Tiger Slaughterhouses and Forensic DNA: How Science is Fighting Illegal Wildlife Trade

Laura: Daniel, thank you so much for joining us. We’re really excited that you could be here with us at our 35th anniversary year, and I’d love to talk about your presentation. I’m very excited to hear about what you’re going to be sharing tomorrow with the group. But before we get started, maybe you could tell us a little bit about yourself and your background.

Daniel: Okay. My background. I started my forensic career back in ‘92, when I joined Czech police. I became the first head of all DNA operations in the Czech Republic or Czechoslovakia and Czech Republic. After ten years, I moved to Bosnia and Herzegovina, where I worked for ICMP (the International Commission on Missing Persons) humanitarian organization mainly sponsored by the United States and Dutch government. After three years, doing, you know, bones from mass graves and having experience with tens of thousands of bones, I returned back home, and set up my own private laboratory specializing in ancient bones because the protocols we’ve developed together with John Davoren, they work also on ancient bones and degraded DNA. And throughout the years, I’ve started with animal DNA typing. And, at this moment, we have the biggest database of physical samples and DNA profiles of big cats. So, almost 1000 samples. And that enables us to do more research and etc., and be more powerful during, you know, cases involving big cats and other endangered species.

Laura: Yes. Your case is so intriguing to me. I wish I weren’t in the studio so I could come and listen to it. So, you’re talking about a shocking example of the illegal wildlife trade that, you know, can happen in Europe, and the Tiger Slaughterhouse is the title. And maybe you can tell us a little bit about the case and then how DNA analysis played a crucial part in exposing it.

Daniel: Yeah. So, the first reports about the case started in July 2018. We started our preparations two years earlier, because we knew that there is such a case and demand from Czech customs and the Czech Environmental Inspectorate. But we needed to be prepared to have the technology in place so that we can start analyzing the samples immediately. So, the setup and all the science behind all of it. In those two years, we were able to develop some basic set of STRs that were quite discriminative for big cats and develop a quantitation system and all the things together. Especially to type lots and lots of tigers, we needed to have reference samples for them. So, we were prepared. And when the case came, we were able to start immediately and to process those hundreds of samples. And out of those samples that were collected by, you know, crime scene officers, we identified some seven different individuals, tigers, plus a bunch of other, you know, protected species. So, it was quite successful from our point of view, you know, as a DNA laboratory.

Laura: Absolutely. And hundreds of samples. I mean, you’re talking about quite a lot of work. What were some of the challenges in obtaining and then analyzing those samples.

Daniel: Actually, it wasn’t so difficult with my background in ancient DNA and also ten years working for the police. And I’ve been, you know, involved in many, many research projects. So, it wasn’t so difficult working with tiny amounts of DNA. It’s a yeah, it’s my daily bread. But yeah, but there are other cases where we have to do some additional research, for instance cases involving tanned skin, because then mitochondrial DNA can be altered by the chemicals used for tanning. So, then it becomes more challenging. And that’s what I love, because I love obstacles so I can climb up.

Laura: That’s very interesting how that would change it. How do you overcome some of those obstacles.

Daniel: When that happens, I’m using my scientific background and I’m talking to my colleagues. And also, being an ex-police officer, I know that it’s not only DNA, but we also have other disciplines I can use to, you know, confirm our results. So, I can use her morphology and et cetera. Or some proteomics. And so, I know the ropes and what to do, you know, to have my results bulletproof.

Laura: So that’s perfect. That’s amazing. I’m sure that you come across highly endangered species in your work. And how does that differ and how you analyze that? Does that make it more challenging?

Daniel: That’s a very good question actually, because with animals, you’re dealing with a different spectrum of samples. The first case included a rhino and rhino skin is extremely, you know, hard to sample and work with. And suddenly, you know, we needed to process feathers and some traditional Chinese medicine, some tinctures. So yeah, what to do and how to proceed? And suddenly we started to validate new extraction methods and et cetera. Especially removal of inhibitors, because suddenly we were facing completely different spectra of inhibitors when compared to human DNA identification. And that’s what I love, because then you have, you know, topics for students and everything for my presentations. And so, I’m not just doing, you know, the same boring stuff, you know, day after another. But we really, you know, on our laboratory meetings we are solving problems, like with human contamination in the last case. So, we’ve developed a suppressor so we can amplify just animal DNA. Any animal, but not human DNA. So, it helps us to analyze more samples in a more fluent way.

Laura: So that’s very interesting. It’s a little bit different, but it’s a mixture.

Daniel: Yeah, it’s, you know, it’s flipped over because normally…

Laura: Right. Different mixture analysis.

Daniel: But even this, you know, human DNA can link the perpetrator to this sample. So, we can do both. But for species identification we need to have, you know, unique approaches.

Laura: Speaking of that, that was actually my next question. When it’s species specific, what kind of innovations have there been or that you’re working on that help?

Daniel: In species identification? I would say, you know, within these past ten years, the technology has evolved significantly. And especially, you know, we have specialized databases that target cytochrome oxidase one gene, mitochondrial gene. And they are verifying the records in the database. So, it’s much more precise. But me being, you know, an ex-police officer, I have to repeat it. So, I know that I must really deliver the perfect product. So, we are also using other databases. We have our own internal database. So, I’m checking everything two or three times just to be sure, you know, it fits. And I’m not releasing, you know, falsely positive or negative results.

Laura: I think that’s really interesting to bring all of the experience you have as an ex-police officer to the work that you’re doing now, because you really understand and speak the same language as you’re working together.

Daniel: Exactly. And I think my biggest advantage is when I go to court to defend, you know, our results with 30 plus years’ experience. And I’m going to court on a daily basis. And even when I was working in Bosnia, I had been doing reports for an International Court Tribunal for the former Yugoslavia. So, I’m quite experienced. And it helps. It really helps, because I’ve also taught at the university for some 30 years. I like the interaction with students, so I’m able to explain things in a simple way. But on the other hand, when the other side tries to somehow crucify me, I can, you know, do the same to them in a very effective way because it’s very helpful.

Laura: We actually talk about that a lot, you know, because there’s a lot of education usually, so people can understand the other side. But you naturally do because you’ve already been there. What impact do you hope, in this case particularly, that it will have going forward with the illegal animal trade?

Daniel: You know, the reason I’ve decided to come to the United States to talk about this case and our identification efforts, is that it’s still not the number one topic of non-specialized conferences, like you have for wild animals and, when I checked the spectrum of presentations on ISHI, it’s mainly genetic genealogy and some rapid DNA and etc. Okay, I understand it, because those guys are working on this on a daily basis, but there are special cases. And they should know what to do. And maybe also it can get people to start thinking in slightly different ways. What can be done? Not just to buy kits, but we are developing all our kits because there is no manufacturer that would, you know, prepare a kit for tigers or other big cats or some parrots. So, we have to do everything ourselves. But suddenly, with my experience three years ago, we were also doing the same, but without the technology in behind. So now we can do everything much faster. We have draft genomes and everything and it’s much easier really. Life is much easier. And within those two years, we’ve really prepared complete identification kits. You know, quantitation, STR typing, including letters. It can be done, because once you know how to proceed and what to do, it’s much easier.

Laura: I think it’s fascinating because there is like a cross-pollination of learning then too. I mean, you’re right, certain topics are trending. And so, we see a lot of that rapid DNA, IGG, things like that.

Daniel: And I remember in Odessa 20 years ago, I’d been reporting about mass graves and bone samples. And I’ve been organizing bone workshops all around the globe and teaching people how to extract DNA out of bones and how to use it. And it’s a big help to others. And suddenly, okay, it’s no longer an emerging topic and everybody is using it. So, okay, now it’s genetic genealogy. But I’ve been using this investigative genetic genealogy 15 years ago. So, it’s nothing new for me. I just wasn’t talking about it. But maybe, and I hope, that this topic and my presentation tomorrow will bring in more attention. And maybe, you know, Promega will invite me to run a workshop next year in Florida. So, that’s what I hope for, because it can be really interesting for others. Because I can see it. You know, with my students, I start with some basics about forensic genetics, history, etc., etc., and normally on the first lecture, you have 100 students and then the number goes down. But when I have a lecture about animal DNA typing, because I always talk about cases I work on, they come back again, and I have even more than those 100 because they bring their colleagues and friends. And so, I can see it’s also fascinating for them. And there are lots and lots of students that would like to work, you know, in my laboratory doing their diploma thesis. But unfortunately, having a private lab without any external governmental sources of money, you know, I can take 1 or 2 of the best students from my class. And that’s good because I can see how they interact. So, it’s some pre-selection and it helps me.

Laura: Well, I was very excited. I remember when we were looking at the abstracts and I was like, absolutely like we need to have we need to have an interview on animal forensics. I think the last interview I did that related to animals was, you know, illegal hunting in Canada. And so, they were trying to tie it back to whoever. So, I was incredibly fascinated by this. I’m so happy you’re willing to come over and share it. It’s going to be a great presentation. I think another interesting part about it is DNA barcoding methods. You know, can you talk about that. Some of the advantages and limitations.

Daniel: Yeah. We have to distinguish… Human identification, individual identification of animals is the same. So we can do, you know, parental testing on some smuggled animals and etc. But then it’s quite easy. But then you have cases where the customer sees some artifacts, traditional Chinese medicine, and they think, “Okay, what about if those Vietnamese Chinese are trying to smuggle some products for endangered species to Asia and that’s my task to say, “Okay, it’s clean. It’s just some pork meat in it or they use tiger and this and that” and with the broad spectrum because those are samples you wouldn’t expect that can contain any biological material. So, we had to develop many assays to be able to answer the questions in a timely manner, you know, within a few days to say, “Okay, but I know it’s not enough” And that’s why we are even working on small instruments that bring you the answers in tens of minutes.

And imagine that in the Prague airport they were, for the whole month, I think it was back in 2016, searching our luggage for presence of biological material and sending this stuff to my laboratory. So it was, you know, a huge amount of samples and everything. A lot of paperwork has been collected, but we really identified many, many endangered species and those, you know, tinctures and other stuff that has been seized. And it was amazing how effective it is when you do proper searches with sniffing dogs and everything and all the technology that’s available at this moment. So, the workload was so high even for customs and the inspectorate. That was just one month and it was the shortest one. It was February. And then the way to process everything till the next year. And we repeated the same. And again, you know, there are many, many cases.

Laura: And yeah, it’s amazing to me.

Daniel: Imagine that they take, you know, tiger bones and they boil them for seven days and seven nights. So, seven times 24 hours of this quite extreme heat. And then they prepare something that we call a solid broth. And they cut it. And then they use it as, you know, some traditional Chinese medicine. But, imagine that even there, there are some tiny amounts of proteins and DNA that can be used to say there is tiger inside. So, four years ago I believed that, you know, such a thing would destroy DNA. But when we did our experiments, we saw that it’s typeable. We can do it.

Laura: Talk about degraded DNA or, you know, DNA that’s challenging. But you were able to do it, and it almost seems like, correct me, I’m probably going to say this wrong, but I mean, yes, you’re kind of coming up with your own rapid solution, you know, when it’s found in a suitcase and trying to help speed that up. So, it’s effective and you can trace it back.

Daniel: We are trying to help. And I really love the interaction with guys from customs and even, you know, police forces, because I need to know what’s necessary to be done, because, as a scientist, I need input. You know, what is necessary to be done. And once I have this piece of information, then it’s much, much easier.

Laura: So, this all seems relatively new. And, you know, we talked a little bit about the logistics of it, but, what else would you like people to know about what’s happening now and how they might apply it in other areas?

Daniel: One important thing is that there are many regulations regarding, you know, biological materials. You know, shipping biological material overseas. But even from continental Europe to England, it’s very difficult and there are many, many limitations. And you must have so-called permits. And so that’s why I would like to see specialized laboratories on every continent. But I do not think it’s possible because there is this question of who will finance it. I think that this illegal wildlife trade is the third most lucrative form for different mafias and smugglers. So, I think it’s worth it to cut their money but it’s a question for our authorities. I’m really free to share the knowledge we have and everything with others because it’s very difficult to make a profit on it, because it’s not human. For tigers, there are maybe several hundred cases per year. It’s not for big pharma companies. But, yeah, for small entities, universities, it can be interesting, you know.

Laura: Yeah. I think it’s incredibly interesting.

Daniel: And, yeah, combine it with some research.

Laura: Not everything should be driven by a profit motive. I mean, there’s an ethical consideration and trying to stop this in other areas with the variety of species. Would you know if you were going to be able to expand this across different continents? How does the variety of species affect that or would it be fairly simple?

Daniel: You know, I would say that every region has its own species that are smuggled. Let’s say out of Africa, you know, ivory. That’s definitely number one. And you have a laboratory in Washington, D.C. that’s specialized on ivory. Then, regarding rhinos, it’s in South Africa. Cindy Harper’s doing an excellent research on individual identifications of rhinos, and also building a database so that you can really trace rhinos to the original, you know, farm or reserve. And surprisingly, my small laboratory in Prague is the reference laboratory for tigers and other big cats because, Vietnam started with their illegal trade with tigers because a dead tiger is much more valuable than a living one. And you can convert it to thousands and thousands of euros or dollars. And that’s why they do this is when they can make some money out of it. It’s like marijuana or, you know, drugs. So, that’s why. And we have the highest population of Vietnamese in the Czech Republic. And that’s why we have so many cases involving big cats.

Laura: It’s amazing that forensic analysis can help solve this problem and really be an integral part of it. Something we couldn’t have imagined, you know, decades ago.

Daniel: But I’ve, you know, snuck out of your question regarding those new technologies for species identification. When we talk about Sanger sequencing, I think it’s, I would say the golden standard, but not always. You know, it’s so easy, because when you have a single source (just a tiger and some medicine) it’s okay. But you can have more animals, and then it’s a problem. So, you have to use some massively parallel sequencing and it’s okay. Fortunately, we have tools we can use. But, we’ve started to research other possibilities. And we just recently published a paper on length polymorphism on the mtDNA control region. And so, it’s also suitable. And there is a similar approach called spindle. It’s a group from Portugal. So, there are also other approaches. And technologically, it’s like we would skip back to the early 90s, you know, using many satellites and etc., but with the knowledge of past technologies, we can do it. But we are also using, for instance, high resolution melting analysis and isothermal amplification. And again, we are trying to develop something that can be used in the field. So that’s what we do and how we play.

Laura: Creative approaches-I love it that you can tailor it. It certainly gets more complex as you add different, you know, species and regions. But where do you see your research going in the future? What’s next?

Daniel: It depends on research grants. Of course, because we are secured to the end of the next year. And what’s going to be next? I don’t know. But having this very valuable database of tickets, I think we will find some sort of financing and some support, even if it’s not from our government. It can be, you know, from abroad.

Laura: Well, I love that you’re able to share that here and really appreciate you sitting down with us so that the video can reach other people who may not be able to attend but would like to know more about this. I think it’s fascinating. I can’t wait to learn more about it. Is there anything that we didn’t touch on that you’d really like to make sure we include in the video?

Daniel: Actually, the list of your questions was great. So, I think we’ve covered all areas that are important.

Laura: Well, thank you so much. And how are you finding it so far? It’s our 35th anniversary, so I’m checking in with everybody.

Daniel: It’s different if I compare it to other conferences like ISFG or the European Academy of Forensic Sciences. But what I love are those small differences. For instance, you know, we’ve exhibited once at ISFG and nobody came to thank us distributors and it’s not fair because, you know, they can organize the conference because they are non-profit, out of our money in a company money. But it’s completely different, you know? Okay. It’s a perfect setup because you have posters, booths, everything, you know, food served in the same area. So, because you’re a company that knows how it should be done, you know. And also, the feedback. So, you’re trying to improve your next and next and next. So, to deliver better and better products. So that’s what I love.

Laura: Well, we are very lucky that you were willing to come and be a part of it.

Daniel: Definitely I promise I will do my best to come next year too.

Laura: That would be fantastic. I hope we get to speak again. I would love to hear what’s next and what’s happened over the last year.

Daniel: Some use cases with me.

Laura: That would be fantastic. Please do. Thank you so much, Daniel. We really appreciate it.