Avian Malaria (& Relatives) Workshop - Part 2 - Indentifying Blood Parasites

Here I am one year into my Master's program at San Francisco State University. Time has flown by so fast it's hard to believe a whole year has gone by already. But at the same time when I look at where I was at this time in 2013 I realize how much my skills and experience have progressed.

One year ago I didn't know how to handle a bird or set up a mist net - now I can handle and take a blood sample from a bird. I have a formal training course under my belt and work at a bird banding station.

One year ago I didn't have a single wildlife permit - now I have three (and IACUC approval from my school.)

One year ago I barely knew how to run a gel or do nested PCR or sequence a DNA segment - now these are integral pieces of my research project.

And one year ago I didn't know a thing about avian blood parasites. Now it's the focus of my project and I've participated in the International Workshop on Malaria and Related Haemosporidian Parasites of Wildlife.

Meet this year's "Haemosporidian Workshop" participants! pic.twitter.com/ui2YKOn5Nk

— Malaria RCN (@MalariaRCN) August 1, 2014

In a graduate program you pick up a lot of your knowledge and tips from your advisor and fellow lab mates. That being said, nothing beats an intensive, concentrated training experience. That's what the Malaria Workshop was - after four packed days of content, I felt that my knowledge about blood parasites had taken a giant leap forward. I even gained skills that I never dreamed I would have... believe it or not, I can now dissect out the salivary glands of a mosquito.

Blood Sampling and Parasite ID

The first two days of the workshop emphasized identifying parasites in blood smears under a microscope. In a past post about Avian Malaria I went into a little detail about the causative agent of malaria, Plasmodium (different species within this genus cause malaria in humans, birds, reptiles, etc.) In addition to Plasmodium there are two other genera of blood parasites within the order Haemosporidia we are concerned with: Haemoproteus and Leucocytozoon.

These three genera each have unique methods of reproduction and are spread by different insect vectors. Once you know what you're looking for, they're quite easy to tell apart under a microscope. Here's what the first two days of the workshop looked like:

First we caught some local birds:

Gray Catbird - Photo by Elin Videvall

And took some blood samples:

Strahil taking a blood sample from the brachial vein of a flycatcher - Photo by Strahil Peev

And made some blood smears - sometimes using the "Polaroid method":

Dovile blotting and drying a smear

But preferably with a fan:

Gediminas Valkiūnas fan drying blood smears - It is important to dry the slides right away because soon as they are exposed to air the parasites begin to change form. Fan drying suspends that transformation and preserves their current form.

Then we fixed the slides in methanol: 

Photo by Elin Videvall

Then we stained the slides with Giemsa stain: 

This process stains the cells purple - the nuclei (bird red blood cells have nuclei) and parasites are stained dark purple while the cytoplasm stays clear - Photo by Elin Videvall

Then we looked under our microscopes!:

Photo by Elin Videvall

We did this for several afternoons, looking at both our local bird slides and some "known positive" slides to practice our parasite ID. Our course instructors were very helpful when we inevitably got stuck:

Photo by Elin Videvall

This is what we saw:

Most of what you see here are avian red blood cells, which are nucleated. Right at the tip of the pointer is a Haemoproteus parasite. There are many many species of Haemoproteus that infect birds but they can be differentiated if you know what to look for. 

We would look for defining morphological characteristics such as:

• Location of the pigment granules (in this picture the small dark dots scattered throughout the cell)
• Location of the parasite nucleus (light pink spot in the top right corner)
• How much of the host cell the parasite takes up and what membranes it touches (in this case the whole cell and both the nucleus and outer cell membrane - this isn't always the case!)
• General size and shape 
• And more

Gediminas Valkiūnas' book has a very helpful key - we used it (as well as his direct instruction) extensively:

And this is Leucocytozoon:

Leucocytozoon parasites are much larger than the other two genera. They're the Jabba the Hutt of the parasite world:

To the untrained eye (us before this workshop) Leucocytozoon looks suspiciously like white blood cells so we had a special lecture on that as well:

Interspersed with all these parasites we also each gave a 5 minute presentation of our research projects. It was so interesting to see the different pieces of the blood parasite world that people had carved out internationally. 

Paulo gives his 5 minute presentation - Photo by Strahil Peev

And this was just the first two days! Stay tuned for the third and final installment - Vectors and Summary

The Third Annual International Workshop on Malaria and Related Haemosporidian Parasites of Wildlife - Part 1 - Facilities

This year I've have two different but equally amazing training opportunities. The first, for the bird banding portion of my master's research, you can read about in Braddock Bay Bird Observatory Spring 2014 Bander Training.

The second, for the blood parasite portion, was a workshop organized and funded by the Malaria RCN, a collaborative network of researchers around the world all working on haemosporidian blood parasites of wildlife. This July was their annual International Workshop on Malaria and Related Haemosporidian Parasites of Wildlife.

Participants in this workshop hailed from all different parts of the world. Represented countries included Lithuania, Mexico, Brazil, Sweden, Australia, India, Bulgaria, Poland, Spain, Colombia, and all corners of the United States.

I broke this update into a couple different entries because it was such a fantastic experience and the content so rich that it deserves an extended post. Between all the people I had the wonderful opportunity to meet and the beautiful campus it was hosted at, it has been a major highlight of my graduate experience so far.

The first installment will cover the gorgeous grounds and facilities. Picture heavy!

The workshop was hosted at the U.S. Fish and Wildlife Service's National Conservation Training Center (NCTC) in Shepherstown, West Virginia. The NCTC is a network of facilities located on a 533 acre property along the Potomac River.

The grounds of the campus were beautiful. Full of wildlife and so green. Coming from California, a state in a severe drought, the green was especially noticeable.  

Some wildlife more intimidating than others. Note the pencil for scale.

The facilities themselves were also beautiful. I stayed in the Rachel Carson Lodge, where every room had a copy of Silent Spring, a seminal book in the environmental conservation movement. 

The Rachel Carson Lodge.

The view surrounding the Rachel Carson Lodge - the green!

Another building we spent a lot of time in was the Commons, where all our meals were held and the location of a beer and wine bar where we would hang out after "class."

The food was another highlight of the accommodations. Each day for both lunch and dinner the entree selection would change; at the deli you could build any desired sandwich; at the grill you could order anything from a grilled cheese to a bison burger; the salad bar was stocked with fresh veggies; and the dessert station served frozen yogurt and a rotating selection of cakes and pies. I have to say as a graduate student living in the pricey Bay Area (ie having very little money) this was a really nice break from ramen noodles and peanut butter.

Stay tuned for Part 2 - Blood Parasite Identification.

Solving the Mystery of Songbird Migration

This June I had the privilege of helping out my friend and fellow lab mate, Allison Nelson, with her research project. She's studying the migration of hermit thrushes. Described by the Cornell Laboratory of Ornithology as an "unassuming bird with a lovely, melancholy song," these birds are well known for their beautiful metallic songs. Despite their popularity, the migration patterns of these birds are not well understood. Different populations with overlapping ranges travel in completely opposite directions - and we have no idea why. Allison aims to answer some of these questions.

To fund this research trip, Allison ran a successful Kickstarter campaign. Kickstarter is a crowd-funding website for creative projects where individuals donate funds at different levels in exchange for small gifts such as a picture or sound recording of a Big Basin hermit thrush.

With the money she raised through Kickstarter Allison was able to purchase 20 tiny geolocators that use light levels to determine the approximate latitude and longitude of the bird. The video below from her Kickstarter campaign describes out the aims of her study:

We spent a week in Big Basin Redwood State Park target netting hermit thrushes and harnessing them up with geolocators. Big Basin is California's oldest state park - the sites we were working in were full of lush trees and undergrowth.

The hermit thrushes here spend the summer in Big Basin and migrate south for the winter. They have high site fidelity, meaning they return to the same sites year after year. Allison color banded birds in Big Basin Park last year and we recaptured 9 of her birds from last year! That means those birds spent the summer of 2013 in Big Basin, flew all the way down to the southern United States or Mexico for winter, and then flew back to the exact same spot in Big Basin for summer of 2014.

Next year we'll return again to try to recapture the geolocator birds to download the data and see exactly where they spent the winter. Given our recapture rates from this year we're confident we'll get quite a few of the geolocators back.

We set up most nets back in the brush but in the final day when we needed to catch the last few stragglers we set up a long net lane along the path - it ended up being successful! Thrushes are ground feeders and took advantage of these walking paths - we caught five birds as they made their way onto the path.

Allison determining the age of a thrush by looking at its feathers and molt pattern.

One indicator is the shape of P10 (the 10th primary flight feather) - pointed (vs. rounded) indicates an older bird.

A geolocator and harness - the loops tucked around each leg with the geolocator extended above the feathers on the thrushs' backs. Each one weighed about a gram or less. 

Ready to go!

The Ups and Downs of Lab Work

Here I am in summer and the field season is pretty much over. I have to do a final tally but I ended up with about 50 complete (blood and audio) samples from male white-crowned sparrows around San Francisco.

I am now entering the analysis phase of my project where I process all of the blood samples I've taken and figure out if the birds are harboring any parasites. Here's a breakdown of the analysis steps that I've undertaken so far:

Collection

When blood is initially drawn from the bird in the field it is placed into a tube filled with lysis buffer. This serves to stabilize the blood at room temperature and also to lyse (break open) the red blood cells so that the DNA can be accessed. Back in the lab the tubes are put into a -80°C freezer for storage.

Extraction

Next comes the extraction phase where the DNA is separated out from the blood sample. Blood is full of many other materials including cell membranes, proteins, serum, etc. but what we're really interested in is the parasite DNA (if it's there.) 

Extraction consists of a digestion phase and a purification phase. Digestion serves to further break down the cells beyond just lysis and then purification separates out the DNA from all the extra "stuff" in the mixture. 

In Digestion we take the frozen blood sample and add it to a tube filled with digestion solution. 

Then it gets incubated at 55°C overnight. 

Allison models the incubator while I reflect. 

Purification consists of several stages of washing and centrifuging to separate DNA out of the solution. In the end we have an aqueous (in water) sample of DNA, called an elution, that is then stored at -40°C until it's next needed.  

PCR 

PCR stands for polymerase chain reaction. The process involves targeting a specific segment of DNA and making many copies of it so we can have a workable quantity for study. It was such a significant contribution to science that the inventors were awarded the 1993 Nobel Prize in Chemistry. 

In my case the segment targeted is one unique to blood parasites. Each of us in the lab has their own PCR kit consisting of all materials needed.

A PCR master mix is made up and pipetted out into individual tubes. Then the elutions from the Extraction step are added.

The tubes are then put into the thermocycler which can be programmed with specific cycles of hot and cool to unwind and copy the DNA segments.

This process usually takes several hours. The end result is a PCR product that can be run through a gel. 

Gel Electrophoresis

Gel electrophoresis is essentially the "read out" step following PCR. It tells me whether or not I have an infected bird or not.

Making up an agarose gel is likely something you did in high school biology. Agarose is dissolved in 1xTBE and distilled water then poured into a mold to set.

I'd like to point out that our very high tech gel cooker is a microwave.

After the gel is set the PCR product is pipetted into the wells along with controls to compare my samples to known outcomes. A ladder with different lengths of DNA segments is also loaded to show DNA is properly moving through the gel. In the picture below the ladder is blue. I've loaded my positive control on both tiers right next to the ladder. 

Then the gel is placed into a liquid bath and hooked up to a gel electrophoresis machine. DNA is negatively charged so when a voltage is applied the segments are attracted to the positive charge and travel through the gel.

The rule for hooking up the wires with the correct charge is "Run towards Red and Black goes in Back." You can adjust the time and voltage you run your gel with to control how far your samples travel. 

Next the gel is stained and washed then placed in an imaging machine.

Here's what it looks like when it's all said and done. This is the printout that I tape in my lab book:

I tested 18 of my samples here. Two of them are positive and the rest are negative. This is both good and bad. Good that the birds are uninfected but bad because I need at least some of them to be infected so I can compare their songs.


I have to say that even though lab work is less frustrating than it was initially I still prefer field work. Nothing beats being outdoors and working with the species you're working to conserve.