Anna Stedman

15
Sep
2010

In the Lab

The week was spent wrapping up my tick project and sending off the Borrelia and Ehrlichia canis positive samples for sequencing.

Our Adventures

This week we were under threat from Hurricane Earl. However, it was downgraded to a tropical storm before it hit the coast of North Carolina… What a disappointment we didn’t get to experience it!

This week-end is called Labour Day week-end with Monday as a bank holiday. It marks the end of summer and everyone goes to the beach or mountains so we decided to beat the traffic and stay at home by the pool near Hannah’s  house for a relaxing few days.

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09
Sep
2010

The sequencing of the ITS gene for the bobcat showed no match to the river otter Babesia which is slightly confusing and difficult to interpret.

Samples from 2 tigers arrived to be tested for Cytauxzoon felis which were negative.

An easier way to test wild animals like bobcats for Cytauxzoon felis, without having to extract the blood which is an invasive procedure and requires sedating the animal, would be to test the faeces.  So I extracted the DNA from poo samples from a cat called Garfield that is chronically infected with Cytauxzoon felis!  If this worked it would revolutionize testing for zoo animals etc. Unfortunately it didn’t but ‘nothing ventured nothing gained’ and even negative results are important where research is concerned.

The sequencing for my tick project returned with 2 positive mother ticks and 5 unrelated tick egg samples for Anaplasma platys. Also 2 different mother ticks were positive for Bartonella.

Our Adventures

We attended a Durham Bulls baseball game where Hannah got a ball to keep as a souvenir. The following morning we did a radio interview with BBC Surrey. They had wanted to do it live which would have meant getting up at 3 am so as it could be broadcasted at breakfast time, however we reached a compromise and recorded it instead at 7am!

This weekend we hired a car again and drove to Wrightsville Beach which is a typical Baywatch beach!

We also saw 3 movies at the ‘Buck 50’ cinema.

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09
Sep
2010

The sequencing for the Babesia PCR product from the bobcat came back from the lab and was a 99% match to a Babesia found previously in a river otter, which is really interesting. To confirm that this is the same species we need to amplify and sequence a different gene from the Babesia parasite in the bobcat sample. To do this we need to order new primers to amplify the ITS gene using a PCR reaction.

Having completed the testing for Bartonella in my ticks and their eggs and sent the PCR amplicons off for sequencing I thought that my mission was complete, only to discover that the ticks were now to be tested for Anaplasma platys and Borrelia and if there are enough DNA samples left to also test for Erhlichia canis.

I have also been given another little project by Dr Breitschwerdt which involves researching into the ‘Blandford fly’. A patient who tested positive for Bartonella reported being bitten by what could be this fly and all the recent media attention in England has highlighted interest in testing this fly for Bartonella.  Its habitat is mainly on the river Stour in Dorset where the local council treats the river banks each year with a selective pesticide as it is an extreme nuisance with many cases being hospitalized following a bite. My search for samples has put me in collaboration with a Professor from the Natural History Museum….

Our Adventures

We hired a car for the weekend and drove to Statesville to see a real country rodeo! It was about a 2 hour drive away into the countryside where there seemed to be a church in every corner. The rodeo was full of local cowboys & cowgirls with the appropriate attire, Stetsons, coloured shirts and boots. Before the rodeo we all had to stand for their national anthem with our hands on our heart, whilst the American flag was brought out by someone riding a horse.

The rodeo then began and we saw various events such as calf roping, barrel racing and bull riding. The bull riding was really good and the cowboys have to be brave and strong as the bull turned on them sometimes, once they had fallen off. In between the events a clown entertained us with Obama and Oprah Winfrey jokes! They also played over the speakers what the announcer described as the redneck anthem ‘sweet home Alabama’. 

Then on Sunday whilst we still had the car we went to a local lake called Lake Wheeler. Anna rows for university team so we went to see if there was anyone she could talk to about going out on the lake and rowing with some people at NC State. We then took a boat out for an hour and Anna taught me how to row.

We also went to the cinema again and saw ‘Switch’ which features Jennifer Anniston. It was really good and the cinema was very full as the movie has just come out.

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19
Aug
2010

The start of something new …

More samples from the bobcat arrived so we tried the PCR again with samples from the liver, spleen and lymph nodes. However, very little DNA was amplified which was a little disappointing. After some thought about things that could be inhibiting our PCR reactions, we decided to measure the DNA concentrations of our samples using a ‘Nanodrop.’

We discovered that the samples were 5 X more concentrated than our controls so we adjusted the PCR reaction mixes to dilute our samples to roughly the same as our controls. The PCR then successfully amplified the target DNA and from further analysis we obtained a preliminary result suggesting the presence of Babesia gibsoni in the liver sample. This is an exciting discovery as Babesia is unknown in bobcats; we must now sequence the PCR product to confirm any findings…however. I eagerly await the results.

I also started my project this week which is to determine if transmission of Bartonella in brown dog ticks can occur maternally. 200 DNA samples from ticks and their eggs arrived from Italy and I ran PCRs on all samples, I will then carry out gel electrophoresis and send off the positive samples for sequencing. Out of 200 so far I have preliminary positives for 3 mothers and 2 eggs. However, the eggs are not from infected mothers!

Our Adventures

Last Sunday I cycled over to Hannah’s and then we went shopping and discovered the ‘Cheese cake factory’ which is a great restaurant that specializes in cheesecakes….not to be missed! Afterwards we caught a movie, Toy story 3.

This week Hannah’s parents and brother arrived and after receiving a tour of the vet college they flew to Washington D.C. on a very small airplane, 36 seater, for a long week-end to go and see the president (well their hotel apparently over looks the White House!) so this week I am writing alone.

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19
Aug
2010

This week I optimised the PCR protocol for my baboon samples to determine the best temperature conditions and number of cycles. This is done using a gradient of different temperatures using Real Time PCR. Once the procotol was optimised we tried varying combinations of primers to achieve a specific test for baboon Babesia.

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This week has flown by! A DNA sample came in from another lab which was from a dead bobcat and we were asked to find out if it was infected with a particular species of babesia called coco.  We used specific PCR primers to amplify babesia DNA. However, the sample we received was small and contained poor quality DNA so another sample has been requested.

5 more samples came in from dogs possibly infected with Babesia conradae which is a relatively new species. First, we were able to view blood smears and see the microorganisms under the microscope which was really cool as not a lot of people have been able to do this for this particular species!  We also ran PCRs on the samples and obtained a preliminary positive result for Babesia conradae. To confirm this result will require sequencing of the PCR product.

I also attended a meeting to talk about the design of 2 posters on Babesia gibsoni and Cytauxzoon felis which one of the PhD students will be presenting in Australia next week.

Our adventures

Last Sunday we went to a local flea market and horse show. It was really hot and we had to walk to the horse show where we met a real southern police man who had written a book! He also gave us a lift. It seems that you need a car to go anywhere in America. After the show we had to walk home in 110F which took over an hour!

On Friday we went to the cinema to see Dinner with the Schmucks which was hilarious. On Saturday downtown Raleigh was ‘wide open’ which is a huge street fair with lots of acts like wrestling, breakdancing, bands, ice cream eating contests and wine tasting. We ate deep fried oreos!

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Baboons, Shrimp and a Heatwave!

This week I did real time PCR on the baboon DNA after first running the ‘house-keeping’ PCR which checks that that DNA has been successfully extracted. Afterwards the amplicons were visualized under UV light following gel electrophoresis; for this ethidium bromide is used as a dye that intercalates the DNA and so enables visualization of the amplified DNA.

There were 19 positive samples out of 31 which was a surprising find. To confirm this, the DNA was sent off to be sequenced and then the nucleotide bases were sorted and matched using a computer programme.  As no specific baboon babesia has been classified, a new species could be discovered and this could lead to further research funding opportunities.

One evening I tried a real southern American dish of shrimp and grits followed by a game of trivia in the bar. On Friday night Hannah and I went out to listen to one of the guys in my lab who plays in a band and afterwards we went on a bar crawl which was interesting!  The following night we went to a restaurant followed by the cinema.

There is currently a heat wave here, 1000F, such that you are told to stay inside with aircon!

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In the beginning…

We arrived on Sunday afternoon and settled into our accommodation. Hannah and I are staying at different places, but both with NCSU students. On Monday morning we went straight into our respective labs. I am working with Professor Ed Breitschwerdt in the Vector Borne Disease Diagnostics Laboratory. The lab mainly focuses on using PCR and sequencing to test samples for the presence of disease causing organisms. For the first couple of days I shadowed PhD students in the lab to learn the different techniques I will need to perform during my stay. A meeting was held on Wednesday to decide my role in the project; I will be using samples from sand flies to test for Bartonella (a zoonosis).

On Wednesday some blood samples arrived from baboons to test if they were infected with babesia so these were given to me to begin my training. Contamination of the samples can easily happen so the correct techniques are imperative. Under strict supervision we began with lessons in pipetting accurately in a safety cabinet before beginning!

The following day I seemed to have locked myself out of my bedroom and as Tracy the PhD student I was staying with was on holiday I had to call out a locksmith to open the door. It cost $110!!!!!!!!!!!!

That evening I met up with Hannah and her mentor, Dr Jody Gookin, to attend a dinner for all the other summer research students who were nearing the end of their projects, as their semesters start in August not October like ours. In America in order to study for a degree in veterinary medicine you need to have an undergraduate degree in science first so all the other summer students are currently undertaking their second degree programme.

The heat here is very oppressive and humid and they have a lot of violent thunderstorms. Apparently we will be here in September when the hurricane season starts!

On Saturday Hannah and I went shopping in Cameroon village near my house which has lots of boutiques and smart shops.

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