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.