Transcription
Pathogens in Hemp & Cannabis
Thank you for joining me today for at a look at pathogens in hemp and cannabis. Think about all the forms of cannabis and hemp; you can have edibles, you could have
Nutraceuticals, plant material, oils, vapes. These are all different types of materials that can be subject to pathogens themselves, or it can be roots for pathogens be transmitted to humans. And usually, the pathogens are deposited in the different materials or the different products through water, through other agricultural products, or through the air.
Around the world foodborne illness is a very large problem. According to the world health organization, 600 million cases of foodborne illness are reported each year. And that means that's one out of every 10 people contract a foodborne illness and this is just the cases that are reported to the world health organization. So, that means there are 420,000 deaths reported each year from foodborne illness worldwide. If we're going to look at the United States, the CDC reports 48 million illnesses each year due to foodborne illness; that's one in six people, and that also results in 3,000 deaths per year. There are many agents for disease for foodborne and waterborne illnesses. In fact, there are over 250 specific agents, and they can range from industrial pollutants to poor sanitation to poor hand washing to food handling. So, you first have your physical contamination this is your large debris your dirt your bugs, then you have chemical contamination where maybe pesticides, heavy metals, or other materials seep into food and water. Then you have parasites, these are worms, nematodes and other types of organisms that enter the food or the water and infect human beings. And then you have your microbiological contamination, your bacteria, your viruses, and your molds.
Consider all the forms hemp and cannabis can take, you could have an edible or an oil, you could have plant material or nutraceutical, or a vape product or an inhalable product, and usually we're concerned about chemical contamination, but we really also need to be concerned with microbial contamination because microbes can actually produce toxic chemicals. So, which microbes well we are most concerned with bacteria viruses and fungi. If we look at a chart of all the different agents that are found around the world that cause disease in the US and worldwide, the top of the list is norovirus. That's a virus which causes 125 million cases around the world of foodborne and waterborne illness, followed by other common bacteria like salmonella and campylobacter and shigella. So, there are a lot of microbial agents that cause disease and depending on where you are in the world the death rate might be different. In the US the list of viruses and bacteria are slightly different there's a slightly higher cases of some other than others around the world those cases are a little different. But what you really should notice is the botulism bacteria, the clostridium bacteria that causes botulism. In the US the death rate is 17.3 percent even though we have usually less than 100 cases a year. On the other side in the worldwide view the aflatoxins actually have the one of the highest death rates at 89.4 percent and there are a lot more cases worldwide, 21,800 cases, worldwide where we haven't, in the US, reported any human cases of illnesses or death from aflatoxins.
So, today we're really going to be concerned with three kingdoms; the fungal kingdom, the bacterial kingdom, and the viruses which aren't quite a kingdom they're in a class of their own, they're not really classified under what we learned in school as your classifications of life. So, let's take a look at a schematic of how you decide what to
examine or what your targets might be. So, you have your products and now you have to decide to target, what's that target going to be you’ll be testing for? Is it going to be a fungus, is it going to be a mold, is it going to be a bacteria, is it going to be a virus? So, what is the actual organism target that you are going to be analyzing for? And then by what method? Are you going to look for a chemical marker, are you going to do a colony count where you're going to plate a live organism and you're going to see how it grows over a set period of time, or are you going to be extracting DNA to get a genetic analysis of your particular target?
Let's look at some of these molecular biological analysis techniques. So, these are your plating techniques and your DNA techniques. So, you have your very classic cellular technique, your culture on media, it can be qualitative it could be quantitative it tends to be semi-quantitative in some cases. It's fairly reliable, it's fairly and sensitive but it doesn’t have a lot of specificity sometimes and it can take quite a long time. There's definitely a lot of labor involved, all of those plates and your agars and things like that they cost money so, there's a cost. And there's a certain expertise to making a successful culture plate. And one of the things that can happen is that you get false negatives or you get results that are a little low because somehow during the sample prep, the cells have died so you don't get active growth on your plates. Then you have your immunological technique. These are the testing of the proteins, ELISA is what we call it, it's qualitative and quantitative, it's easy, it's fairly high throughput. Again, it has some low specificity, and it can have some issues with levels of detection. Then you have the nucleic acid or the DNA methods, these are your PCR and your qPCR methods. Again, you could have them qualitative or quantitative, and high throughput you do not use cultured organisms so your samples do not necessarily have to be alive to do your
your PCR, but they do have some costs for enrichment and purification because you
have to purify your samples.
Now we prefer PCR when we talk about a lot of our pathogen testing. It's a fairly straightforward process it's been in use for decades now and simply put you denature a strand of DNA, usually between 94, 98 degrees centigrade, you anneal it, meaning you use primers and nucleotides to section an area of DNA that you wish to copy, and usually there are some target genes for each of the different species or each of the different organisms that get targeted to make sure you are reproducing the DNA for the that particular organism. And then you elongate, that means you create longer
strands of that copied DNA. So, usually go through several cycles of this where you denature, anneal, extend, denature, anneal, extend, and this is controlled by temperature So, high temperature denatures, cooler temperatures anneal, and then mid-level
temperatures to extend that DNA. You do this over and over again, and each time you do a cycle your DNA amount doubles, so you need very little DNA in order to get an accurate PCR or qPCR. So, you can have as little as 10 copies of DNA and be able to
successfully amplify that DNA and determine what organism you're dealing with. And then there are several ways you can do your PCR. You can do endpoint PCR; this is where you amplify it to a certain point and then you would usually run gel electrophoresis to see if you have the DNA sequences and the quantity that you
were amplifying. Then you have real-time PCR, this is a way of quantifying your DNA sequences. So, you put fluorescent markers or tags on your DNA sequences and as they are amplified, you will watch the quantity of DNA as it increases.
Now we're going to talk about some of the targets you'll be looking for in your cannabis species. What are the bacteria and what are the molds. I'm not going to go through the viruses because there are not as many there are definitely more molds and bacteria that are possible with your cannabis species. First, we're going to look at bacteria. These are single cell organisms and they're prokaryotes meaning they don't have that nuclear membrane envelope that we're used to seeing in a cell. They live in all habitats, they are the ultimate survivors so from deep ocean vents to the top of frozen mountains, they have the ability to survive. And likewise. they live in all extremes; they live in in deserts, they live in cold, they live in heat, so the hottest places on earth and the coldest places on earth you will find bacteria. And they're needed for life, but they can also be pathogens and the ones that are concerned with cannabis are things like Salmonella, E. coli, Bile Tolerant Gram Negative Bacteria, staphylococcus, and Enterobacteriaceae.
If we look at Salmonella, most of the states in the United States require some sort of Salmonella testing on recreational and medicinal cannabis. The regulations for hemp, especially products which can be consumed, or edibles, they are going to be very
Similar. So, you're going to have either no tolerance for Salmonella in your culture, or you're going to have very low tolerance, either less than one cfu per gram, or some states are a little more generous, like Connecticut, New Mexico and Utah, and you have 10 grams of with no detection as your limit.
So, these are the states that have regulations when it comes to E. coli for cannabis. Again, this is recreational and medicinal with very similar, if not the same, regulations for hemp edibles and hemp nutraceuticals. So, you have different types of E. coli. So,
you have general species of E. coli, and then you have the Shiga toxin producing
E. coli the STEC. So, depending on which pathogen the state requires, you can see they're either dark or light purple. So, the dark purple are the general E. coli species plural, and the lighter purple is the STEC, and then you can see there are a few states that actually have requirements for both. So, something like Pennsylvania has requirements for both.
So, the fungal kingdom is very interesting. When the first classification of organisms started hundreds of years ago, we first classified them as plants. So, the early scientists saw that they grew, they didn't move, they kind of looked like plants, so they put them with plants. But, as the science has moved forward over the centuries, we've recognized that they are not plants, but they're not quite animals either. They have characteristics, they're a blend of plants and animals. They have chitin in their cell walls like plants do. But they're heterotrophs, and they have digestive enzymes. So, that's kind of animal-like. There's no photosynthesis, again very animal-like, no photosynthesis. And the only growth we see is through the growth of the organism itself or the release and growth of spores and that's very plant-like. And finally, another plant-like characteristic is a being a decomposer. The fungal kingdom has nine phylum, and that goes from the macro fungi, the big fungi that we think about, down to the little fungi.
So, on the macro fungi scale we have mushrooms. You can think of all the different phylum of mushrooms. So, these are our traditional looking cap stems growing out in the woods, growing in places in the garden, these are our very large macro fungi. And then you start going down the scale and you get smaller yeasts, and finally mold as well, and those are more in the micro fungi range. And the ones that we are most concerned
about when we talk about cannabis and hemp are the molds and the yeast the mycotoxin producing forms of the fungi.
Some of the most common mycotoxins are these four groups here, the Aspergillus, the Penicillin, the Fusarium, and the Claviceps. But for most cannabis products they are most focused on these two groups here, the aspergillus products and the penicillin products, the aflatoxins the ochratoxins. Now if you are testing recreational or
medicinal cannabis in the United States it's being tested for in several states
and you can see that sometimes they test for four species of Aspergillus, sometimes they test for three species, sometimes it's a total of all four species, sometimes it's only for inhalation products. So, the regulations can widely vary, and the Aspergillus tends to
be more highly volatilized and inhaled so vapes and plant material that is going to be smoked is going to have the biggest impact for some of this Aspergillus. That's why some of the regulations say for inhalation or inhalable products instead of edible
products. Now one way that you can detect Aspergillus and, as I said, one of the
ways that we prefer to test Aspergillus is with PCR detection. So, we have the different species of Aspergillus, and we have internal controls, and we use qPCR in order to
amplify the DNA of the different strains of Aspergillus. Here is a comprehensive list of all the different mycotoxins, all the fungi that are associated with them, and their
health effects and the sources. And you can see a lot of commonalities, cereals, grains things like that. Now somebody might say “well I'm dealing with cannabis so why should I care?”. Well, if you are producing a nutraceutical and you're blending it with maybe some sort of cornstarch, then you're going to want to know that aflatoxins can be in corn. If you're making a food or edible product, again you're going to want to know what
potentially can be part of the product and what should I be testing for. As for cannabis, when it comes to testing, they're mostly looking for the Aspergillus, the penicillin species and, the affluent ochratoxins. I've had to explain several times, what's
the difference between like the testing for Aspergillus or the testing for mycotoxin,
aren't they kind of like the same thing? Well, I like to equate them to the actual
organism is like the spider. So, you can have the spider, but you don't necessarily have the poison. And the poison, or the venom, is the mycotoxin so what are you looking for, are you looking for the spider or are you looking for the venom.
So, when you're doing cannabis testing for recreational or medicinal cannabis or cannabis products, or hemp products, are you looking for the spider are you looking for
the venom? Well, the answer is it depends, what state you're in, what their requirements are. Some states you're looking for different species, some states you're looking for the total of all Aspergillus species, some states you're looking for the actual mycotoxins and you might be having a concentration limit for each one, or you might have a concentration limit for all five of them in total. And usually that total is in the range of
about 20 parts per billion. So, we're going to look at mycotoxin by the numbers. It's been estimated that about 25 percent of all food is infected with some form or some amount of mycotoxins. And one of the biggest mycotoxins is the aflatoxins. The World Health Organization considers it to be life-threatening. Anything above one ppm, or one milligram per kilogram, is considered to be life-threatening. Out of the aflatoxins, B1 is the most, toxic between 0.3 and 18 micrograms per kilogram of body weight a day. And food items should have less than 20 micrograms per kilogram in foods, it's less than 20 ppb. And then you have your ochratoxin, which is also toxic and has a limit by the FDA
of five milligrams per kilogram of body weight a day. So, I'm going to end this presentation with a look at some of the famous cases in history of mycotoxin poisoning.
The first one we have is the curse of the pharaohs. There has been some belief that the
curse of the pharaohs, the reason why all of these archaeologists died, was possibly exposure to mold when opening these tombs. So, the mold became airilized they were breathing it in, and they suffered from mycotoxin poisoning.
Salem Witch Trials, there has been a line of thought since the 60s and 70s that possibly the Salem Witch Trials were due to ergot poisoning. Their main source of bread was grain, the rock grain rye and that could have been contaminated with the ergot mycotoxins and that would cause hallucinations, muscle spasms, so then you would have all of this very odd behavior and that might have been the genesis of the Salem Witch Trials.
In 2004 there was an outbreak of mycotoxin poisoning there were 125 people who died and over 200 that were sickened from grains that had been poisoned with mycotoxins.
And you have the pet food recalls, especially the dog food recall, as late as last year. When there were mycotoxins found in pet food and in dog food that caused sickness and death.
And of course, we've always heard about the dangers when you're buying a house, or you're living in an apartment, or you have a water leak, of black mold, that you don't want black mold. That mold could get you sick, and black mold, if you breathe it in, could give you respiratory problems or it could kill you. That's because a lot of the mold that
constitute the general phase black mold are those aspergillus molds or penicillin molds. So, that's why they say to be aware of the black mold, it's because of the mold and
the mycotoxins that they produce.
I would like to thank you all for attending the presentation, and I hope you found it of value.
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