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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19
Aug
2010

In the lab this week, I’ve been extracting tissue from slides and carrying out PCR followed by gel electrophoresis in order to determine if Enterococcus species are present in the sick and healthy kittens.

I had a tour of the small and large animal hospital this week too. Derek, who works in my lab, is also a large animal vet and he took me down to the cow barn along with some vet students to learn all about cows. I learnt how to listen to the heart and lungs and learnt what signs are abnormal in an ill or unhealthy cow. I also got to palpate a cow which I’ve always wanted to do. It was very warm and the contractions of the muscles were so strong, it made my arm ache a little.

Our adventures

This week Anna and I went to a shopping mall. It had lots of expensive shops and even sold Hunter wellies for $120! So they are a lot cheaper in England. There is also an outdoor pool nearby so we spent one day sunbathing and chilling in the pool. It was very relaxing and a nice way to cool down as it’s so hot at the moment. Saturday night we went to watch Salt featuring Angelina Jolie at the local cinema.

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In the lab this week I have been carrying out F.I.S.H on some slides in order to show where and if bacteria are adhering to the intestinal wall of infected animals. In this technique, fluorescent probes attach to the bacteria, and they can be identified under different wavelengths of light.

I’ve also been carrying out PCRs on the slides with the tissue and adherent bacteria in order to amplify the bacterial DNA, giving us lots of copies to work with. The DNA was analysed by gel electrophoresis to identify what species of bacteria was present.

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This week I have read lots of research papers, in order to come up with a protocol for my experiment. I’m going to grow bacteria isolated from approximately 100 samples and use them to infect some polystyrene wells, in order to determine if they can form biofilms. I have also been looking at Gram stains using a microscope which have shown that in these 100 samples, Enterococci and E. coli bacteria have adhered to the intestinal wall, and in many cases, these adherent bacteria may have caused diarrhoeea in the samples.

Unfortunately, the stems cells didn’t grow so next week we are going to attempt to culture some more by changing the solutions used, amount added etc.

I have watched gel electrophoresis being carried out and it’s very interesting to see what happens and the results in real life compared to learning and reading about it in lectures. It all makes a lot more sense now.

The people I am working with in the lab and I went for an ice cream where they have over 90 flavours! There was one called ‘cold sweat’, which contains lots of chillis and peppers, unfortunately, no one tried it.  A waiver form has to be signed in order to eat it and it made the morning news here!!!!!

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On Monday I had a lab meeting where I met with everyone who works in the gastrointestinal physiology lab which is run by Dr Jody Gookin. The personnel in the lab all discussed what their various projects were with me and any plans they had to improve their projects. That lunchtime we all went out for a Mexican in a restaurant; I had never eaten in a Mexican restaurant before, so I had beef fajitas which were lovely.

During the week, I observed how to run a PCR to detect Tritrichomonas foetus in cat faeces. The lab has various pieces of equipment to diagnose Tritrichomonas foetus, a protozoan parasite of cats which causes diarrhoeea and can be easily spread to other cats, especially in shelters and multiple cat households. I also learnt how to passage pig cells, and have my own culture of pig intestinal cells growing in the incubator which I need to check on every 2 days and replace the media.

One of the PhD students in the lab is attempting to grow some cat colon stem cells so I observed how she attempted to isolate crypt cells from the colon. The crypt cells are where the stem cells are found in the colon. It was also my birthday on Friday so all those in the lab and I went for an Italian at lunchtime. I had a bag of presents and cards from home which I was strictly told not to open until my birthday.

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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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