Petco carries products for dogs, cats, fish, reptiles, amphibians, birds and small animals.

“As a trusted partner for pet parents, we believe this is the right thing to do, and we’re proud to take this step in the best interests of pets,” Petco Chief Executive Jim Myers said, in a written statement. Myers is a graduate of John Carroll University. “What we feed our pets matters, and this milestone supports the company’s steadfast commitment to putting our customers, partners, animals and the communities we serve first.”

Petco, based in San Diego and founded in 1965, said it hasn’t carried or sold dog or cat food from China for several years, and that extending that stance to pet treats “allows Petco to expand the assortment of safe and healthy alternatives that are made in the U.S. or in other regions around the globe that support complete pet health.”

Raw milk risks: a science perspective from Europe

The European Food Safety Authority (EFSA) concludes that raw milk can carry harmful bacteria that can cause serious illness. Implementing current good hygiene practices at farms is essential to reduce raw milk contamination, while maintaining the cold chain is also important to prevent or slow the growth of bacteria in raw milk. However, these practices alone do not eliminate these risks. Boiling raw milk before consumption is the best way to kill many of the bacteria that can make people sick.

Spew milkConsumer interest in drinking raw milk has been growing in the European Union (EU) as many people believe it has health benefits. Under EU hygiene rules, Member States can prohibit or restrict the placing on the market of raw milk intended for human consumption. Sale of raw drinking milk through vending machines is permitted in some Member States, but consumers are usually instructed to boil the milk before consumption.

In their scientific opinion on public health risks associated with raw milk in the EU, experts from EFSA’s Panel on Biological Hazards (BIOHAZ) conclude that raw milk can be a source of harmful bacteria – mainly Campylobacter, Salmonella, and Shiga toxin-producing Escherichia coli (STEC).

The Panel could not quantify the public health risks associated with drinking raw milk in the EU due to data gaps. However, according to Member State data on food-borne disease outbreaks, between 2007 and 2013, 27 outbreaks were due to the consumption of raw milk.

Most of them – 21 – were caused by Campylobacter, one was caused by Salmonella, two by STEC and three by tick-borne encephalitis virus (TBEV).  A large majority of the outbreaks were due to raw cow’s milk, while a few of them originated from raw goat’s milk.

“There is a need for improved communication to consumers on the hazards and control measures associated with consumption of raw drinking milk,” says John Griffin, Chair of the BIOHAZ Panel.

Infants, children, pregnant women, old people and those with a weakened immune system have a higher risk of falling ill from drinking raw milk.  

Abstract

Raw drinking milk (RDM) has a diverse microbial flora which can include pathogens transmissible to humans. The main microbiological hazards associated with RDM from cows, sheep and goats, horses and donkeys and camels were identified using a decision tree approach.

raw.milkThis considered evidence of milk-borne infection and the hazard being present in the European Union (EU), the impact of the hazard on human health and whether there was evidence for RDM as an important risk factor in the EU.

The main hazards were Campylobacter spp., Salmonella spp., shigatoxin-producing Escherichia coli (STEC), Brucella melitensis, Mycobacterium bovis and tick-borne encephalitis virus, and there are clear links between drinking raw milk and human illness associated with these hazards.

A quantitative microbiological risk assessment for these hazards could not be undertaken because country and EU-wide data are limited. Antimicrobial resistance has been reported in several EU countries in some of the main bacterial hazards isolated from raw milk or associated equipment and may be significant for public health. Sale of RDM through vending machines is permitted in some EU countries, although consumers purchasing such milk are usually instructed to boil the milk before consumption, which would eliminate microbiological risks. With respect to internet sales of RDM, there is a need for microbiological, temperature and storage time data to assess the impact of this distribution route. Intrinsic contamination of RDM with pathogens can arise from animals with systemic infection as well as from localised infections such as mastitis.

Raw-Milk-Card-FrontExtrinsic contamination can arise from faecal contamination and from the wider farm environment. It was not possible to rank control options as no single step could be identified which would significantly reduce risk relative to a baseline of expected good practice, although potential for an increase in risk was also noted. Improved risk communication to consumers is recommended.

Summary

Following a request from the European Food Safety Authority (EFSA), the EFSA Panel on Biological Hazards (BIOHAZ) was asked to deliver a scientific opinion on the public health risks related to the consumption of raw drinking milk (RDM). In particular, the BIOHAZ Panel was requested to identify the main microbiological hazards of public health significance that may occur in RDM from different animal species, to assess the public health risk arising from the consumption of RDM, to assess the likelihood of RDM being a significant source of antimicrobial resistant bacteria/resistance genes, to assess the additional risks associated with the sale of RDM through vending machines and via the internet and to identify and rank potential control options to reduce public health risks arising from consumption of RDM.

According to European Union (EU) legislation, “raw milk” is defined as milk produced by the secretion of the mammary gland of farmed animals that has not been heated to more than 40 °C or undergone any treatment that has an equivalent effect (Regulation (EC) No 853/2004). A top-down four-step decision tree was used to identify the main microbiological hazards associated with RDM of different milk-producing species in the EU. Microbiological hazards that can be transmitted to humans through milk and which were reported from cows, sheep and goats, horses and donkeys and camels in the EU were listed. Those hazards which could be transmitted via milk but were not reported from milk-producing animals in the EU were excluded from further consideration. Microbiological hazards identified as potentially transmissible through milk and present in the EU milk-producing animal population included the bacteria Campylobacter spp. (thermophilic), Salmonella spp., shigatoxin-producing Escherichia coli (STEC), Bacillus cereus, Brucella abortus, Brucella melitensis, Listeria monocytogenes, Mycobacterium bovis, Staphylococcus aureus, Yersinia enterocolitica, Yersinia pseudotuberculosis, Corynebacterium spp., Streptococcus suis subsp. zooepidemicus,the parasites Toxoplasma gondii and Cryptosporidium parvum and the virus tick-borne encephalitis virus (TBEV). Those hazards transmissible via milk of one species and present in the EU were also considered to be potentially transmissible by milk of other species if present in the EU.

napoleon milkEvidence for RDM as an important risk factor for human infection in the EU was based on epidemiological evidence that the hazard has been associated with illness from the consumption of RDM in the EU, the extent of occurrence of the hazard in different milk-producing species in the EU, the prevalence of the hazard in milk bulk tanks or retail RDM in the EU, and expert opinion. Between 2007 and 2012 there were 27 reported outbreaks in the EU involving RDM. Of these, 21 were attributed to Campylobacter spp., predominantly C. jejuni, one to Salmonella Typhimurium, two to STEC and three to TBEV. Four of the 27 outbreaks were due to raw milk from goats, the rest being attributed to raw milk from cows. The published literature was also considered, which highlighted additional outbreaks of TBEV and outbreaks of B. melitensis, M. bovis and STEC, although some of these were prior to 2007. No outbreaks attributable to L. monocytogenes in RDM were reported between 2007 and 2012.

STEC, Salmonella spp. and Campylobacter spp. are essentially ubiquitous pathogens and are likely to be found in milk-producing animals and their milk throughout the EU, as indicated by prevalence data from raw milk testing. TBEV was also considered to be a main hazard based on outbreak data, together with evidence of spread in Europe and the virus being detected in raw milk. B. melitensis and M. bovis have been associated with outbreaks involving raw milk, but these are less common and more geographically restricted than the other pathogens and control programmes in Europe have generally been successful in reducing human disease from these pathogens.

For other hazards, epidemiological evidence of illness was either historical or limited to reports from outside Europe. L. monocytogenes infection is associated with a high mortality rate in vulnerable groups, and the organism was as frequent as Campylobacter and STEC in raw milk. The lack of robust epidemiological data (including outbreaks) linking listeriosis to consumption of raw milk in Europe meant that it could not be considered a main hazard. The ability of L. monocytogenes to grow at chill temperatures, coupled with its prevalence in raw milk, suggests that further study in relation to RDM may be justified, particularly as several risk assessment models outside Europe have already been developed for this pathogen.

colbert.raw.milkThere is a clear link between drinking raw milk and human illness with Campylobacter spp., S. Typhimurium, STEC, TBEV, B. melitensis and M. bovis, with the potential for severe health consequences in some individual patients. Owing to the lack of epidemiological data, the burden of disease linked to the consumption of raw milk could not be assessed. Published quantitative microbiological risk assessment (QMRA) models from Australia, New Zealand, the USA and Italy, for Salmonella spp., Campylobacter spp., STEC O157 and L. monocytogenes in RDM from cows, were reviewed to identify their strengths and limitations. No QMRAs were available for RDM of other species. The risk estimates provided by the QMRA models reviewed cannot be extrapolated to the European situation as a whole. The outputs from the Australian and New Zealand risk assessments for STEC O157 and Salmonella spp. estimate a high level of milk contamination, which contrasts with the outputs from the risk assessment for these pathogens in RDM in one region of northern Italy, where the risk associated with STEC O157 was estimated as very low because of model uncertainty. Similarly, the Australian and New Zealand risk assessments predicted a higher risk for Campylobacter spp. than the risk assessment conducted in one region of northern Italy, largely as a result of differences in the extent of faecal contamination. From the model used in the Australian study it can be concluded that improving on-farm hygiene leads to a decrease in the number of predicted cases of illness due to Campylobacter spp., Salmonella spp. and STEC O157 from the consumption of RDM. A QMRA could have helped in further estimating the public health risks and evaluating the effect of the mitigation options in Europe for these hazards, but could not be undertaken because country and EU-wide data are limited.

Antimicrobial resistance has been reported in several EU countries in isolates of Campylobacter spp., Salmonella spp., STEC and S. aureus from raw milk or associated equipment such as milk filters, and may be significant for public health. Such isolates have been primarily associated with raw milk from bovine animals, which may reflect the more limited screening of milk from other species. Strains of Campylobacter spp., and particularly C. jejuni, exhibiting resistance predominantly to tetracyclines but also to some other antimicrobials have been reported in two Member States (MS). There have been no reports of antimicrobial resistance in isolates of Salmonella spp. from outbreaks associated with raw/unpasteurised in the EU in countries other than the UK. In the USA, there has been a report of a raw milk-associated outbreak caused by multidrug-resistant (MDR) S. Typhimurium, with a single fatality ascribed to resistance of the organism to antibiotics. Despite STEC O157 being the organism most commonly associated with RDM-related outbreaks of STEC gastrointestinal illness in several EU countries, little information is available about the occurrence of antimicrobial resistance in such outbreak strains. Antimicrobial resistance has been reported in a water buffalo raw milk-associated STEC O26 outbreak in one MS in 2008 and in raw milk-associated STEC outbreaks in the USA. Antimicrobial resistance in isolates of L. monocytogenes from raw milk and raw milk dairy products has only rarely been reported in EU countries.

Meticillin-resistant Staphylococcus aureus (MRSA) has not been isolated during outbreaks of infection associated with RDM in EU countries. Although not typically regarded as a food-borne pathogen, there have been increasing reports of the isolation of MRSA from dairy farms and bulk tank milk in several EU MS. Although identified in E. coli in bovine animals in some MS, extended spectrum beta lactamase (ESBL)/AmpC gene-carrying bacteria have not been reported in RDM in EU MS. In the USA, a range of Salmonella serovars with ESBL/AmpC genes have been identified in raw milk surveys.

Sale of RDM through vending machines is permitted in some EU MS, with considerable variation in the number of machines in different countries. There is little indication of RDM other than cow’s milk being sold through vending machines. Although vending machines dispense drinking milk in a raw state, consumers are usually instructed to boil the milk prior to consumption. If consumers were to comply with these instructions, the microbiological risks associated with raw milk would be eliminated. The temperature of RDM in vending machines is generally kept below 4 °C and therefore variability in milk temperature is more likely to arise between the farm and vending machine and between the vending machine and point of consumption by the consumer. One study in Italy demonstrated that temperature variability in the supply chain from farm to consumer could potentially result in the multiplication of L. monocytogenes, S. Typhimurium and STECO157:H7.

Fresh and frozen RDM of different species (cows, goats, sheep and camels) is available via internet sales although there are no data on the microbiological or temperature controls for these milks from the bulk milk tank through to the point of consumption. The variability in temperature control and duration of storage by consumers would contribute to the multiplication of some pathogens if these are present in the milk.

The steps in the production to consumption chain for RDM present many opportunities for contamination by microorganisms, some of which may be transmissible to humans. Intrinsic contamination of milk can arise from systemic infection in the milk-producing animal as well as from localised infections, such as mastitis. Extrinsic contamination of milk can arise from faecal contamination and from the wider farm environment associated with collection and storage of milk. Observance of good animal health and husbandry, together with the application of good agricultural practices (GAPs) and good hygienic practices (GHPs), are essential to minimise opportunities for contamination of RDM with pathogens in the production to consumption chain for RDM. No single step could be identified which would provide a significant reduction in risk relative to a baseline of expected good animal health and welfare and good agricultural and hygienic practices. Therefore, it was not possible to rank control options with respect to risk reduction since any deviations from the expected “best practice” baseline are likely to result in an increase in risk.

The reviewed QMRA models identified on-farm hygiene control and maintenance of the cold chain as factors influencing the outcome of the models for some pathogens. Although L. monocytogenes is not considered to be one of the main hazards associated with RDM in the EU, the reviewed QMRAs from outside the EU do show that the risk associated with L. monocytogenes in raw cow’s milk can be mitigated and reduced significantly if the cold chain is well controlled, the shelf-life of raw milk is limited to a few days and there is consumer compliance with these measures/controls.

The BIOHAZ Panel identified several recommendations arising from the opinion. There is a need for a better evidence base to inform future prioritisation and ranking approaches and studies should be undertaken to systematically collect data for source attribution for the hazards identified as associated with RDM and collect data to identify and rank emerging milk-borne hazards. Because of the diverse range of potential microbiological hazards associated with different milk-producing animals, hazard identification should be revisited regularly. There is a need for validated growth and survival models for pathogens in RDM of different milk-producing species, particularly in relation to the temperature and storage time of RDM from the producer up to the point of consumption. Finally, the Panel recommended that there should be improved risk communication to consumers, particularly susceptible/high risk populations, regarding the hazards and control methods associated with consumption of RDM.

STECs in Sweden on beef and leafy vegetables

This study investigated the occurrence of Shiga toxin-producing E. coli (STEC) in beef and leafy greens available on the Swedish market. New data are required for assessing the public health risk of STEC in food, which could be used for developing risk management strategies.

beef.stecFood samples were collected at retail stores, importers, outlets and the markets. Samples of minced or whole meat from cattle collected were fresh or frozen from 2010 to 2011. The beef sample collection included products from the most common countries or regions exporting beef to Sweden. The collection of leafy greens consisted of domestic and imported products that were available on the Swedish market from 2012 to 2013.

Detection of virulence genes (stx 1, stx 2, eae) and genes specific for different serogroups (O26, O103, O111, O145 and O157) was performed by real-time PCR followed by isolation of bacteria from the stx -positive enriched samples by use of immunomagnetic separation. STEC bacteria were overpriced isolated by an immunoblot thing method. All STEC isolated from the food samples were serotyped.

STEC was isolated from 23 (13 percent) of the 177 imported beef samples tested. Approximately 3 percent of the beef samples contained STEC on positive stx 2 and eae, both of which are important markers for the probability of the bacteria to  cause severe disease. In total, 27 STEC were isolated, belonging to 14 different serogroups. STEC O26 was most common (approximately 2 percent of the beef samples), whereas STEC O157, frequently implicated in STEC-related foodborne outbreaks in Sweden, was found in two (one percent) of the beef samples.

swedish-chefThe enrichment broth of 11 (approximately 2 percent) of the 630 samples from leafy greens were tested positive for stx 1 and / or stx 2 by PCR analysis; however, no bacteria were isolated. Presumptive STEC was detected in co-enriched samples from bothering domestic and imported products. E. coli was found in 68 (39 percent) out of 174 and 14 (30 percent) out of 46 samples of imported and English leafy greens, respectively, indicating that the proportion of stx -positive E. coli into the samples was low.

Nanopore sensor for botulinum toxin detection

By Dr. Yong Wang, Nanopore Single Molecule Sensing Group, Biological Engineering and Dalton Cardiovascular Research Center, University of Missouri-Columbia, writes in Nanowerk:

Botulinum_toxin_3BTABotulinum neurotoxins (BoNTs) are the most poisonous substances known to humans, with a median lethal dose (LD50) of 1ng per kg of body weight and are the cause of the life-threatening neuroparalytic illness botulism. BoNTs are regarded as possible biological warfare agents that could be used for bioterrorism attacks on the food chain.

Of the seven known serotypes of botulinum neurotoxins (BoNTs), designated A-G, BoNT-A, -B, -E and -F are toxic to humans. They are mainly produced by Clostridium botulinum and released from this bacillus as single polypeptide chains.

After cleavage by bacterial or host proteases, these polypeptides adopt an active di-chain form of toxin, which consists of a 100 kDa heavy (H) chain linked to 50 kDa light (L) chain by a disulfide bond. H chains control cellular internalization of the toxins via receptor-mediated endocytosis, allowing them to preferentially attack the nervous system. Upon endocytosis of the holo-protein, the L chain is translocated from the vesicular lumen into the cytosol, where it acts as a Zn2+-endopeptidase to specifically cleave components of the soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) complex, and thus hampers exocytosis and neurotransmitter release.

Currently, world-wide geographical distributions of BoNTs, which are dictated by the type of foods that serve as the toxins source, have started to blur owing to the ever increasing international trade of food products. Moreover, the majority of new cases of BoNT intoxication have a trend to be associated with drug use, in particular that of black tar heroin.

Owing to the ease of distribution of BoNTs, along with their potency, the Center for Disease Control and Prevention (CDC) in Atlanta, Georgia, lists BoNTs as one of the six most dangerous bioterrorist threats. On the other hand, due to their specific actions, BoNTs are the first toxins licensed for human use in the United States to treat muscle dysfunctions/spasms and associated skin wrinkles.

Both bioterrorism defense and medical attention require an early and rapid detection of BoNTs. Traditionally, this is accomplished by looking for signs of botulism in mice which receive an injection of human serum or stool samples.

While this bioassay remains the most sensitive detection approach, it is costly, slow (takes days), associated with legal and ethical constraints, and is restricted to the CDC along with some state health department laboratories.

Besides the use of living animals in detection, various biochemical assays, such as electrophoresis and immunoblot analysis have been used to detect the cleavage of specific SNARE proteins. These in vitro detections are faster (within a day), but with reduced sensitivity, compared to the mouse bioassay.

The subsequent development of time-resolved fluorescence-based enzyme-linked immunosorbent assays (ELISA) allowed utilizing lanthanide (Eu3+)-tagged antibodies to detect BoNT-A and -B. ELISA sped up the outcome of BoNT assays to hours.

Within a similar time-frame, the BoNT-A activity can also be monitored using the substrate SNAPtide™, a fluorescently conjugated synthetic peptide that contains the native cleavage site for BoNT-A.

Recent developments in mechano- and fluorescence resonance energy transfer (FRET)-based sensors offer a reduced detection time and/or increased sensitivity. The micromechanosensor uses force spectroscopy to detect BoNT-B. This toxin cleaved synaptobrevin 2 (Sb2) is attached to a bead, which was suspended off a cantilever via Sb2 interaction with syntaxin 1A attached to the cantilever. The detachment of the bead from the cantilever marked the cleavage of Sb2, allowing detection of low nanomolar range of BoNT-B within 15 minutes.

Additionally, in vitro detection using FRET probes that contain fragments of SNARE proteins as toxin substrates shows picomolar sensitivity within 4 and 16 hours.

Although the two later assays are very promising for practical use, they require expensive and technically complex equipment. Overall, there is a need for further development of assays for detection of BoNTs.

In recent work, Dr. Yong Wang and his collaborators described a nanopore-based assay for detection of BoNT-B.

Illustration of the principle for nanopore BoNT-B detection. Lp-Sb2(1-93) (based on structure PDB ID code 2KOG in the Protein Data Bank (www.pdb.org)) would not affect the pore current, as shown by the hypothetical trace below the structure. After the cleavage of Lp-Sb2(1-93) by BoNT-B/Zn2+ (arrow), the short digested product Sb2(77-93)d would generate a distinct modulation of pore currents, as shown by downward transient blocks in the hypothetical trace, while the long product Lp-Sb2(1-76)d would not affect the nanopore current. Dotted lines are levels of zero current. Analytes would be applied from the cis (pore vestibule) side of the bilayer that partitions the recording chamber. (Reprinted with permission by American Chemical Society)

“We utilized the emerging aerolysin pore to track the BoNT-B digestion of its substrate, a derivative of the synaptic protein Sb2 (also known as VAMP2). The dynamic change of the specific digestion product reported the existence of the toxin at sub-nanomolar (500pM) concentration within minutes,” says Dr. Yong Wang.

This system also demonstrates usefulness in investigating biophysical mechanisms for peptide translocation and interaction with the nanopore. The aerolysin protein pore can be used to elucidate the interaction of the nanopore with a synaptic protein Sb2 derivative and its BoNT-B cleavage products.

“We determined that only the small C-terminal BoNT-B digest peptide in its native state can interact with the pore to generate current blocks, while other native peptides/proteins in the reaction mixture and serum cannot produce observable blocking events,” notes Wang.

“This research is motivated by the danger posed by BoNT which is one of the most potent bio-toxins,” he adds. “Rapid and sensitive detection of this toxin, due to drug-use, food-poising or terrorism, is a priority in toxicology and biodefense. This is an area of future interest and a fertile ground for improvements of the present BoNT-B nanopore detection scheme. This approach could be adapted to the selective peptide detection for biosensing and investigating biological processes, including the detection of activity of Clostridial toxins, and biophysical mechanisms for biopolymers interaction and translocation through the nanopore.

This important peer-reviewed scientific result was published in ACS Applied Materials & Interfaces online on December 16, 2014 (“Nanopore Sensing of Botulinum Toxin Type B by Discriminating an Enzymatically Cleaved Peptide from a Synaptic Protein Synaptobrevin 2 Derivative”).