cc  >> Nadia Dominguez: Thank you  for joining me  for Wednesday Nite at the Lab  at the UW-Madison  Biotechnology Center.  Tonight we welcome  Dr. Bruce Christensen,  a professor in the Department  of Pathobiological Sciences  here at UW-Madison.  Dr. Christensen received his BS  in biology from the University  of Wisconsin-River Falls.  He then earned his PhD in  medical entomology and  parasitology from Iowa State  University.  He began his tenure  at UW-Madison in 1982  as an assistant professor.  Since then, he has served on  several editorial and scientific  advisory boards and has even  been the president of the  American Society of  Parasitologists.  Currently, he is a professor in  the Department of  Pathobiological Sciences.  He researches the relationship  between the mosquito vectors and  the parasitic etiologic agents  required for the natural  transmission of vector born  diseases.  So please join me in welcoming  Dr. Bruce Christensen to  Wednesday Nite at the Lab.  [APPLAUSE]  >> Bruce Christensen: Thank you. 

 We're going to speed  right into this, okay?  I know you.  You used to do my eyes.  You visited my garden, too.  Okay.  Cool.  Right?  I think so.  Never mind.  So for those of you that don't  know, mosquitoes aren't  mosquitoes aren't mosquitoes.  There are lots of different  mosquitoes.  There are about 3,000 of them in  the world. 

 The United States doesn't have  very many, and Wisconsin even  has fewer.  But we still think of these  because we always get these  warnings about West Nile virus  or La Crosse encephalitis, and  these are indeed important  diseases, but I'm going to talk  about some things that I'll  argue might be a bit more  important, we just don't know  about them.  So a little bit about the  biology of mosquitoes.  They're all aquatic.  They are what we call  holometabolous insects, so  they're just like butterflies.  They have a larval stage, they  have a pupal stage, and an adult  stage.  And so the larvae are kind of  interesting in that they have to  have this breathing tube.  They have to stick up through  the water and get gaseous  oxygen.  That's why they used to be able  to control mosquitoes by putting  oil on the top of the water, but  we don't do that anymore because  it's not cool to put oil on top  of the water, especially in  marshlands.  And then when the pupal stage  appears, they have these  respiratory trumpets and they  still have to get their gaseous  oxygen. 

 And then the adult emerges.  And this is an Aedes aegypti  emerging out of this pupal  exuviae we call it and going to  sit on the water for a while,  dry out, tan its cuticle and fly  away and do mosquito things.  Now males are very kind and  gentle.  They're flower feeders just like  males of most species.  Females are voracious blood  feeders.  So these actually are the  disease transmitters.  And the reason females, most  species of mosquitoes, the  females blood-feed is because  they need this protein to make  eggs.  So they get the blood from the  protein.  Otherwise you can keep a female  in the laboratory for several  months just on a sugar solution.  But she'll never make eggs. 

 There are few exceptions to the  rules, but we're not interested  in those because those that  don't take a blood meal, don't  transmit diseases so I don't  have any interest in them.  I don't study mosquitoes  because I think, well I think  they're cool, but I study them  because they transmit diseases,  not for the biology themselves.  I'm interested in the biology  as this relates to disease  transmission.  And there's three broad  categories of pathogens that  mosquitoes transmit.  The viruses, or more technically  they're called arboviruses, not  viruses of trees but arboviruses  refers to viruses transmitted by  arthropods.  So this is West Nile virus and  St. Louis encephalitis  and western equine encephalitis,  eastern equine encephalitis,  all of these in the  United States.  In the world, though, it's  probably dengue virus and yellow  fever virus are the important  ones.  A disease called filariasis,  which is actually a roundworm,  you may know this from mosquito  transmitted filarial worms  because of dog heartworm.  It's the same type of nematode  parasite transmitted.  And then, of course, malaria  which is a protozoan parasite. 

 So let's talk about these three  biggies here.  These are not trivial diseases.  So we're talking about, there's  6.8 billion people in the world  and 2.5 billion of them are  at risk of malaria and about  half a billion are infected  at any time.  About a million deaths.  This varies from 800 million to  3 million depending upon who's  making the estimate.  But the sad thing is the vast  majority of these are children  under the age of five in  sub-Saharan Africa.  So think of the sort of outrage  if you were dealing with the  disease that killed a million  kids in Europe and North  America.  This is a child in Malawi and  this picture was taken by Terry  Taylor at Michigan State  University and this child is  suffering from cerebral malaria.  So they go into a condition  that's almost like lockjaw.  They get rigid. 

 Lymphatic filariasis is also not  trivial.  About 120 million people  infected.  40 million of these with severe  disease.  And this disease is oftentimes  classified as elephantiasis  because of the blockage of the  lympatics and we'll talk about  that a little bit more.  Dengue fever is not quite so  severe, but the problem is now  is that where it used to be sort  of this break bone fever that  hurt you a lot, now in most  parts of the world the  hemorrhagic form of dengue is  around so people are dying and  this is getting to be a  situation that is kind of scary.  And there will be a dengue  outbreak in the United States.  It's not a question if, it's  going to be a question of when.  So we're getting outbreaks in  Mexico and pretty close places.  This is what I'm going to spend  my time on today, though.  Because I've spent most of my  life working with this disease. 

 And I can't cover them all.  We'll touch on malaria a little  bit in Papua New Guinea because  it's a same species of mosquito  transmitting both so there are  some implications there that I  want to talk about.  So I'm going to try and, with  filariasis, give you a  background on what the disease  is because probably most of you  don't know what it is, show you  what it looks like, how it  cycles, and then we'll talk  about a global program to  eliminate lymphatic filariasis  and then how our research on,  of all things, mosquito immunity  actually plays into this.  So if you're not familiar with  DALYs, DALYs are  disability-adjusted life years,  and filariasis falls within this  category of diseases we call  neglected tropical diseases.  They're called neglected because  nobody cares about them because  they don't kill people.  But if you look at the  disability-adjusted life years,  hookworm doesn't really kill a  lot of people, but it has a  major impact in the normal  physical and mental development  of children throughout much of  the tropics.  These are really quite bastardly  diseases, but we don't hear much  about them.  I would venture to guess that  most of you haven't heard of  most of these diseases.  Some of you might have.  So it falls in that category. 

 Peter Hotez, actually, that is  sort of the advocate out of  George Washington University for  neglected tropical diseases was  just on campus a little while  ago talking about that very  subject.  He's a pretty cool guy.  So what about lymphatic  filariasis?  It's a parasitic roundworm.  It's an actual nematode.  The vast majority of cases are  caused by this parasite called  Wuchereria bancrofti, probably  95%.  But we know not quite so much  about this.  The problem with it is the only  host for this is humans.  So it's kind of hard to work  with in the laboratory.  It's hard to bring humans in.  If you want to work with the  parasite, you go to where the  parasite is. 

 Brugia malayi you can put in  gerbils or in cats and in dogs.  So you can work with that.  Brugia timori is a very small  segment of the population  infected.  But it's different than malaria.  Malaria is transmitted by an  Anopheles mosquito.  This is transmitted by a whole  bunch of different genre in  species of mosquitoes depending  on what part of the world you're  in.  Which makes it much harder to  figure out transmission dynamics  and what you need to do.  The distribution, this is truly  a tropical or subtropical  disease.  The vast majority of cases occur  in the subcontinent of India and  sub-Saharan Africa.  With focal hot spots. 

 Haiti is a hot spot.  Around Recife, Brazil, is a hot  spot.  Papua New Guinea is a huge hot  spot.  But the vast majority of these  120 million cases are in  sub-Saharan Africa and India.  Parasitologists do life cycles.  It's what you do.  The cool part about parasites  is their life cycles.  They're very complex and you  wonder how they ever evolved.  In this particular one, the  adult worms live in the  lymphatics of humans and they  give rise to living offspring,  little worms that we call  microfilariae.  And these then circulate in the  blood and in the lymph. 

 Mosquitoes then come along and  pick these up when they feed.  But it's very interesting in  that the microfilariae don't  always circulate in the  peripheral blood, the blood on  the surface, all the time.  They only do it at certain times  of the day and these times that  they do that coincides with when  a mosquito feeds.  So the mosquitoes pick this up  in a blood meal, take it into  their midgut, the worms burrow  through the midgut, crawl to the  flight muscle in the mosquito,  burrow into the cells for the  flight muscle, undergo two  molts, nematodes have to grow by  molting because they have a  cuticle on the outside, until  they reach the infective stage  or third stage larvae.  And then when the mosquito  feeds, they crawl out of the --  and actually crawl in the holes  that the mosquitoes made when  it's feeding.  So it's a very hazardous life  cycle and very inefficient life  cycle.  But the adult lives for five  years so they don't have to have  many successes in a year to  survive.  This is that periodicity.  So here we have a nocturnal  periodicity so that you only  find them in the peripheral  blood at night.  And this is with things like  Culex that are night feeding  mosquitoes. 

 Now this has a huge impact when  you're doing a survey.  Because if you're going to go  out and do a survey by  determining if somebody has  microfilaria, you have go at  night, you have to do what is  called night bleeds.  So you go around and you knock  on houses and villages at night,  wake the people up, take a blood  sample then.  In French Polynesia where you  have Aedes polynesiensis as the  vector, they're daytime feeders  and there you have a diurnal  periodicity.  So it kind of makes you believe  in evolution.  Things kind of work in a  co-evolutionary status.  So for those of you that don't  know what an internal anatomy of  a mosquito looks like, here's a  cartoon.  So they bring these parasites  in.  They go into this midgut.  Burrow through the midgut. 

 They have what they call an open  circulatory system, a body  cavity, and they crawl through  that, get into these flight  muscles, and they burrow right  intracellularly.  And then after their first molt,  the mouth opens and so then they  eat mosquito tissue.  And then the second molt, their  anus opens so then they're like  a real tube within a tube  nematode, and then they crawl  out and go up here in the  proboscis to be transmitted.  So here are some pictures.  This is a microfilaria as you  would find it in a thick smear  of a blood smear.  They have this sheath which is  actually part of the membrane or  yolk membrane.  And this is a scanning electron  micrograph we took a long time  ago.  It's like you are floating in  the body cavity of a mosquito  and looking down.  This is the mosquito midgut.  This is actually a worm that  just burrows its way right  through. 

 It's kind of a brute force  thing.  No receptors required on this  baby, it just charges through.  And these were taken by some of  my colleagues in Egypt where  we've been working for quite a  while.  And this is a first stage larva.  These are sections through the  muscle and shows this first  stage larva.  And this is a second stage  larva.  And then when a mosquito feeds,  if you haven't seen a mosquito  feed, the next time one lands on  you in the summertime, just let  it feed and watch it.  It's kind of cool.  Because this labium here of the  mosquito bends, and then these  structures here are the stylets,  and they have two tubes so they  drive these in, and one tube  squirts in saliva to keep your  blood from clotting, and the  other one sucks in blood.  So it's kind of a two-tube  arrangement. 

 Well, these worms are lying in  here and they have to actually  break out.  And now this is a silly--  Okay, here we go.  I got to do it this way.  So Sarah, my one doctoral  student, now you can see how  these worms get out.  This is the only film like this  in existence.  These are really active.  And these are pretty big.  These are almost a millimeter in  length now.  So when they're actually feeding  and these things come out,  there's actually a drop of the  mosquito's blood called  hemolymph that comes out and  they're actually in that.  And so then when they pull the  stylets out, they they crawl in  the hole that the stylets made. 

 So if this hemolymph dries up,  the parasite dies.  So transmission is terribly  inefficient.  All right, so let me see if I,  okay where do I, okay I might  need some help here.  Okay.  >> Slide show.  >> Slide show.  Where is slide show?  Up on top?  >> To the right.  >> To the right, right, right,  right, right.  Cool, okay.  Oops. 

 That didn't work.  Excuse me.  All right.  So when it gets into a human  then, there's usually what we  call a prepatency period.  It's hard to tell exactly what  it is because you can't  experimentally infect a human  and then check to see when you  see microfilaria in the blood.  But it's probably something in  the neighborhood of three to  five months, something like  that.  Probably 80 million of the  people of the 120 million  infected don't show any  symptoms.  So they look normal, yet they  can have a really good  microfilaremia.  But some of them get occlusion  of the lymphatics.  And you get occlusion of the  lymphatics, of course, you  compromise drainage of fluids,  especially out of the extremity. 

 So you get this build up in this  lymphedema which gets the name  elephantiasis.  It can be mild like this in this  lady or in this child.  It's not just an adult disease.  This is a 14-year-old boy.  And then it can get extreme.  So this is when you start to get  this term of elephantiasis  because you actually get skin  that actually has an elephant  skin texture to it.  And what we found out is that  much of this is associated  with secondary infections,  both bacterial and fungal.  Also, something I didn't show a  picture of because I wasn't sure  if I should or not, is commonly  is hydrocele in males, which is  a swollen scrotum.  And some of these, if you look  at some of the old textbooks,  the scrotum is so large that men  literally have to use a wheel  barrow to carry their scrotum  around because it can almost  touch the ground.  So there's some old Japanese  woodcut when filariasis used to  be there, and I've got a copy of  one in my office where,  actually, the man with the  hydrocele was in the back and  they had a sling to a man in  front and then that carried his  scrotum so he could walk through  the village. 

 And that is absolutely true.  It's not exaggeration at all.  The burden of lymphatic  filariasis, to me, is quite sad.  That the morbidity is very  serious with these  disability-adjusted light years.  The socionomic losses are, well  estimates for India is a billion  dollars, it's probably more than  that.  But it's a psychosocial and  psychosexual sort of lost  life opportunities.  So this woman is never going  to get married.  Some children in India, it was  just like the old leper times  when the family would lock the  child in the back room and not  bring him out because it's  embarrassing.  So it's a nasty disease.  And needs to be dealt with. 

 And there is a way to deal with  it.  It's called the Global Program  to Eliminate Lymphatic  Filariasis.  And I'm going to talk about  successes and some nonsuccesses  but how it works.  There are two principal goals.  And the first one is to  interrupt transmission.  We don't have any drugs that  will kill the adults in the  lymphatics but we do have some  drugs that, given once a year,  will knock down microfilaria and  keep these suppressed for close  to a year.  So the strategy is you're going  to give these drugs for at least  five years.  And your strategy is not to just  give it to infected people.  But if you have a village where  somebody is infected, it's much  cheaper to just go into a  village and give everybody  drugs.  So that's the strategy it's  called mass drug administration. 

 You use either, I don't know if  you recognize these.  How many of you have dogs?  Any of you have dogs?  So ivermectin is heart guard,  that's what you give once a  month, when you give to prevent  heartworm infections.  Diethylcarbamazine is what you  used to give in flarabits that  you give every day.  We use two different drugs  because if you're dealing in an  area that also has a parasite  you know of, Onchocerca, which  causes river blindness, you  can't use diethylcarbamazine  because it's contraindicated.  You can really make people  terribly sick and kill them if  you give DEC if they're also  infected with Onchocerca or if  they're infected with a parasite  that's really cool called the  African eye worm.  So in areas where I work in  Egypt and in Papua New Guinea,  we use diethylcarbamazine,  it's probably better.  So we kill the microfilariae,  keep them down.  If you do this for five years,  basically interrupt  transmission, by that time the  adults die, you have no more  disease and you're happy campers  and you go home.  The other part of the program  is to alleviate suffering.  And what they've found out is  you can actually do a lot of  relief by treating this  lymphedema as you treat any  lymphedema: elevation and, in  this case, doing some very  thorough cleaning with  antibacterial and antifungal  compounds and you can make a  huge difference in the  well-being. 

 So what progress have we made in  this?  Well, what's really cool about  this is we can complain about  drug companies, but once in a  while drug companies show some  altruistic behavior that's a  little bit perplexing at times.  And they started out there was a  gentleman we brought in that got  an honorary degree at Wisconsin  by the name of Bill Campbell  who worked with Merck.  And he's the one that really was  instrumental in determining what  ivermectin does.  And he got the idea because it  kills horse Onchocerca that it  would kill filarial worms of  people too.  And he convinced Merck that they  should do this.  And the guy that he convinced,  his boss, new full well that if  they did this, they knew there'd  be no profit.  These people don't have any  money to pay for a drug.  This is a true orphan drug  program, if you're going to  define an orphan drug program.  But, fortunately for him, it did  work and the person that was his  boss became the CEO of Merck and  they went to the stockholders  and they said cool.  So to date now in both the  control for river blindness and  lymphatic filariasis, Merck has  probably given away market value  of close to a billion dollars  worth of ivermectin. 

 And then GlaxoSmithKline is  giving away all of the  Albendazole.  And if you combine Albendazole  with these others, you may have  some anti-adulticide activity,  plus you're killing two birds  with one stone because the  Albendazole kills all the  gastrointestinal parasites like  hookworms and whipworms and  roundworms that have such a  devastating effect on kids.  So you really do a lot of good,  as long as you're there giving a  drug, give them the other drug  too.  The other thing that was  developed by Gary Wihl at Wash  University was an antigen  detection kit for diagnosis so  you don't have to go at night.  It's fun to go at night.  On the Nile Delta the people are  so friendly.  But the problem is getting  anything done because you wake  them up and it's 11:00 and they  invite you in but then they want  to make tea.  And then they have to make tea,  and then you have to drink tea.  So it's like an hour before you  get out, and you say, I can't do  this.  But you don't want to upset them  by not taking their offer of  tea. 

 So this makes it nice.  You can go through in the day  and just do a finger prick, and  you put it on a card, positive.  The only ones you have to go  back and bleed to see if the  have microfilaria are those that  are positive on the card test,  which might only be one and a  half percent of your population  of 10,000 people.  So that's a lot better than  doing all the bleeds.  And then the effective  management of clinical diseases.  So when you're in the field, you  don't weigh people, that's kind  of hard to do.  So oftentimes you use a stick.  And this is marked with one and  two and three.  That means for this person  you're going to give them two  tablets of ivermectin.  So it's a nice easy way that you  can train anybody to work for  the ministry of health to go out  and give these things. 

 This is Dr. Maggot, isn't that a  wonderful name for my Egyptian  colleague, doing a night bleed  because of this.  But this is an antigen detection  kit that was developed by  Gary Wihl at Wash U  in St. Louis.  So this is very nice.  You get two bands,  it's a positive;  one band, it's negative.  It's great.  Results in 10 minutes.  And a Brazilian medical doctor,  Dr. Gerusa, is the one that  really turned the whole world on  to the fact that you can make  a big difference in the health  and well-being of these people  just by thorough cleaning  of these things and  killing the bacteria and fungi.  And you can have a huge impact  in terms of reducing this  deformation.  So really cool things that have  happened.  The other thing that has  happened is that we developed  molecular techniques to diagnose  infections in mosquitoes. 

 And the reason you have to do  this, you can dissect an  individual mosquito to determine  if it's infected, but when you  have a control program going on,  you keep reducing the prevalence  of infection.  So to determine if you have  transmission, you have to look  at more and more mosquitoes.  So when you get to the point  where you have to look at 50,000  or 100,000 or 200,000 mosquitoes  to find out, you can't dissect  that many.  So what you do is you actually  pool them, collect them, pool  them and then we use a molecular  technique where we amplify DNA  that's specific for that  parasite.  And we can detect a single  infected mosquito in a pool of  50 mosquitoes, and it's pretty  cool.  It has limitations, but it's  pretty cool.  So here's trapping.  This is actually some work we're  doing in upper Egypt near the  Sudan border.  So this is a trap where you're  actually collecting ovapositing  mosquitoes.  You put water in there and then  you put a hay infusion that  attracts them so they want to  lay their eggs. 

 Then when they go to leave,  they've got this fan that sucks  them right up in there.  And then most of the time you  set a CDC light trap here, and  that's baited with dry ice.  CO2 is the attractant for  mosquitoes.  And this is one of my doctoral  students, Sarah Erickson,  sucking mosquitoes out of this  bag so that we can do analysis.  And she's the one that really  did the work that showed that  the diagnosis of DNA for  parasites in mosquitoes is good  to see if they've ingested a  parasite, but it doesn't tell  you anything about whether or  not they're transmitting because  even a mosquito that ingests a  parasite and then kills all of  the parasites will still show  positive because of that  residual DNA.  So she did some cool stuff.  So where is this working?  Well, I think it's going to work  really well in Egypt.  Now you look at Egypt and how  many of you have been to Egypt  and seen the pyramids?  This looks really nice but you  know all this out here, that's  smog because the city of Cairo  comes right up to the pyramids.  It's never as pristine as what  you think in the tourist books.  But it's sand so you think,  mosquitoes?  But you've got the delta. 

 And since they built the Aswan  Dam so you don't have flooding  and then droughts, you have  water all the time, you actually  increase the prevalence of  lymphatic filariasis, since the  Russians built that in the '60s,  to levels that were never seen  before that.  Not just lymphatic filariasis,  but also snail transmitted  disease called schistosomiasis  or blood flukes.  Why do I think it's going to  work in Egypt?  Because the Nile Delta is like  an island.  It's like controlling on an  island.  So here's the delta.  No mosquitoes live here, here,  here.  They sure don't live in the  Mediterranean.  You basically have this isolated  area plus the prevalence of  infection is quite low.  If we can't control it there,  it's not going to work any place  in the world.  But I think it's going to work  there. 

 The mosquito that transmits it  in Egypt is Culex pipiens.  This is the same species as we  have here that's called the  northern house mosquito.  But our mosquito likes to feed  on birds, their mosquito likes  to feed on mammals.  But it's the same species but  very different biology because  they've been separated for a  while.  Culex pipiens and another  species are really good urban  vectors of lymphatic filariasis  because they're sewage breeders.  They like this.  This is a canal.  This is where they put all of  their sewage.  All of their garbage, everything  else goes here.  And this is teaming with  mosquitoes. 

 You go down and collect and  you've got gas bubbles going up.  You have a dead cat laying  there, a dead goat there, and  these mosquitoes are just thick  all over.  So in urban areas in the  developing countries, the water  you have to breed in as a  mosquito is basically sewage  water.  The open water is where you  collect sewage.  They don't have the nice sewage  treatment plants.  They do breed out in these more  rural areas.  But they really like this.  This is actually a sewage pit  that you pull a rock off and you  lower a dipper down in there.  It collects the sewage from all  the village huts.  There's no sewage treatment. 

 And that's teaming with  mosquitoes too.  This is Gary Weil.  Now you talk about people  getting along.  Gary Weil is at Jewish hospital.  He's a staunch Jewish gentleman  that does most all of his work  with Muslim people in Egypt and  we have a grand time.  I think the world could get  along if you just kind of dealt  in diseases and health.  I'll quit preaching, sorry.  So here he's giving the drugs.  You actually make sure and give  the drugs to each individual to  make sure that they take them.  Because it's hard sometimes  because sometimes they don't  feel good after taking the drug,  and if they're showing no  symptoms of the infection, it's  kind of hard to get compliance  at a level you have to have  compliance to make this  elimination program work. 

 And that's what might goof it up  in Egypt is compliance.  You probably have to have at  least 85% compliance within a  community to have this work.  Okay, so there's been progress  but what are the uncertainties?  Will this strategy interrupt  transmission in all endemic  areas?  And we're going to take you to a  different endemic area.  Is it possible to really define  and quantify what we call  epidemiologically valid end  points?  You got to know when to stop.  There's limited resources in  these countries, how do you keep  doing a control program?  You want to know when you can  stop because they need resources  for so many different things.  Duration of treatment and drug  coverage that you need.  We don't know in certain areas.  What are some of the guidelines?  We're not concerned about these  because I don't do this, we're  concerned about them but this is  what I'm going to talk about  because these are defined a lot  by the vector.  So this is what we'll talk a  little bit about.  So there are two major  thresholds. 

 If you stop and think about it.  It's not rocket science.  It's the threshold as far as  whether or not a vector is  transmitting the parasite to a  human, mosquito to a human, or  from the human to the mosquito.  So if you disrupt either of  these, underrun either of these,  you're going to eliminate  transmission and no longer have  disease.  So how do you define these  end points?  To do this, you really have to  understand local filarial  parasite, mosquito host  infection processes to be able  to do this.  You have to be able to build  really effective and accurate  mathematical models that tell  you, okay, we can stop now and  lymphatic filariasis will never  come back.  You don't want to do what  happened in India with malaria  control.  They had malaria control so that  they only had a couple thousand  cases a year.  And then they stopped.  They had to use their resources  for other things. 

 I think much of it went towards  nuclear armament because  Pakistan had that so they had to  put money into it.  And now they have malaria  problems much worse than what  they ever had been they even  started the control program.  So you want to make sure if  you're going to do this, you're  going to do it right.  And this can be highly variable  in different endemic areas,  because of the diversity  of vectors, difference in  facilitation and limitation.  I'll talk a little bit about  facilitation.  And what we term vector  competence, how well is the  association between the parasite   and the mosquito that's  transmitting that parasite?  Is it is good association  or bad association?  That's vector competence.  And this is why.  This is just a little sampling.  I'm not going to give you a quiz  afterwards, you don't have to  remember these.  These are all distinct and  different mosquitoes that are  responsible for the transmission  of different filarial worm  parasites in different parts of  the country. 

 Actually, quinquefaciatis,  transmitting  Wuchereria bancrofti in Brazil  is quite different than  the one transmitting in India,  is quite different than the one  transmitting in Haiti,  is quite different than the one  transmitting in rural areas  of sub-Saharan Africa.  So they're very different.  So how do you, you're basically  faced with a situation of having  to define all of these different  entities.  So mosquito vector competence is  really governed by sort of the  genetic factors of the mosquito  and the parasite and how these  interact.  So whether they're going to  transmitted or not transmitted.  You've got to realize that the  vast majority of those 3,000  mosquito species transmit  nothing.  They're not disease vectors.  Why is that?  That's kind of an important  question.  We do know that the immune  response of mosquitoes plays a  major, major role in resistance.  So this may seem strange to you,  but I've made my reputation, for  the most part, being what we  call a mosquito immunologist. 

 It's not that I study our immune  response to a mosquito bite,  but I study how a mosquito  actually responds to a foreign  entity that it ingests.  Now they don't have what we  would call an immunologic memory  or anything like that, but they  have a very good innate immune  response.  They can recognize foreignness  with a fair degree of  specificity and oftentimes kill  it.  Then these are other things that  are little pie in the sky that I  don't believe too much in.  And you may have read some like  can you engineer mosquitoes to  be resistant and release them  into the wild to displace  natural populations?  And I think there's lots of  problems with that.  Most of them dealing with the  genetic heterogeneity of the  parasite that people tend to  ignore.  So this is what I do.  And I'm not going to bore you  with it but I'm going to tell  you a little bit about it.  I can't explain why it's  important.  So what is innate immunity in  mosquitoes?  Well sometimes it's simple  things like the cuticle of a  mosquito keeps something from  going in. 

 You have an integument on the  gut that functions as a barrier.  Just like our gut functions as a  barrier for pathogens.  But they do a lot of things that  mammals do.  They have phagocytosis and  encapsulation.  We call these hemocytes, but you  could replace hemocytes with a  white blood cell or a  macrophage.  It's kind of the same thing.  They serve the same purpose.  They produce the reactive  intermediates of oxygen and  nitrogen just like we do to kill  a pathogen.  They have lots of proteolytic  cascades that we do.  One of them leads to something  that we'll talk about that you  don't have happening in mammals. 

 You synthesize antimicrobial  peptides which, of course,  vertebrates do.  Lots of stress associated  proteins, which we do.  And then opsonization  and iron sequestation,  molecules, all of these things  we do as or initial response  against pathogen and so do  mosquitoes.  I study this.  If I'm famous for anything it's  like we know more about  melanotic encapsulation of  mosquitoes against parasites  than anybody in the world.  Myself and CC Chen, who you'll  see a picture of.  He's in Taipei, Taiwan.  Much of our work in the lab has  involved this mosquito called  Armigeres subalbatus.  It's a natural vector for this  Brugia pahangi parasite, which  is a parasite of cats and dogs.  It's not a human parasite. 

 Although the old editors suggest  the Brits actually infected some  people with it and got it to  take.  We don't do that anymore.  But what makes this very  interesting is that if you feed  this mosquito a blood meal that  contains microfilaria Brugia  pahangi, they develop just fine.  You take this very closely  related human pathogen,  Brugia malayi, and feed it,  it kills all of them.  In fact, these are so hard to  tell the microfilaria between  these two species the Japanese  used to determine by feeding the  blood of an Armigeres, and if it  developed they knew it was  pahangi, if it didn't develop,  they knew it was malayi.  So this is a really nice model  system to study how a mosquito  kills something.  Because you have this nice  control.  So our experimental animal is a  mosquito, albeit a small  experimental animal, but we do  things with this just like other  people do with mice, and we  don't have to worry about animal  care committees too much.  They don't care if we abuse a  mosquito.  So we actually anesthetize a  mosquito. 

 This is a bent 19 gauge syringe  needle.  So a hypodermic needle that we  have a vacuum on so we can  anesthetize a mosquito.  Hold it on a vacuum, inoculate  things into it, take things out  of it and they'll survive.  They survive just as well if  they're not inoculated.  So we've gotten pretty good at  this.  We can bleed them, get mosquito  blood, look at it, do all these  things.  We just have to practice more  before we're able to do it.  So my students and postdocs have  to be a little more patient.  So this is what the response  looks like.  This is a section  of a mosquito. 

 This is actually the salivary  glands.  This is where Mr. Malaria or  Mr. Arbovirus would be real  happy.  But here's one of these  microfilaria, and you can see  here the cells.  If we dissect this out, here's  the worm, here are all these  hemocytes or immune reactive  cells, and this material that's  building up around it is a  combination of the pigment  melanin probably -- with  proteins.  Very similar to the way a  mosquito tans its cuticle  or an insect.  I think they also tan their eggs  the same way.  So I think what they've done is  taken advantage of a  biochemistry that they use for  all different kinds of  physiology to use it for  defense.  Again, kind of cool evolution.  So this is just some electron  micrographs that shows the worm.  And this is a great big  granulocyte, one of these  hemocytes. 

 This is some melanin.  And this is one that's been  totally killed.  All of this dark pigment,  all the cellular remains  have encapsulated.  So pretty efficient means of  killing these parasites.  And so we've done all of this.  So basically we're responsible  for determining all of this  biochemical pathways.  Cloning all the genes.  Looking at gene regulation.  All of this and we always come  back to the same thing,  like so what.  So what does this in the  laboratory really mean in the  real world of disease  transmission?  So specifically, for our  laboratory, do mosquito parasite  interactions and melanization  immune responses influence  the transmission dynamics of  filarial worms in endemic areas?  Which is a terribly important  question if you're going to try  and determine when you're going  to stop these control programs  or how you're going to measure. 

 So that takes us to a  biologist's delight.  Which is Papua New Guinea.  And this is really a phone call  from a vector biologist there by  the name of Moses Bockarie we  talked to.  So my friend CC Chen and I have  gone there.  Now I'm not trying to insult  your intelligence here as far as  where Papua New Guinea is,  but before the first time I went  I called M&I Bank, because they  have an international monetary  thing there.  And I said, is it possible for  me to buy some kinas because I'd  like to have some when I go to  that country so I could buy my  visa, and she told me, no, sir,  we don't sell any African money.  So I thought I should probably  point out that it's a long, long  ways from Africa.  So this is where it is.  It's a fascinating, fascinating  country.  And you may know some about it  but I've learned more since  being there. 

 There's an enormous diversity in  geography, ecology and human  biology because we have this  equatorial type thing that has  these huge mountains  and then these coastal areas.  It just kind of comes up  like this.  You have cool areas up here.  You have real tropical areas  there.  The human population  is only about 5.1 million.  And they hold about 14%  of the languages of the world.  So you have, can you imagine  that over 850 different  languages in a population  of 5.1 million?  So you can understand how  difficult it might be to  establish some national  healthcare policies.  It's hard anyway because of  transportation, but then the  language difficulties  can make it difficult.  But it needs it.  It needs a lot of help. 

 This is by far the worst.  So think a population of  5.1 million, which is probably  about the same of Wisconsin,  maybe a little bit more.  So think if we were losing  30,000 children each year  under the age of 5.  Probably wouldn't be very  accepted.  Life expectancy is 25 years less  than its neighbor Australia.  Probably the major healthcare  problems are pneumonia and  malaria.  HIV is ramping up.  They did a smart thing in 1968.  They have such limited resources  they established this  Papua New Guinea Institute  of Medical Research.  And the whole purpose of this is  to try and do at least limited  studies to know where it might  be best to put their limited  resources. 

 Plus, they were smart in that  they get people like the Welcome  Foundation and the European  Union and the National  Institutes of Health to  contribute money for researchers  such as me to go there and do  stuff.  So they really, most of the  money to run the institute  is not Papua New Guinean money,  most of it is European and  United States and Canadian  money.  So what is the filariasis  in malaria?  I'm going to cover a little bit  of malaria here.  You've got about a million  people infected and three  million people at risk of  lymphatic filariasis, in other  words, 20% of the population  is infected with lymphatic  filariasis.  Every species of malaria,  malaria is caused by a parasite  called plasmodium, and there are  four distinct species, maybe  five but we won't count that  one.  And the important one is  falciparum because it kills  people.  Vivax and malaria are the ones  that you relapse on.  Usually they don't kill you.  You might want to die,  but they usually don't.  Ovale is terribly rare,  rarely found. 

 But in parts of  Papua New Guinea you can find  six percent of the population  infected with ovale.  So there's like malaria  going all over.  Mostly in the lowland regions  but other places.  All of these parasites are  transmitted by a complex of  mosquito within the Anopheles  punctulatus and  Anopheles farauti group.  And there's lots of different  sibling species.  The only way you can tell them  apart is by molecular  diagnostics.  So you actually look for some  difference in sequence between  that.  But they have different  biologies.  So here for example,  this is my good friend,  one of my best friends, CC Chen,  from Taipei, Taiwan.  He's at National Yang-Ming  University. 

 So here's Anopheles punctulatus.  We can collect it in these ponds  here.  I can guarantee this water must  be 90 degrees to 100 degrees  Fahrenheit.  It's almost hot to put your hand  in it.  And there and there.  But it you go for farauti, which  is a another major vector, you  only find that in these cool  pools that are in brackish  waters very near the coast.  So even though they both  transmit the same parasites,  their biology is very  different so you have to look at  them very different and  separately if you're going to  understand transmission  dynamics.  So if we look at transmission  dynamics, we're really looking  at the parasite interactions,  and in Papua New Guinea  it's one we call facilitation.  In other words, it's a pretty  simple concept.  As you get more microfilariae in  the blood, you tend to have more  parasites develop in the  mosquito. 

 But what happens in Papua New  Guinea is that when you get to  higher levels, all of the sudden  you get lower numbers.  And it's really strange.  So there's a reduction in the  intensity in the mosquitoes.  And Sarah got me these graphs  from a paper in 2006.  So normally Anopheles does this  which is like more microfilariae  in the circulating blood, more  parasites are going to develop  in the mosquito that feeds on  that blood.  The one where you always have  facilitation or limitation is in  this Culex mosquitoes where as  you get higher and higher  numbers, you tended to reach a  plateau and you never got more  parasites in the mosquito.  What's happening in Papua New  Guinea is many of the Anopheles    behave this way.  So it's really weird.  So that's why we went there,  is to figure out why.  So they have a lot in certain  villages. 

 You can have as many as 98% of  the people in a village infected  with lymphatic filarial worms  with Wuchereria bancrofti.  And in many cases, if they have  a high microfilaremia, this will  be correlated with lymphedema.  And this is correlated with  transmission intensity.  The transmission potential in  some areas is phenomenally  large.  Think of this, living in an area  some people are exposed to 2,300  infected stage larvae each year.  But what's amazing  is it should be more.  If you sit and look at this it  should actually be more.  So the question is, why not?  So we went there to do this.  We were talking before the talk  about the roads and this  gentleman here, who has traveled  in Papua New Guinea, and I think  you said you were on the road to  Goroka in this area.  This is basically the highway  system of Papua New Guinea. 

 So you fly into Port Moresby,  and then you stay in your hotel  there until you get to the  airport to fly out.  And then you feel pretty safe.  You don't wander the streets of  the Port Moresby for lots of  reasons.  So what we did in two places we  work.  One is in Madang because the  Papua New Guinea Institute of  Medical Research has their  research lab in vector biology  there.  But where they have a field  station is, the first time we  went and where we went a couple  times to collect, is we would  fly to Wewak and then drive to  Maprik, and out at Maprik is  where they have a field station.  Driving is, you were asking  about the roads, well this is  the condition of the roads  during the dry season.  In the wet season it's kind of  bad but it's okay.  You just bounce a lot.  It's worth it for the scenery. 

 The scenery is spectacular.  It's primarily rain forest  except where they're burning  it off to grow some bananas.  The research facilities are  quite nice.  They have good vehicles.  This is their research lab  in Maprik.  They have nice living facilities  that don't cost us anything.  We have to cook our own food.  So you buy it all in Wewak,  to carry there, because there's  no place really to buy it  much in Maprik.  And, fortunately, when you go  with CC, they have a wok there  and if he has a wok,  he can cook anything.  And Bruce doesn't cook,  but CC does. 

 I buy food, he cooks,  we get along grand.  When we go to the field and we  need to work with the people of  the villages, we usually stop at  a little stand.  This is true subsistence sort of  living.  There's not grocery stores where  they get commercial sort of  canned goods.  So normally you can stop and buy  bananas and banana leaves and  some cigarettes, and then these  items here are called  the betel nut palm.  This is betel nut that they like  to chew.  And they do the same thing in  Vietnam.  They do the same thing in  Taiwan.  It's a psychoactive alkaloid.  And oftentimes what they do  is they dip it in lime. 

 Not citrus lime  but the powdered lime.  It enhances the psychoactive  effectiveness.  And you can tell people that  chew it because their teeth get  stained red.  And everybody chews it, even the  children chew it.  So here you can see the powder  from the lime on this little  girl.  And this is our research  facility, and I show this  because these are betel nut  palms right by the research  facility.  Now the government is trying to  keep people from chewing this  because it's kind of like  chewing snus and what not.  It can cause some oral cancers  but everybody does it.  So if you want people to be  happy when you go into the  villages, the one thing you can  bring them that they love is you  can bring them several kina  worth of betel nuts and you give  it to them.  And they greet you very happily. 

 So they're all going to get  their betel nuts.  We give them to Henry,  Henry takes his little stash for  himself, and then he distributes  it, supposedly, equitably to the  village.  So this is a village complex.  We have to hike back to some of  these villages.  The people are extremely happy  and very, very nice.  Malnutrition is very apparent.  It's pretty obvious that some of  the children are having some  severe problems, but they're  happy people and they're very  cooperative people.  And they keep a very nice  village.  And so what we do in these  villages is we actually collect  resting mosquitoes in the  morning.  And we cheat. 

 In this case, we went into those  houses to collect mosquitoes  that we knew at least one person  had an active microfilaremia  of lymphatic filariasis.  This kind of defines, as the  press secretary for the Premier  Grand Chief of Papua New Guinea  who happens to be his daughter,  we had dinner with her, and  she said Papua New Guinea runs  economically on three things:  land, pigs and women,  not necessarily in that order.  That's sort of the bartering  that goes on between various  tribes.  Pigs are terribly important  to all of the villages.  So this is Henry.  When we finish up it's usually  pretty dark, and CC and  yours truly, I still have  my Brewers hat.  So in the morning then we  collect all our mosquitoes,  bring them back to the lab, and  then it's just really basic  biology.  We just dissect them and we see  what's happening to those that  are infected.  Henry is a marvelous dissector,  as well as a great driver.  Speaks very good English. 

 Of all the countries I've gone  to, I think the people  in Papua New Guinea have  a better grasp of English  than any other country.  And it's probably because of the  influence that Australia has  had.  So the one foreign language most  of the people that speak another  language is English.  You can always find somebody  that speaks a bit of English.  So we come back to the lab and  spend hours and hours and hours  dissecting mosquitoes.  And what we've found is actually  these very good vectors of this  parasite actually do melanize  these parasites.  And this just shows an example  of the type of thing that you  can find.  And much greater than what we  had ever thought.  So we dissected 418  Anopheles punctulatus,  and 72 of these were infected.  So you know we cheated and went  into an area and collected  these from some place where  there were infected people. 

 And most of the time what  happens is they rest inside  after they feed on a person,  they go and rest inside the  house.  So once a mosquito gets a big  blood meal, it doesn't want to  go flying away.  How am I doing?  Okay, I'll hurry.  I'm about done.  So we recovered 242 worms and  about 50 of the mosquitoes that  we dissected harbored melanized  worms, and 22% of the entire  population of worms were killed.  And 20%, basically, of the  mosquitoes killed them all.  So any of these that would have  been diagnosed by molecular  techniques would have shown up  as a positive mosquito, but yet  they have nothing to do with  transmission.  Now there are all kinds of  questions that were raised from  this, and fortunately having  these data the Fogarty at the  NIH also thought they were  really good questions so they  just funded us for three years  of work answering some of these  questions.  We know that this has a huge  impact on trying to determine  elimination thresholds, but our  question is, what about all of  the other members of this  Anopheles punctulatus complex?  To test these we really have to  develop a situation where we can  get blood from people, put it in  a membrane feeder and feed them  so we have a known exposure.  The big question that a lot of  people have is because these  mosquitoes oftentimes transmit  both parasites, both malaria and  filarial worms, and many, many  of the people are infected with  both. 

 So if a mosquito comes and feeds  on somebody, they're picking up  filarial worms, plus they're  picking up a malaria parasite.  Well the filarial worm goes  through the midgut within an  hour or two.  The malaria parasite sits in the  midgut and has sex and does some  other things and it's about 24  or 36 hours before it goes  through.  So the big question is, when you  activate the immune system by  this transmission of the worm  through the midgut, does this  then activate the immune system  such that it can actually have a  negative impact on malaria?  So the question is, if you can  control lymphatic filariasis,  could you actually make  transmission of malaria worse?  And that's a very serious  question that has to be  addressed in a lot of different  areas.  And then is it density dependent  and other things, and  other things, and other things.  So lots of stuff to do.  So, anyway, these are people  that work in my lab.  These are the people,  both close, Shelly Michalski at  Oshkosh, far away in Taiwan and  Papua New Guinea and Cairo.  And the National Institutes  of Health gave me enough money  so that I can survive.  And now thank you. 

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