BFRG Highlights 2016

Welcome to the BFRG Highlights, our new feature, to celebrate our success of the past year and to reflect on some of the exciting developments in the Brain Function Research Group!

We started off 2016 with the very first BFRG Research Day, and hosted collaborator Romy Parker from Cape Town as our invited speaker and judge. It was great fun and a huge success, and we hope to do the same again next year. Congratulations to Anna Haw on taking home the prize for best speaker.

On the student front, many of our students made excellent progress this year:

Honours student Kristin Nel, who conducted research on ethnic variations in autonomic skin wrinkling with Peter Kamerman in the Pain Lab, says: “I truly loved the atmosphere in the BFRG lab and would choose to work with this group of professionals over and over again – thank you.”

PhD student Arista Botha from the Wildlife Conservation Physiology (WCP) team started her study on the nutritional ecology of free-living buffalo in the Kruger National Park by having 13 buffalo implanted with miniature temperature-sensitive data loggers in February 2016. Since then she’s been going back every two months to collect blood samples and carry out vegetation analyses. Arista is looking forward to removing the loggers and getting her data early next year. Arista also wrote some great blog articles on her experiences with buffalos in the bush, and on her lab work.

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A large buffalo bull can weigh close to a tonne. Photo by Arista Botha

Another WCP PhD student, Nora Weyer, was awarded a prestigious Oppenheimer Memorial Trust scholarship this year. She was also invited by the Zoological Society / Zoological Garden in Frankfurt, Germany, to present a public talk on the responses of aardvark to the hot and dry conditions in the Kalahari semi-desert. Read about Nora’s PhD research in Afrotherian Conservation and the Diamond Route Newsletter, or follow her aardvark news on Twitter. Nora finished her data collection after spending six months at the University of Cape Town analysing aardvark scats and prey abundance samples in the lab of Professor Mike Picker. She is now back in Joburg working hard at analysing her data and writing up. “It’s been another phenomenal year doing my PhD with the BFRG, and I hope that 2017 will be just as exciting!”  Good luck for the final stretch!

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Another postgraduate student  in the WCP team, Wendy Panaino, also has been conducting research in the Kalahari, on the ecophysiology of ground pangolins (read more on her blog). Aside from the blog article, she had great media coverage on her pangolins, such as this CNN video . On her highlight this year, Wendy reports that “just being surrounded by amazing people in the field of wildlife and research has been such an incredible experience in terms of how much I have learned from these people and how much I have grown.”

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A pangolin looks up from its ant meal. Photo by Wendy Panaino

Led by Nora and Wendy, our WCP team recently joined an interesting collaboration with Dr Frédéric Delsuc (University of Montpellier, France), who studies the evolution of the gut microbiome of free-living aardvarks and pangolins, and other ant- and termite-eating mammals in the Kalahari.

News from the Sleep Lab is that Chloe Flinn has been recruiting participants for her MSc research on the effect of sleep disturbance on pain sensitivity. Chloe has also had some great media attention: JoburgToday.tv visited her at Wits to film a short piece on her research project. Watch the video here, and follow Chloe’s research on facebook to find out more!

Prinisha Pillay, from the pain lab started a longitudinal project for her PhD at a new site this year and submitted her second paper from her PhD research (fingers crossed!). Next year she is looking forward to completing her data collection early in the year and submitting her thesis by December 2017.

Several of our postgraduate students successfully completed their studies in 2016:

Anna Haw, research officer, veterinarian and PhD student in the WCP team completed her PhD (read about her project here), while MSc students Stephanie de Lange (who graduated in 2015 from the University of Pretoria, co-supervised by Andrea and Anna, WCP team) and Tanusha Dukhan (Fever Lab) who graduated this year had their graduation ceremonies. Both Stephanie and Tanusha graduated with distinctions. PhD student Sean Chetty from the Pain Lab completed his PhD and will join the graduation ceremony in December. MSc students Dershnee Devan (Pain Lab) and Kirsten Redman (Sleep Lab) are in the process of completing their degrees following examination. We wish them all the best, and hope to see them on the graduations list soon!

Edward (“Ned”) Snelling, from the University of Adelaide, joined the WCP team in April as a postdoctoral fellow and was very quick off the mark in publishing papers. One of his publications had social media buzzing. Ned also started the BFRG “coffee club”, which led to the Illy coffee shop on the 4th floor of Med School doubling its profits! All of the coffee club members were delighted by the arrival a month ago of our new coffee machine in the Richard Hellon room!

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Another postdoc in the BFRG, Gaëlle Ngassa, who joined the Pain Lab last year, started recruiting patients for her project investigating epigenetic changes of HIV-associated sensory neuropathy in an African population. She has already submitted her first paper from this project to the Journal of Acquired Immune Deficiency Syndromes, and published two others from her previous work. Next year, Gaëlle looks forward to starting her epigenetic analysis.

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Toni Wadley completed her 3-year Hillel Friedland Postdoctoral Fellowship with the Pain Lab and, following the departure of Anna Haw to Stanford for an MBA programme, took over as our BFRG Research Officer and Lecturer in the School of Physiology. In her own words: “If I had to design my best job, this would be it!” Toni has kept the BFRG running smoothly and we wish her all the best for the coming year. Her big paper from her postdoc also had great media coverage.

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Patricia Price, honorary BFRG member and collaborator in the Pain Lab, paid us a visit earlier this year and presented a talk to the School of Physiology about “An integrated study of immune recovery in HIV/HCV patients beginning ART in Indonesia”. Peter Kamerman will reciprocate next year and visit Patricia at Curtin University in Perth.

The Pain Lab has some really exciting collaborations and joint projects planned for next year. We look forward to reporting more on that in our 2017 blogs!

In the Sleep Lab, Karine Scheuermaier was on sabbatical this year giving her the opportunity to work on some collaborative research this year. She spent two month in Berlin, Germany working with her former Harvard colleague, Dr Mirjam Münch. They re-visited performance data from a study they had run together on the impact of evening light exposure on healthy older adults who complained of disrupted sleep. She also started a collaborative research study with Dr Katinka de Wet from the University of the Free State on sleep and HIV interactions. The sisters Mosilo and Mampho Machere started data collection in the Tseki clinic in Phuthaditjhaba (Qwa Qwa), braving the cold weather.  Finally, she was given an opportunity to publish with Dr Duffy on nocturia in a special circadian rhythms and aging issue of Current Aging Science.

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Lois Harden, from the fever group, was also on sabbatical this year. She undertook several collaborative projects with researchers Dr Helen Steele, from the Department of Immunology at the University of Pretoria, Prof Shabir Madhi and Dr Gaurav Kwatra, from the Respiratory and Meningeal Pathogens Research Unit (RMPRU) based at Chris Hani Baragwanath related to fever and sickness behaviour and immune responses to Groups B Streptococcus. She also helped several postgraduate students in the fever group with data collection and analysis and worked on 12 publications including both human and animal based studies.

The BFRG members also travelled and networked extensively this year. Some of the local conferences attended included the PainSA congress in Umhlanga, where Prinisha took the joint first prize for best oral presentation, the Oppenheimer De Beers Group Research Conference in Johannesburg, the Mammal Research Institute 50th Anniversary Conference at Mopani Camp in Kruger National Park, the South African Wildlife Management Association Symposium in Tzaneen, and the South African Society of Sleep Medicine conference in Johannesburg. Several of our students also participated at the Wits Health Sciences Research Day.

PainSA Group photo

Toni Wadley (far right) and the rest of the team, (from the left) Sean Chetty, Peter Kamerman, Prinisha and Dershnee Devan at the PainSA congress. (Photo by Toni Wadley)

International conferences included the Society of Experimental Biology conference in Brighton, UK, the Elsevier Inflammation symposium, Miami, Florida, the International Congress of Vertebrate Morphology, Washington, D. C., and the European Sleep Research Society Meeting in Bologna, Italy. Toni and Peter attended the World Pain Congress in Yokohama, Japan, where Peter was elected as Vice Chair of NeuPSIG, the Neuropathic Pain division of IASP (International Association for the Study of Pain).

There were also personal highlights for many members of the Group. Robyn Hetem and Maartin Strauss welcomed their second child, Ayden John, and a few days later our Honorary Research Fellows (and previous postdocs) Ian Murray and Hilary Lease introduced their son Osirus to the world. Duncan Mitchell was delighted by the arrival of his second grandson in New Zealand.

To round off the year, the Swanepoel family hosted a BFRG staff braai, and everyone joined in to celebrate a productive 2016 at the BFRG publications tea!

Next year, we will be welcoming many new members into our group. We are also excited for Duncan to be plenary speaker at the International Union of Physiological Sciences Congress in Rio de Janeiro, Brazil, and at the International Mammalogical Congress in Perth, Australia. There will surely be lots of exciting news in 2017, so follow us here on the blog, and on Facebook and Twitter to keep up with us throughout the year.

The BFRG Team wishes everyone a Merry Christmas and a Happy New Year!

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Human Immunodeficiency Virus – the sneaky sleep thief

Kirsten Redman

 

Stepping into the field of physiology was not something I took lightly. It was largely intimidating; hugely intimidating, in fact, especially given that I came from a microbiology background! When the option came for me to exercise my knowledge of virology in my Honours year, I jumped at the opportunity.

 

Research had already been widely conducted on HIV and sleep in other parts of the world. Indeed, with the success of highly active antiretroviral therapy (ART), HIV has increasingly taken the position as a chronic illness. And in fact, quality of life aspects have become more prominent as HIV-related life expectancy has increased. As more was discovered elsewhere, it was a shame for me to realise that not much research on sleep and HIV was being done here in South Africa. So that is where I fitted myself in – well, my supervisors created this niche, but for the purposes of this story I’ll say that I inserted myself.

 

During my Honours year, I had run a cross-sectional study in a treated HIV cohort of South African patients, which unexpectedly revealed a relationship between higher CD4 counts and poorer sleep quality. Just to give some background, having a low CD4 count is one of the markers of AIDS, and the aim of ART is, by various means, to increase CD4 counts.
This relationship was seemingly counterintuitive, because one would expect there to be better sleep quality with better immune status (higher CD4 counts), as has been shown in studies in other countries, which found that lower sleep quality was associated with lower CD4 counts. Our cohort at the time differed from other studies’ cohorts as our patients had started ART later in their disease and their baseline CD4 counts were very low. We then hypothesized that during immune reconstitution, there may have been an increased immune activation in these patients, which may explain why the higher CD4 counts were associated with worse sleep quality. So we designed a longitudinal study to see how sleep quality progressed from an ART naïve state, up until a few months on treatment. In this longitudinal study, surprisingly, we found that patients did not complain about their sleep per se but seemed to start off with high daytime sleepiness which reduced (got better) across the study. In this new study, we found no relationship with CD4 counts and sleep quality, but instead found that people who had low viral loads at the time of initiation onto ART had worse sleep than those who had high viral loads.

 

So what happened between my honours and my masters research studies?

 

First of all, my cross-sectional study patients had been diagnosed for seven years and treated on average for four years, very different from the longitudinal cohort who had just been put on treatment and for whom we had followed up ‘only’ until the 18th month, though it is possible that sleep disruption occurs after our cut-off time period. Second, as mentioned before, patients in my cross-sectional study had started their treatment late in the disease when CD4 counts had dropped below 100 cells/μl (AIDS is defined by <250 CD4 T cells/μl and below 100 cells/μl, there are higher chances of developing immune reconstitution inflammatory disease when put on ARV treatment). In our longitudinal cohort, the guidelines had changed and patients started ARVs on average around 250 CD4 counts/μl. So it is possible that what we observed in the first study was a unique effect of starting treatment so late.  In fact, current ‘reservoir’ studies (which follow HIV infection in CD4 memory T-cells) show that immune reconstitution is associated with a higher HIV reservoir load in CD4 memory cells. It is also associated with CD4 activation when treatment was started at lower CD4 counts, while there is nearly no HIV reservoir and very low CD4 activation when treatment is started when CD4 counts are still above 500 cells/μl.

 

And what about this new finding on the relationship between viral load and daytime sleepiness?

 

Currently we postulate that there is an underlying immune-mechanism affecting sleep quality. Indeed cytokines such as TNFa, Il-6, and Il1 have been shown to affect sleep quality. It may be that those who showed lower viral loads may have had a higher immune response against HIV, with concomitant higher production of cytokines, which may have led the patients to experience higher daytime sleepiness. Conversely, those with high viral loads may be those who are not able to build such a strong immune response and would have lower cytokine secretion, leading to lower daytime sleepiness. So my next step is to perform some cutting-edge (not-really) totally awesome (yes, really) cytokine analyses (actually I just ran it last week!)  and flow cytometry, assessing the different activation of the CD4 T-cells as people start treatment up until over a year on treatment and investigate its association with sleep measurements. As you can see from the picture below, I’m already on the task of telling the T-cell’s story.

 

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Kirsten hard at work in the lab.

 

Believe me, nobody is more excited than me to find out what the answer is… but we’ll have to wait and see. On to PhD!!

 

 

The African Buffalo

Arista Botha

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A large buffalo bull can weigh close to a tonne. Photo by Arista Botha

 

The African buffalo is notorious for being one of the most dangerous animals of the African bush. A large bull can weigh close to a tonne and their horns can grow more than a metre wide. Not only are they big and strong, but also fearless. Many online videos, such as this one, show buffalo attacking lions to defend their calves or fellow herd members:

 

Buffalo are also known for being one of the deadliest large mammals for humans to come across in the African bush. In recent news a SANParks ranger was attacked and seriously injured by an African buffalo.

 

I had my first encounter with buffalo during my Masters’ research. Dr Hilary Lease was the postdoc who was involved in the project and she was also my supervisor and friend. We did fieldwork together at Mokala National Park. Even though my study was focussed on black and blue wildebeest, Hilary and I would often encounter buffalo when out on foot, tracking our study animals. While other large ungulates, such as gemsbok or eland, would run away at the sight of a human on foot, a buffalo would move in for a closer look…

 

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Not only are they very dangerous animals, buffalo are also very inquisitive. Photo by Piet Rossouw.

 

They have this intimidating glare that, even when you are seated in a vehicle, makes you wonder how safe you really are. During our fieldwork, we would often climb a hill, just to find a buffalo looking at us inquisitively from the other side. The buffalo would take a few steps towards us, and we would retreat back to our car.

 

One day, we were struggling to locate a particularly elusive wildebeest bull in a very hilly area. Due to the uneven terrain around us, the tracking transmitter signal was bouncing around. We followed a signal in one direction, convinced that the wildebeest must be just on the other side of the next hill, only to find that when we got there, the signal was suddenly coming from the opposite direction.

 

Staring intently at the tracking receiver, we walked along the crest of a hill. In our frustration with the bouncing signal, we let our usual vigilance slack. Suddenly, there was a loud rustling behind us. We turned around to face an enormous and very surprised buffalo bull only a few meters away from us. For several frozen seconds humans and bull just stared at each other. Then the realisation dawned on us that WE NEED TO GET OUT OF HERE. Slowly, we backed away down the slope, too scared to turn our backs on this large beast. The minute we were out of the buffalo’s sight we ran for it!

 

With great speed and pumping adrenaline, we made our way down the hill and all the way around to the other side. In our haste, we flushed an African wildcat out of a bush! That was my first sighting of this elusive cat species. Although not much more than a blur, with a ringed tail that sped away in front of us, it left me in breathless amazement.

 

We arrived at the safety of our vehicle with a fresh respect for buffalo and a new cat species to tick off our sightings list. Laughing in nervous relief and still trying to catch our breath, we drove off and decided to go and look for our wildebeest elsewhere.

 

Nowadays I am privileged enough to study the impressive buffalo for my PhD – fortunately from a safe distance and with support from SANParks’ very experienced wildlife staff. Yet even from the other side of a fence, every time I am scrutinised by that intimidating glare, I can’t help but feel the need to slowly back away.

South Africans with HIV-related pain are surprisingly active, but not due to their resilience.

Antonia Wadley

When one thinks about chronic conditions that are commonly painful, HIV doesn’t typically spring to mind. However, more than 50% of HIV-positive individuals experience painful conditions like headache, chest pain or neuropathy, and that pain is frequently experienced as moderate to severe in intensity.

 

What struck researchers from the Brain Function Research Group (BFRG) at Wits University as odd was that despite this high burden of pain in HIV, a couple of papers have emerged suggesting that, having asked patients, functional interference (having difficulty with things like walking or going to work) was not as great as they might have expected. One of these papers was from the BFRG and had been completed locally in Johannesburg, South Africa.

 

To investigate whether pain does actually affect function in HIV (as it does in many other clinical conditions), researchers Dr Antonia Wadley, Emeritus Professor Duncan Mitchell and Associate Professor Peter Kamerman from the BFRG, based in the School of Physiology, Faculty of Health Sciences at Wits, conducted a cross-sectional study.

The results from the study, titled: Resilience does not explain the dissociation between chronic pain and physical activity in South Africans living with HIV, are published today, 13th September, in the journal PeerJ.

 

To explain why pain may not affect function, the researchers first put it down to African patients being resilient – the ability to cope with adversity.

 

Explains Wadley: “Nobody’s assessed resilience in people living with HIV and chronic pain before. We hypothesised that people living with HIV would generally be pretty resilient and those who were more resilient, would be more active and report lower pain intensity.”

 

Measuring resilience in HIV-patients objectively for the first time

For the study, the researchers recruited HIV-positive patients from an HIV clinic in Johannesburg: half with chronic pain (defined as having had pain most days for at least three months) and half without. They then assessed resilience and, as well as asking patients about their activity, the researchers measured it objectively for the first time in a subset of patients using accelerometers, which are like sophisticated pedometers. They also asked the patients about their day to day worries.

 

“It turns out,” Wadley says, “that we were right on one thing, HIV-positive patients in our study were really resilient, but our hypothesis was wrong: being more resilient didn’t associate with being more active or having lower pain intensity. In fact, the activity results astounded us. Not only was patients’ activity not as affected as one might expect, it wasn’t affected at all.”

 

There was no difference in activity intensity, duration, or time spent at different intensities of activity between those with and without chronic pain. “This is something you just don’t see in other types of long term pain,” she adds.

The researchers then looked at how frequently patients worried about their health, money, food and family. “We thought that if patients were worried about money and having enough food that pain might be relegated to a lower priority.” They found that patients in chronic pain worried more frequently about each of these things compared to their pain-free counterparts and that health was lowest down the list.

“So it really does appear that if you are poor, pain may be relegated to a lower priority. Indeed, our analysis showed that worrying more about food associated with higher levels of activity,” says Wadley.

 

Going forward

The researchers also asked the patients in pain what else they worried about and who they had told about their pain.

 

“It turns out that HIV-related stigma is a real problem and that half the patients in pain had not told their closest friends and some not even their family about their pain, for fear that it might reveal their HIV status.”

 

Wadley says it thus seems that economic stresses and fear of HIV-related stigma may drive people to maintain high levels of activity, even when they are in severe pain.

 

“What’s not clear is whether this kind of level of activity in the face of pain is helpful or harmful and that’s something we will be looking into next,” she adds.

PainSA Congress 13th -15th May 2016, Umhlanga

Toni Wadley

 

Last month, I headed over to Umhlanga with some of the other Pain lab members for the annual PainSA Congress. PainSA  is the South African chapter of the International Association for the Study of Pain and the Congress is a meeting of health care professionals and scientists from across the country who work with pain.

 

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Me (far right) and the rest of the team, (from the left) Sean Chetty, Peter Kamerman, Prinisha and Dershnee Devan at the PainSA congress. (Photo by Toni Wadley)

 

In the pain lab, we’re always going on about how pain is under-recognised and under-treated in African HIV+ patients. Every PainSA congress, the sentiment that access to opioids in Africa for severe pain is woefully inadequate is repeated. Yet at this year’s congress we heard the opposite angle. US.  Professor Tracy Jackson, medical director of outpatient pain clinics at Vanderbilt, gave hard hitting talks about the ineffectiveness of current chronic pain therapies despite the US spending $630 billion a year on the problem. She spoke about the over-reliance on opioids in a country that uses 80% of the world’s stock and where opioid overdose is the leading cause of accidental death.

 

Tracy is a big advocate of non-pharmacological interventions including functional rehabilitation programmes. These programmes are residential and involve an entire multidisciplinary team of healthcare professionals aimed at getting patients back to being functional, even if that means their pain staying the same. Her new take on these programmes is to offer ‘Relief retreats’ where restoration programmes are held at retreat centres rather than in a typical clinic setting.

 

Tracy is really entertaining to listen to. Follow this link for a TEDx talk she gave in Nashville very recently on the topic of non-pharmaceutical treatment of chronic pain.

 

The theme of getting patients with chronic pain back to work was echoed throughout the congress. Dershnee, an MSc student in the pain lab, gave a workshop on the topic. Dershnee has a background in occupational therapy. Through her current work in medical insurance she knows only too well that absenteeism from work costs South Africa R12 billion a year.

 

Peter, the head of our pain lab, gave a plenary talk on “Neuropathic Pain: so many people, so few drugs”. Sean Chetty, a pain lab PhD student and anaesthesiologist, gave a plenary on pain management being a human right. In the largest research paper session a PainSA congress has had, I spoke about the effect of HIV stigma on pain, and Prinisha presented some of her PhD data about the pharmacological treatments that are actually being used in the clinic for HIV-associated sensory neuropathy. Prinisha won the joint first prize for best talk along with Tory Madden a new postdoc at UCT.  Well done Prinisha!

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Prinisha giving her winning talk. Photo by Toni Wadley

Combating the ills of opioids (and saving rhinos)

Anna Haw

 

A field of brightly-coloured, flowering poppies is as spectacular to the brain as it is to the eye. Opioids, drugs derived from the opium poppy, act on receptors in the brain to bring about sensations such as euphoria and pain relief. Just as the wind through a field of poppies can capture your gaze and keep you mesmerized for hours, so too can opioids hook your brain; a phenomenon well understood by heroin addicts. Sadly, euphoria and pain relief are not the only opioid-induced effects. Another side-effect blocks the drive to breathe, resulting in the death of an estimated 69,000 people worldwide each year.

 

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Opioids were first isolated from poppy seeds and are famous for their effect on the brain. Photo by Anna Haw.

 

Opioids block pathways in the brain that regulate breathing and dampen the feedback loops that tell the brain to take deeper breaths or more breaths when oxygen levels are low. Without enough oxygen in our blood, cells die and ultimately the heart will stop beating.

 

While opioid use in humans, with the subsequent consequences, is well recognized, opioid use in wildlife is perhaps not so widely understood. Have you ever wondered how wildlife veterinarians capture, treat and transport enormous wild animals such as rhinoceros and elephant? How do vets make these two-ton (or more) animals sleep by firing a measly dart into them? The secret lies in those fields of brightly-coloured poppies. Potent opioids, such as a drug commonly referred to as M99, are an essential component of a wildlife vet’s armory and the only class of drug capable of bringing about a deep sleep (or immobilisation) in large herbivores like the rhino and elephant. A major advantage of opioids is that their effect can be reversed with an antidote. When a vet has finished working with an animal, she injects the antidote and within seconds to minutes, the sleeping beast is awake and wandering back into the African bush.

 

However, the picture is not always so rosy. Sometimes the animals don’t get up, or sometimes they get up, only to suffer consequences of the event in days, weeks or months to come. It so happens that the white rhinoceros, currently needing the most hands-on interventions in the face of escalating poaching levels, is one of the species that is most severely affected by M99’s ill-effects. Just as opioids can kill humans, so too can they kill wildlife, with the white rhino being particularly susceptible to this fate.

 

White rhino are so severely affected by M99 that vets have been experimenting with different treatment options in an attempt to improve the oxygen levels in the immobilised rhino’s blood and reduce the risk of death. However, with many different vets trying variations on a similar theme, one standard approach, scientifically proven to improve oxygen levels, has been lacking. Therefore, with a team of researchers from the University of the Witwatersrand (Wits), University of Pretoria and SANParks, I systematically tested different available treatment options, firstly in boma-housed rhino and then in free-ranging rhino within the Kruger National Park. In the bomas, we were surprised to find that giving a high volume of oxygen through the rhino’s nostril made the physiological imbalances caused by M99 even worse and did not improve breathing. Another treatment option, currently used by many veterinarians, is a drug called butorphanol. Butorphanol partially reverses the effects of M99, and it is believed that the negative side-effects are reversed more than the positive effects, such as immobilisation. However, the advantages of butorphanol are not clear-cut and, invariably, some arousal occurs together with improved breathing. In a two-ton, confused wild rhino, unwanted arousal can be problematic. We also found that butorphanol did not fully correct the oxygen levels in the rhino. So we combined the administration of oxygen through the nostril with an injection of butorphanol. To our delight, this treatment option completely corrected the dangerously low oxygen levels, thus significantly improving the safety of rhino immobilisation.

 

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Immobilised white rhino receiving oxygen through its nostrils. Photo by Andrea Fuller.

 

As the fight against poaching intensifies, more rhino need to be immobilised for procedures such as translocation and the fitting of tracking devices. Moreover, vets are increasingly faced with severely injured rhino that have survived a poaching incident. These compromised animals require repeated immobilisations for intensive veterinary care and often cannot survive any additional physiological stress. Thankfully, as a result of our research, rhino capture need no longer be such a risky procedure and wildlife vets across South Africa have already adopted the approach of administering oxygen and butorphanol to immobilised rhino.

 

References:

Haw AJ, Meyer LCR, Fuller A. 2016. Nalbuphine and butorphanol reverse opioid-induced respiratory depression but increase arousal in etorphine-immobilized goats (Capra hircus). Veterinary Anaesthesia and Analgesia.

Haw AJ, Hofmeyr M, Fuller A, Buss P, Miller M, Fleming G, Meyer LCR. 2015. Butorphanol with oxygen insufflation improves cardiorespiratory function in field-immobilised white rhinoceros (Ceratotherium simum). Journal of the South African Veterinary Association, 86: Art #1276.

Haw AJ, Hofmeyr M, Fuller A, Buss P, Miller M, Fleming G, Meyer LCR. 2014. Butorphanol with oxygen insufflation corrects etorphine-induced hypoxaemia in chemically immobilized white rhinoceros (Ceratotherium simum). BMC Veterinary Research, 10:253.

How to do an ELISA (or A day in the Fever Lab)

Arista Botha

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An example of a 96-well ELISA plate. Photo by Arista Botha

 

When you are writing up your PhD protocol, your experiments all seem simple. Then, when it gets to actually carrying out the experiment, things are often a lot more complicated than what they appeared like on paper. For example, my methods description was strewn with all of the blood chemistry tests one could possibly think of. Once my protocol and my ethics were approved, I had to start making it happen. How hard could it be?

 

As it turns out, finding an ELISA kit for any animal other than humans, rats or mice is tricky, so I ended up sending about twenty emails back and forth to various companies. Finally I found a company that could send me the right ELISA kit. The second surprise came when I saw what ELISA kits cost! I never knew that such a small thing could be so expensive. Luckily, my supervisor had a grant that was just about the right amount to cover my ELISA kits. So I had a product, a supplier, a quote and a grant. Satisfied, I placed my order. I was so excited when, a few days later, my kits were delivered. Like a little kid opening a Christmas present, I unpacked my kits, marked them clearly with my name, and stored them neatly into the fridge.

 

I stood back and thought: Now what!

 

Panic started to set in. During my Master’s degree I had spent most of my time in the field, tracking my study animals and doing behavioural observations. The result being that I have not spent any time in a laboratory since my undergraduate degree! How did I get myself into this?

 

Luckily Tanya and Lois from the Fever Lab were doing ELISA’s for their research, and they agreed that I could tag along and they would teach me how to do an ELISA. So I joined the Fever Lab for a day.

 

First off, I have to say that the Physiology Department have the most amazing equipment in Monica’s assay lab! To start off with, Tanya used an ultrasonic homogenizer (also called a sonicator) to prepare rat brain tissue before performing an ELISA for detecting rat IL-1β protein. A sonicator is similar in purpose to a homogeniser (or the blender in your kitchen) except that a sonicator uses ultrasonic sound waves to disintegrate the tissue. ULTRA-SONIC SOUND WAVES! That sounds like something from a science fiction movie…

 

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An ultrasonic liquid processor (sonicator) is used in our lab to sonicate the samples. Photo by Arista Botha

 

And then, they have this “super-pipette”, which we call the “Pipette-man”! The first part of doing an ELISA is pipetting your sample and other solutions provided by your ELISA kit into the sample wells. This little piece of equipment makes a big difference in speeding up the whole process.

 

Pipette

This pipette allows you to pipette simultaneously into eight wells, saving a lot of time during the ELISA process. Photo by Arista Botha

 

After incubating your samples the wells have to be washed out properly. You can either manually empty each well and then wash it out with wash buffer using an ordinary pipette (repeat times five!), or (the better option) you can use an automatic well washer, such as the one shown below:

 

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The automatic well washer saves you hours of repeatedly pipetting wash buffer into each well. Photo by Arista Botha.

 

After washing your samples, you add a substrate solution (consisting of colour reagents), which makes your samples turn blue, if for example, IL-1β is present in your sample. After leaving it to incubate for a set time period you add the stop solution, which changes the colour from blue to yellow.

 

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Adding the stop solution changes the sample colour from blue to yellow. Arista Botha

 

The final step in the ELISA process is, of course, getting the results! The concept is that the darker yellow your samples, the higher the concentration of the substance you want to measure in your sample (in this case, IL-1β). You put your 96-well plate into a microplate reader, which is set at a certain wavelength, and which will give you optical density (OD) readings. Further calculation of these OD readings ultimately will give you your sample concentration.

 

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The ELISA microplate reader measures the optical density of each well, which you then use to calculate the concentration of your sample. Photo by Arista Botha

 

After spending the day with the Fever Lab, I feel a lot more confident (and excited) about doing my own ELISAs. I am so glad that we have these equipment and skilled people in our research group and that we can learn from one another. This is why the BFRG is a team and why we do research TOGETHER!