15
Sep
2010

In the Lab

This week we ran the PCR for E.Hirae on our samples. The gel showed that 3 out of the 24 animals were infected with E.Hirae.

However, our positive control didn’t show up suggesting there may have been a problem with the PCR. So, we will run a GAPDH PCR which will tell us if any cat DNA is in the sample, such as that of the tissue, which will tell us if we can extract enough DNA from the samples using the new extraction method. If you can’t successfully find and extract cat DNA in the samples then there is no chance of finding any bacterial DNA.

I also went up to the internal medicine rounds room where I observed what happens in that side of the vet hospital. A very interesting case had come in which was troubling the specialists.

A dog had low red blood cells, white blood cells and platelets. It was thought that the dogs own cells, such as the macrophages, might be destroying the blood cells. This is a very rare case and the vets think it may be hemophagocytosis. There have only been 24 cases reported in dogs and very few in humans, so little is known about this disease.

A scientist has come up with a drug that could potentially kill the cells that are killing the red, white and platelet cells. It will be a trial run, but as the prognosis doesn’t look good the vets are going to see if the drug works, as little else can be done.

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15
Sep
2010

In the Lab

This week I finished the DNA extractions and so now they are going to be put in the PCR machine to amplify millions of copies of DNA. We will first PCR the samples for E. Hirae as we think this is the most likely bacteria we are going to detect. We know for sure that the bacteria are Gram positive cocci adhering to the gut epithelium.

If some samples come back negative for E. Hirae then we will run them through a PCR for E. faecalis/E. faecium. Then any samples negative for this will be run through a SOD PCR. SOD is super oxide dismutase, which is a gene found in all bacteria. We will then send off the DNA for sequencing to determine what bacteria species were present.

We then finished off the F.I.S.H for E. coli in order to show that E. coli is adhering to the epithelium. We originally decided to perform F.I.S.H for E. coli as ram stains for these samples demonstrated Gram negative rods adhering to the epithelium and so we believed these were most likely E. coli. In order to confirm this we used an E. coli probe which binds to a sequence of DNA that is specific to E. coli.

Out of the 9 samples we observed Gram negative rods adhering in 8/9 samples and these fluoresced for E. coli when tested using FISH and then examined using a florescent microscope. Also these 8 samples showed that the E. coli was adhering to the tissue. Interestingly, all 9 samples were taken from unhealthy kittens that had been euthanized or died due to intestinal problems such as severe diarrhea.

Finally, this week I went to a rounds meeting one morning. It is where qualified vets who want to specialize in a specific area meet to discuss cases that have been seen at the hospital with the qualified specialist neurologists, cardiologists etc.

They go through the tests results carried out and the symptoms presented by the patient. They then list various differential diagnoses as to what could cause the symptoms and decide how to treat it.

The qualified specialists are there to ensure the residents are correct in what they think and say and to ask them challenging questions. It was really interesting to see how many different differentials could cause polyarthritis (a whole white board full!!!).

From these possible diagnoses they then decided what is most probable and relevant, relating back to the results, symptoms and history of the patient.

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15
Sep
2010

 In the lab

A fairly quiet week compared to previous weeks…

We examined necropsy slides from a deceased dog infected with Babesia. Babesia sp. are protozoal organisms that parasitize erythrocytes such that they can evade the immune system by hiding inside the red blood cells.

All the slides showed tissues and blood vessels almost entirely occluded with the parasite (lungs, brain, liver etc.), except for the kidneys which might be because of the osmolarity. It seems amazing that the animal was able to survive up to this point with this extent of infection.

I had a promise for samples of the ‘Blandford Fly’ to be shipped from the U.K. Hopefully they will arrive in time.

Our Adventures

On Friday evening we went to the Wake County Speedway to watch motor racing. One race was children driving miniature cars.

The Vet College hosted its annual dog Olympics show in downtown Raleigh on Saturday which had classes such as the howling contest and musical sit. The event encourages the general public to adopt the dogs kept by the vet college as blood donors.

On the same day was ‘Bug fest’ where besides lots of stalls you could dine on bugs, some even chocolate coated! Needless to say Hannah and I did not indulge.

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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

This week we discovered that the DNA extraction from the paraffin embedded blocks was successful. We therefore managed to extract E. Hirae bacteria successfully from the tissue sections. Compared to previous times we used a microwave to melt the paraffin together into a ring around the tube rather than using xylene, as it is thought that the xylene damages the DNA and may inhibit the PCR.

We also carried out F.I.S.H on 5 of the slides which corresponded to 5 different animals, presumably healthy. F.I.S.H works using specific fluorescent probes which bind to a specific sequence of the organism that is being searched for. On these sections we added an E. coli probe which fluoresces red under UV light. We discovered that all 5 animals had E. coli present and more importantly they were seen adhering to the epithelial cells.

More F.I.S.H is going to be carried out on more slides from individual animal this week.

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09
Sep
2010

My 6th week (Hannah)

We are trying a different protocol in order to extract good quality DNA from the tissue section slides. Instead of scraping the slides with the mounted tissue, we shaved off 10, 15 and 20 sections of the tissue which is embedded in paraffin, rather than getting histology to mount them onto slides.

We think that the large amount of xylene that we add to remove the paraffin may be damaging the tissue, so we placed the tissue embedded in paraffin into the microwave to clump all the paraffin together. We also cut out the specific section of the tissue where we saw large amounts of bacteria adhering to it and again added this to a tube and micro-waved it. The results for the gel that we run will be ready in the next blog.

Our Adventures

My parents came to visit me for a week last week. At the weekend we flew out to Washington on a 36 seater plane, it only took 30 minutes but as the plane was so small it was a little scary when there was turbulence. We spent the weekend just looking around and sight-seeing. We saw the white house and all the various war memorials and president memorials. It reminded me of a European city. Then during the week they had a tour of the vet school given by Jody Gookin and I got to show them what I’ve been doing so far over the summer.

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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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