Tag Archives: animal research

Nobel Prize 2015 – Protecting People against Parasites!

The 2015 Nobel Prize in Physiology or Medicine has been awarded to scientists whose research has led to therapies that have saved hundreds of millions of people around the world from parasitic diseases that can otherwise cause disability and death.

William C. Campbell and Satoshi Ōmura shared one half of the award “for their discoveries concerning a novel therapy against infections caused by roundworm parasites”, while Youyou Tu was awarded the other half “for her discoveries concerning a novel therapy against Malaria”.

Portraits of the winners of the Nobel medicine prize, shown on a screen at the ceremony in Stockholm. Photograph: Jonathan Nackstrand/AFP/Getty Images

Portraits of the winners of the Nobel medicine prize, shown on a screen at the ceremony in Stockholm. Photograph: Jonathan Nackstrand/AFP/Getty Images

In the press release announcing the award the Nobel Assembly at the Karolinska Institute highlighted the impact of the therapies that were developed thanks to the work done by these three scientists, Avermectin in the case of William C. Campbell and Satoshi Ōmura, and Artemisinin in the case of Youyou Tu.

The discoveries of Avermectin and Artemisinin have fundamentally changed the treatment of parasitic diseases. Today the Avermectin-derivative Ivermectin is used in all parts of the world that are plagued by parasitic diseases. Ivermectin is highly effective against a range of parasites, has limited side effects and is freely available across the globe. The importance of Ivermectin for improving the health and wellbeing of millions of individuals with River Blindness and Lymphatic Filariasis, primarily in the poorest regions of the world, is immeasurable. Treatment is so successful that these diseases are on the verge of eradication, which would be a major feat in the medical history of humankind. Malaria infects close to 200 million individuals yearly. Artemisinin is used in all Malaria-ridden parts of the world. When used in combination therapy, it is estimated to reduce mortality from Malaria by more than 20% overall and by more than 30% in children.

For Africa alone, this means that more than 100 000 lives are saved each year.

The discoveries of Avermectin and Artemisinin have revolutionized therapy for patients suffering from devastating parasitic diseases. Campbell, Ōmura and Tu have transformed the treatment of parasitic diseases. The global impact of their discoveries and the resulting benefit to mankind are immeasurable.

Animal research played a key part in the development of these therapies, as the Nobel Prize press release has pointed out.

Image: NobelPrize.org

Image: NobelPrize.org

In the case of Avermectin, after  Satoshi Ōmura had identified a series of bacterial cultures that produced a variety of antimicrobial agents, including the bacteria Streptomyces avermitilis which  showed promise against parasitic roundworm infection in mice, in 1979 William C. Campbell and colleagues identified a particular component produced by S. avermitilis called Avermectin B1a which had a broad efficiency against roundworm infections in a wide range of domesticated animal species, including cattle, sheep, dogs and chickens. Following this the team developed a modified form of Avermectin B1a known as Ivermectin, which was entered into clinical trials following positive tests in animal models of parasitic infection, and has since gone on to become a key treatment for parasitic infections – particularly the nematode worm infections that cause River Blindness and  Lymphatic Filariasis (the extreme manifestation of which is known as elephantitis) – and is on the World Health Organization’s list of Essential Medicines.

Image: NoberPrize.org

Image: NobelPrize.org

Ivermectin, and other members of the Avermectin family of therapies, are also widely used in veterinary practice, and their development and use is a good example of the One Health principle in action. You can learn more about the discovery of the Avermectins and Artemisinins in the advanced material Avermectin and Artemisinin – Revolutionary Therapies against Parasitic Diseases produced by the Nobel Assembly.

In 2011 we took a look at Professor Youyou Tu’s research that led to the development of Artemisinin therapy for malaria, and the key role played by mouse models of malaria infection,  in a post entitled “George is OK: Thank the men who stare down microscopes!”

While the news reports don’t state which drugs Cloony took to beat malaria, It is most likely that he was treated with artemisinin-based combination therapies (ACTs), which became available in the late 1990s and are now in widespread use.  If that is the case, he has benefited from mouse studies done in China the late 1960s and early 1970s when over 100 traditional herbal remedies were screened in a rodent model of malaria for anti-malarial activity (1). Eventually “Project 523” scored a hit when Professor Tu Youyou identified an extract of the plant qinghao, scientific name Artemisia annua, which had good anti-malarial activity, leading to the development of the artemisinin-based anti-malarials which have become the first-line treatment for malaria in the past decade.

We congratulate this years Nobel laureates in Physiology or Medicine, their research has improved the lives of hundreds of millions of people across the world over the past 3 decades, and will continue to do so. We hope that their success continues to inspire scientists around the world to rise to current and future public health challenges!

Speaking of Research

Guest Post: Why science needs to improve

Jeremy BailooToday’s guest post is from Jeremy D. Bailoo, PhD, a developmental psychobiologist in the Division of Animal Welfare at the University of Bern, Switzerland. He is currently involved in research which examines the manner by which we house and care for animals and its relevance to animal welfare and how it affects experimental results. He is particularly interested in providing empirically based procedures for refining animal housing.

Why science needs to improve

In a recent article in the Huffington Post, Professor Marc Bekoff and Dr. Hope Ferdowsian outlined their reasons for believing that science does not need mice. Their article was written in response to an editorial in the New York Times which advocated for the need for female mice in laboratory research. Bekoff and Ferdowsian made a number of interesting points and cited relevant supporting literature. However, their response presented only certain aspects of the issues involved. In this piece I will deconstruct the arguments levied by both sides. I will refrain from critiquing information that was not accompanied by a citation in either article, as these constitute unsubstantiated opinion.

The authors of the New York Times editorial described a new study published in the journal Nature Neuroscience which suggested “that research done on male animals may not hold up for women. Its authors reported that hypersensitivity to pain works differently in male and female mice….If these differences occur in mice, they may occur in humans too. This means a pain drug…might appear to work in male mice, but wouldn’t work on women.” These authors then state that failure to consider gender or sex in research is well recognized and cite the work of Zucker and Berry (2010) as well as the repositioning of interests statement of the National Institutes of Health (NIH) specifying sex as a biological variable in NIH funded research (see here and here).

The NYT editorial framed a well-articulated argument and did not overstate any of the claims that it made. The issue of the underrepresentation of females in biomedical research has been repeatedly highlighted (e.g., here, here, here, here and here) with little change in US science funders’ policy until now. It is important to note that nowhere in this article is it stated that all research in mice is ungeneralizable to females. Indeed, whether a scientific result is generalizable to both sexes is dependent on the phenomenon being studied; and this seems to be the case in particular for pain research in mice.

Mice in a research laboratory. Image courtesy of Understanding Animal Research.

Mice in a research laboratory. Image courtesy of Understanding Animal Research.

In their argument against the use of mice in research in the Huffington Post, Bekoff and Ferdowsian state that “numerous experiments on male and female non-human animals (animals) fail to reliably hold up in humans, and many prominent researchers have argued we need to develop non-animal models in order to learn more about serious diseases from which numerous humans suffer.” It is without question that some (not all) experiments in male and female rodents fail to replicate their results when that same experiment is performed on humans. However, as the ability to falsify and to replicate an experimental result are the cornerstones of the scientific method, failure to replicate an experimental result does not imply poor generalizability of an animal model to the human condition. I have recently co-authored an article on this topic demonstrating that meta-analytic studies have revealed that the reporting of criteria related to experimental design and conduct in some biomedical animal experiments is poor. The reasons why the result of an experiment conducted in non-human animals may fail to be replicated in humans is a consequence of complex processes that cannot and should not be trivially summarized by the statement “we need to develop non-animal models in order to learn more about serious diseases from which numerous humans suffer.”

In support of their argument, Bekoff and Ferdowsian cite the article “Mice Fall Short as Test Subjects for Some of Humans’ Deadly Ills”. In summarizing this article, Bekoff and Ferdowsian imply that because C57BL/6 mice (a single strain of 16 classified as Tier 1 in priority for investigation) do not seem to be able to model sepsis in humans, then all mice fail as a model of human disease. This is a logical fallacy, and a quick google search leads to very interesting responses to this article. Some are in favour of this piece (e.g., here) while others quickly identify flaws with the logic (e.g., here and here). Indeed, in the original article, the authors state “The study’s findings do not mean that mice are useless models for all human diseases.”

Next, Bekoff and Ferdowsian make the claim that the former director of the National Institutes of Health, Elias Zerhouni has lost confidence in the use of mice to model anything that is related to humans (see here). Bekoff and Ferdowsian fail to cite the clarification or perhaps are unaware of the clarification that was given (see here) in which Mr. Zerhouni states, “In short, animal models remain essential to the basic research that seeks to understand the complexities of disease mechanism.” As my colleagues at the website Speaking of Research have put it: “Animal models are essential to developing new medicines. They are, obviously, not sufficient on their own – cell cultures, human studies and computer models (among others) are also crucial methods used alongside animal models.”

The next paragraph with a citation states “Even experiments involving similar nonhuman species have shown that studies in mice, rats, and rabbits agree only a little more than half of the time (please see Hartung and Rovida 2009)”. Careful reading of this citation, however, does not yield this information. Indeed, nowhere in this article are any of these claims made. More interestingly, the cited article states, “no acceptable alternatives to reproductive-toxicity testing (in animals, my emphasis) have emerged, or are likely to be validated by 2018. Computational approaches are also limited by the complexity of reproductive toxicity and because half of the REACH chemicals are mixtures, inorganic, salts or contain metal atoms, rendering toxicity less predictable”. Thus, rather than supporting Bekoff and Ferdowsian’s arguments, it would seem that Hartung and Rovida advocate for the use of animals in toxicological research because there are no good alternatives.


Laboratory mouse. Photo courtesy of Understanding Animal Research.

Bekoff and Ferdowsian then state, “Attitudes toward animals are also changing, and now is the time for action. As per a recent nonpartisan Pew Research Poll, a solid 50 percent of people surveyed now oppose the use of animals in laboratory experimentation — an all-time high in the public opinion research literature.” This is indeed alarming and is the reason I have spent many hours researching these data. It is time that active scientists speak up for their science and break the cycle of misinformation that is spreading throughout our society.

In their penultimate paragraph Bekoff and Ferdowsian indicate that many may be incredulous in realizing “that mice and rats aren’t animals but a quote from the federal register does in fact read, “We are amending the Animal Welfare Act (AWA) regulations to reflect an amendment to the Act’s definition of the term animal. The Farm Security and Rural Investment Act of 2002 amended the definition of animal to specifically exclude birds, rats of the genus Rattus, and mice of the genus Mus, bred for use in research” (Vol. 69, no. 108, 4 June 2004).” It is worthwhile to note the date of this citation, June 2004 – 11 years ago. Much has changed in those 11 years and much will continue to change in the future. As science progresses, the type of animals used in research, the manner in which they are used, and their care will be continually scrutinized by scientists and the public. As a result, animal care, use, and corresponding regulations will continue to be adjusted. Moreover, animals used in research (including birds, rats, mice) are covered by Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals since 1985 while guidelines for the care and use of laboratory animals have been critically considered since 1963 and have been continually updated as new information becomes available. Ferdowsian and Bekoff are either ignorant of current US regulations governing research or are deliberately being disingenuous.

These authors conclude that “there are numerous non-animal alternatives that are extremely reliable (please also see), and it’s about time they are used.” Again, where is the evidence for this? As I have outlined in this commentary, Bekoff and Ferdowsian have not provided sufficient evidence to come to this conclusion. Moreover, the statement that many non-animal alternatives are currently available and reliable requires careful deliberation. An example of such deliberation can be found here. The unsubstantiated statement that alternatives exist and are reliable does not make it so. Currently, such research and methods complement, rather than replace, research in non-human animals.

Thus, it would seem that the argument levied by Bekoff and Ferdowsian that science does not need research with mice is misleading. Poor reproducibility of experimental results is a problem in biomedical research. Indeed, it is a problem with science in general (e.g., here, here and here). To address the question “does science need mice”, one would have to: 1) examine the fields of science which use mice, 2) identify whether the science is performed with experimental rigour (design and conduct), and then 3) evaluate whether the findings obtained from these rigorous experiments are reproducible. By and large, the scientific community is still at step 2. As I mentioned previously, many fields which conduct research using mice report results that are irreproducible. The current cause ascribed to these failures is poor experimental design and conduct. This insight is gained by analysing whether information related to experimental design and conduct in published manuscripts and experimental applications are reported. For many fields of study employing the use of rodents, we cannot even begin to evaluate the effectiveness of a model because the manner in which the study was reported was poor. It is worth emphasizing that poor reporting of aspects of a study related to experimental design and conduct does not necessarily imply that a study was conducted poorly. Ascertaining this information would require interviews for each published article in question; a Herculean, if not impossible, feat. As highlighted in my recent paper, many solutions have been put forward to improve the manner in which we execute and report experiments but until these are endorsed and enforced, science in general will not improve. And that also applies to research using humans as subjects.

Jeremy D. Bailoo, Ph.D.

The opinions expressed here are my own and do not necessarily reflect the interests of the the University of Bern or the Division of Animal Welfare at the University of Bern.

Truvada prevents HIV infection in high-risk individuals! A clinical success built on animal research

In the past two weeks we’ve learned of a major advance in ongoing efforts to halt the spread of  HIV, two separate clinical studies have reported that a daily regimen of a pill called Truvada as a pre-exposure prophylaxis (PrEP) is highly effective in preventing infection in high risk groups. This success is a result not just of the dedication of the clinicians who conducted these trials, but also of a series of pivotal studies conducted in non-human primates more than a decade ago that laid the scientific foundations for them.

In the first study of more than 600 high-risk individuals conducted at Kaiser Permanente in San Francisco, which was published in the journal Clinical Infectious Diseases, researchers found that Truvada – a combination of the anti-viral drugs tenofovir and emtricitabine – was 100% effective in preventing infection.  In the 2nd  study, called the PROUD study and published online this week in the Lancet, of more than 500 high-risk men undertaken in 13 sexual health clinics in England Truvada reduced infections by 86%.

Truvada prevents HIV transmission in high-risk individuals. Image: AFP / Kerry Sheridan

Truvada prevents HIV transmission in high-risk individuals. Image: AFP / Kerry Sheridan

These results have been greeted with enthusiasm in media reports, with headlines such as “Aids vanquished: A costly new pill promises to prevent HIV infection” , “A pill designed to prevent HIV is working even better than people thought” and  “Truvada Protected 100 Percent Of Study Participants From HIV: This is exciting!”. It’s worth noting that these are not the only trials to show the potential for Truvada to block HIV infection, earlier trials in Kenya, Uganda and Botswana also showed that it could substantially reduce infection rates, including in heterosexual couples where one partner was HIV positive and the other was not. There has been some concern that those taking Truvada would be less likely to take other safe sex measures – such as using condoms – but the results of the PROUD study showed no difference in acquisition of other sexually transmitted infections between those who started Truvada treatment immediately and those who delayed for 1 year, suggesting that they did not engage in riskier behavior as a consequence of taking Truvada.

Thanks to a multi-pronged approach to preventing HIV infection, combining barrier methods such as condoms,  Highly Active Antiretroviral Therapy (HAART) to lower viral load in infected individuals, and the use of antiviral medications to prevent mother-to-child transmission, the spread of HIV infection has slowed dramatically in many regions of the world, and pre-exposure prophylaxis with Truvada certainly has the potential to help reduce it further.

As we applaud the researchers who conducted these first real-world evaluations of Tenofovir in high-risk populations, it is also a good opportunity to remember the researchers whose work led us to this point. One of those pioneers is Dr. Koen Van Rompay, a virologist at the University of California at Davis who played a key role in the early development of Tenofovir and  its evaluation in pre- and post- exposure phophylaxis in macaque models of HIV infection. In 2009 Dr Van Rompay wrote an article for Speaking of Research explaining how important animal research was to the early development of such HIV prophylaxis regimes, and how important it continues to be as scientists develop ever better treatments, which we share again today:

Contributions of nonhuman primate studies to the use of HIV drugs to prevent infection – Koen van Rompay

Since the early days of the HIV pandemic, as soon as it was clear that an effective HIV vaccine would still be years away, there has been considerable interest in using anti-HIV drugs to reduce the risk of infection following exposure to HIV (so-called prophylaxis). Animal models of HIV infection, especially the rhesus macaque, have played a major role in developing and testing these treatments.

The development of HIV drugs to treat HIV-infected persons has shown that many compounds that are effective in vitro (i.e., in tissue culture assays) fail to hold their promise when tested in humans, because of unfavorable pharmacokinetics, toxicity or insufficient antiviral efficacy. The same principles apply to the development of drugs to prevent HIV infection. The outcome of drug administration is determined by many complex interactions in vivo between the virus, the antiviral drug(s) and the host; with current knowledge, these interactions cannot be mimicked and predicted sufficiently by in vitro studies or computer models.

Testing different compounds in human clinical trials is logistically difficult, time-consuming and expensive, so only a very limited number of candidates can be explored in a given time. Fortunately, the development of antiviral strategies can be accelerated by efficient and predictive animal models capable of screening and selecting the most promising compounds. No animal model is perfect and each model has its limitations, but the simian immunodeficiency virus (SIV) of macaques is currently considered the best animal model for HIV infection because of the many similarities of the host, the virus and the disease. Non-human primates are phylogenetically the closest to humans, and have similar immunology and physiology (including drug metabolism, placenta formation, fetal and infant development). In addition, SIV, a virus closely related to HIV-1, can infect macaques and causes a disease that resembles HIV infection and AIDS in humans, and the same markers are used to monitor the disease course. For these reasons, SIV infection of macaques has become an important animal model to test antiviral drugs to prevent or treat infection.

Studies in rhesus macaques first indicated that Tenofovir could block HIV infection. Photo: Understanding Animal Research

Studies in rhesus macaques first indicated that Tenofovir could block HIV infection. Photo: Understanding Animal Research

Different nonhuman primate models have been developed based on the selection of the macaque species, the particular SIV strain and the inoculation route (e.g. IV injection, vaginal exposure) used (reviewed in (33)). These models have been improved and refined during the past two decades. For example, SIV-HIV chimeric viruses have been engineered to contain portions of HIV-1, such as the enzyme reverse transcriptase (“RT-SHIV”) that the virus requires in order to multiply or the envelope protein (“env-SHIV”) that the virus needs if it is to escape from a cell and infect other cells, to allow these models to also test drugs that are specific for HIV-1 reverse transcriptase or envelope (28, 35).

Many studies in non-human primates have investigated whether the administration of anti-HIV drugs prior to or just after exposure to virus can prevent infection. The earliest studies indicated that drugs such as the reverse transcriptase inhibitor zidovudine (AZT), the first approved drug treatment for HIV, were not very effective in preventing infection, but a likely reason for this was the combination of a high-dose viral inoculums used, the direct intravenous route of virus inoculation, and the relative weak potency of drugs at that time (2, 4, 13, 19, 20, 36). The proof-of-concept that HIV drugs can prevent infection was demonstrated in 1992 when a 6-weeks zidovudine regimen, started 2 hours before an intravenous low-dose virus inoculation that more accurately represented HIV infection in humans, protected infant macaques against infection (29). These results were predictive of a subsequent clinical trial (Pediatric AIDS Clinical Trials Group Protocol 076), which demonstrated that zidovudine administration to HIV-infected pregnant women beginning at 14 to 34 weeks of gestation, and continuing to their newborns during the first 6 weeks of life reduced the rate of viral transmission by two-thirds (10).

Since then, a growing number of studies have been performed in macaques to identify more effective and simpler prophylactic drug regimens. These studies generally used lower virus doses, sometimes combined with a mucosal route of virus inoculation that mimics vaginal or anal exposure responsible for the majority of human HIV infections. These studies demonstrated that administration of some newer anti-HIV drugs, including the reverse transcriptase inhibitors adefovir (PMEA), tenofovir (PMPA), and emtricitabine (FTC) that prevent the virus from multiplying in the infected cell, and the CCR5 inhibitor CMPD167 that stops the virus from binding the CCR5 receptor on the cell surface and entering a cell in the first place, starting prior to, or at the time of virus inoculation, was able to prevent infection, though with varying success rates (3, 4, 16, 24, 25, 31, 34, 35). Only very few compounds such as the reverse transcriptase inhibitors tenofovir, BEA-005 and GW420867, and the CCR5 inhibitor CMPD167, were able to reduce infection rates when treatment was started after virus inoculation. For those drugs that were successful in post-exposure prophylaxis studies, a combination of the timing and duration of drug administration was found to determine the success rate, because a delay in the start, a shorter duration, or interruption of the treatment regimen all reduced the prophylactic efficacy (5, 11, 21, 22, 26, 27, 31) , information that has guided the design of subsequent clinical trials.

While some of the compounds such as GW420867 that showed prophylactic efficacy in the macaque model are no longer in clinical development (e.g., due to toxicity or pharmacokinetic problems discovered later in pre-clinical testing), the very promising results achieved with tenofovir have sparked further studies aimed at simplifying the prophylactic regimen. Several studies in infant and adult macaques have demonstrated that short or intermittent regimens of tenofovir (with or without coadministration of emtricitabine) consisting of one dose before and one dose after each virus inoculation were highly effective in reducing SIV infection rates (15, 30, 32).

The demonstration at the beginning of the 1990’s that anti-HIV drugs can prevent infection in macaques has provided the rationale to administer these compounds to humans to reduce the likelihood of infection in several clinical settings. Antiviral drugs are now recommended, usually as a combination of several drugs, to reduce the risk of HIV infection after occupational exposure (e.g., needle-stick accidents of health care workers) and non-occupational exposure (e.g. sex or injection-drug use) (6, 7). As mentioned previously, drug regimens containing zidovudine and more recently also more potent drugs such as nevirapine have proven to be highly effective in reducing the rate of mother-to-infant transmission of HIV, including in developing countries (10, 14, 17), and save many thousands of lives every year . Because the short nevirapine regimen that is given to pregnant HIV-infected women at the onset of labor frequently induces drug resistance mutations in the mother that may compromise future treatment (12), tenofovir’s high prophylactic success in the infant macaque model has sparked clinical trials in which a short tenofovir-containing regimen was added to existing perinatal drug regimens to reduce the occurrence of resistance mutations and/or further lower the transmission rate (8, 9, 18, 30, 32).

Scanning electron micrograph of HIV-1, colored green, budding from a cultured lymphocyte. Photo: C. Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus

Scanning electron micrograph of HIV-1, colored green, budding from a cultured lymphocyte. Photo: C. Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus

Because an efficacious HIV vaccine has so far not been identified, the concept of using pre-exposure prophylaxis also as a possible HIV prevention strategy in adults has gained rapid momentum in recent years. The promising prophylactic data of tenofovir (with or without emtricitabine) in the macaque model (23, 32, 35, 37) combined with the favorable pharmacokinetics, safety profile, drug resistance pattern and therapeutic efficacy of these drugs in HIV-infected people, have pushed these compounds into front-runner position in ongoing clinical trials that investigate whether uninfected adults who engage in high-risk behavior will have a lower infection rate by taking a once daily tablet of tenofovir or tenofovir plus emtricitabine. The results of these ongoing trials are highly anticipated. An overview of the design, status and challenges of these trials which are currently underway at several international sites and target different high-risk populations can be found on the website of the AIDS Vaccine Advicacy Coalition (1, 23).

In conclusion, nonhuman primate models of HIV infection have played an important role in guiding the development of pre- and post-exposure prophylaxis strategies. Ongoing comparison of results obtained in these models with those observed in human studies will allow further validation and refinement of these animal models so they can continue to provide a solid foundation to advance our scientific knowledge and to guide clinical trials.

Koen van Rompay DVM Ph.D. is a research virologist at the California National Primate Research Center at UC Davis.

Cited literature
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Guest Post: How do birds see the world?

Professor Aaron Blaisdell

Professor Aaron Blaisdell

Today’s guest post is from Professor Aaron Blaisdell and graduate student Julia Schroeder in the Department of Psychology at the University of California Los Angeles. Prof. Blaisdell’s area of research is animal learning and comparative cognition. He received his Ph.D. in Experimental Psychology and Behavioral Neuroscience at Binghamton University in 1999. Julia Schroeder is a graduate student in the Psychology Department at UCLA. This project is the basis of her dissertation research which she hopes to complete by May, 2016. She received a BS in Psychology at Whitman College where she compared rational decision processes in pigeons and humans. You can support their research through their crowdfunding campaign.

How do birds see the world?

How do birds fly around objects without crashing into them? Their object perception must be similar to ours, despite having a dramatically different brain and separate evolutionary history. Birds and mammals share a last common ancestor roughly 275 million years ago! Nevertheless, most birds and mammals, especially primates, rely on sight to navigate their world, find mates, avoid foes and predators, seek food and water, and care for their young.

Vision, both sensation and perception, has been one of the top areas of research in experimental psychology and neuroscience, going back to the visual psychophysics scientists of 19th century Germany. Visual perception and cognition is currently a dominant area of study in cognitive neuroscience. Much of what we’ve learned about human vision actually comes from research in nonhuman primates, especially the macaque monkey. This makes sense, since the human visual system is like that of just monkeys and apes.

One picture that has emerged is that, when we open our eyes, we see a world populated with objects. Our object-centered view of the world is also shared with the rest of the primates.

Pigeon in a test of comparative cognition.

Pigeon in a test of comparative cognition.

What about birds? They also navigate their world using vision. Flying puts high demands on the ability to rapidly detect and process visual information. The last thing a birds wants to do is to fly into an object because it couldn’t see it in time! This suggests that bird brains also engage in visual computational processes similar to that of the primate. But we currently don’t know much about how they do so. We want to know if birds solve the incredibly complex computational process of object perception the same way that primates do.

In our next research project, we plan to test whether bird brains handle object perception the same way that the human brain does. Pigeons will play a video game where they have to rapidly peck objects as they appear on a computer touchscreen located in a Skinner box. As soon as the object is pecked, a small food reward will be delivered to the pigeon from a hopper located below the screen. The faster the pigeons peck at the object, the sooner they get fed. The speed of their responses will tell us how the birds see the objects.

Specifically, we will show the pigeons four different objects, A, B, C, and D, one at a time (actual objects are different colored geometric shapes). The objects will appear in one of four locations on the screen (see a demo here). The objects will appear in a specific order that repeats. The locations in which the objects appear will also repeat. This will allow us to test how pigeons bind features into objects. If pigeons integrate features as humans and other primates have been shown to do, then they should learn that specific objects always appear in a specific location. This is called object-place learning. The object’s identity and location become bound as shared properties of a unique, coherent object. After the pigeons learn to play the game, we can then test for object-place learning by presenting special non-reinforced probe test trials. On these test trials, we will change the order of some of the objects, locations, or both.

Stimuli in learning sequence.

Stimuli in learning sequence.

Changing only the object or location should break the object-place association. Changing both together, however, preserves the object-place association, even though the sequence order has changed. If, like humans, pigeons bind object and location information together into perceptual memory, then changing only the object or the location order should be more disruptive than changing both!

What is life like for a laboratory pigeon?

Like all other vertebrates in research, housing and laboratory conditions for pigeons are well regulated. All research protocols go through the same stringent processes of review by the University’s IACUC, and the health and welfare of each pigeon is overseen by the Division of Laboratory Animal veterinarian staff. They receive the best possible care. In a typical pigeon laboratory, the pigeons are maintained as part of a flock in a vivarium. Birds are typically individually housed in large, comfortable cages, with constant access to water and grit. Feeding times are typically restricted to the afternoon after all subjects have completed their behavioral training. This keeps them motivated to work for food reinforcement in the operant chamber, and maintains subjects at a healthy weight similar to that of pigeons in the wild. Despite being housed in individual cages, the birds can see, hear, and smell the birds in the surrounding cages, thereby simulating a flock as it would be found in the wild. Unlike most mammals, or even parrots, pigeons do not engage in much touching or grooming of each other. Rather, pigeons in a flock hang out in close proximity to one another.

While some labs acquire wild-caught pigeons from their local area, we purchase ours from a vendor that breeds pigeons and other fowl for research purposes. Pigeons are a domesticated species, having lived in human environments since the dawn of agriculture in the Mediterranean region of Europe, Asia, and North Africa. Darwin was a known pigeon fancier, and bred pigeons as part of his own experimental investigations into the process of evolution by natural selection! To this day, there are pigeon fanciers and clubs around the world that breed pigeons for show, racing, and aerial acrobatics.

Why is this research significant?

The bird brain has a very different organization than the brains of humans and other mammals. Birds don’t have a visual cortex, for example. Thus, our research can lend insight into how a brain of such different structure solves the same computational process as does the mammalian brain.

Also, the brain of a pigeon is the size of your thumb! So how can birds, like pigeons, see objects the way that we do with far fewer neurons than in the human or monkey brain? Knowing how birds see the world can tell us a lot about what is unique about human vision, and what we share with other species.

Finally, we can also use our knowledge of how small bird brains efficiently create visual objects out of messy input to find new and powerful ways to build artificial visual systems for small mobile devices, such as drones and robots.

Many neuroscientists believe object perception is one of the most important and central processes of human vision. Nevertheless, object vision has been incredibly difficult to build into robot vision using AI approaches. Perhaps we can reveal the secrets to complex object perception in the small pigeon brain that will allow for breakthroughs in computer vision. This would be a huge win for human society!

Aaron Blaisdell and Julia Schroeder

Behind the Scenes of Zebrafish Research

Today we have the 2nd in a series of articles by Jan Botthof, a PhD Student at the Cambridge University Department of Haematology and the world renowned Wellcome Trust Sanger Institute. Following his first article “Zebrafish: the rising star of animal models”, Jan discusses here how Zebrafish used in scientific research are housed, cared for and bred.

Today I am going to look at some of the things that have to happen in the background to allow scientists to carry out their research. These things include the rules and regulations covering zebrafish use in research, general care and daily work in the fish facility.

Zebrafish research in the UK is covered by the same laws that govern research on all other vertebrates, as outlined in the Animals (Scientific Procedures) Act (ASPA), originally instated in 1986 and recently revised to implement the provisions of the new EU directive. This means that the standard of care is just as high for fish as for mammals. All institutes housing fish need a licence ensuring that standards are met, every research project is evaluated for possible harm to the animals and all of the people involved in research or care for the fish receive mandatory training in order to ensure that the fish are treated correctly. Everyone takes utmost care to ensure that the fish lead a comfortable life in the zebrafish facility.

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish housing
Now that we have covered the basic legislation, let’s talk about essential zebrafish care. Nowadays, fish are usually housed in special rooms, unlike the beginning of their use in research back in the 1970’s, when they were commonly just kept in a few tanks on a shelf in the lab. These rooms are designed to keep a constant temperature (between 24 and 28°C depending on the institution) and the lights are programmed to give a constant light-dark cycle to simulate the sun (usually around 16 hours of light and 8 hours of darkness).

Various commercial fish housing systems exist, but most of their features are very similar. The basic components of such a system are the fish tanks, racks to hold them, an integrated water supply, as well as water filtration and monitoring components.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

The tanks are designed to allow a constant inflow of fresh water, easy removal from the rack and convenient access for feeding. These tanks used to be made of glass, but currently different kinds of plastics are much more popular due to the lower weight, making it much easier to handle them. Unless a procedure requires identification or separation of a specific fish, they are always kept in groups, not only for practical reasons, but also because zebrafish are very social animals and need interaction with other fish.

The water filtration and monitoring system ensures that the water is free of contaminants, has the right pH, salinity, hardness, enough dissolved oxygen and does not contain too much nitrite and nitrate stemming from waste products (i.e. fish excretions, excess food). Apart from the constant flow of fresh water, tanks are cleaned regularly to prevent the accumulation of waste products, as well as microbial and algal growth.

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

A separate quarantine room is also very important. This is where incoming fish from outside facilities are kept on a separate water system to prevent the introduction of parasites and diseases into the main facility. These fish are preferably received as early embryos, which are disinfected before shipping to kill any germs.

Just like there are different housing systems, there are different possible food sources, ranging from commercially available dry fish flakes to adult or larval brine shrimp. This diet is often supplemented with paramecia (small single celled organisms) to achieve optimal growth and survival rates when the fish are raised to adulthood. Exactly which diet is chosen depends on the individual facility. At the Sanger Institute, fish are fed adult brine shrimp, which are very rich in protein, soft and easily digestible (especially compared to brine shrimp cysts) and they are able to survive and swim even in fresh water. This is much closer to the natural diet that the fish would obtain in the wild than most commercially available diets.

Brine Shrimp. Image: Hans Hillewaert

Brine Shrimp. Image: Hans Hillewaert

Disease prevention
During the daily cleaning and feeding tasks, all tanks are monitored for diseased or injured fish, which are then humanely euthanized to minimize suffering. Euthanasia is usually carried out using an overdose of a common fish anaesthetic followed by destruction of the brain to ensure the death of the animal. Detailed records are kept to identify recurrent problems, such as potential parasitic infections. Dead fish are also removed from the tanks and their data recorded. This monitoring is especially important for fish that have been treated with drugs or that carry mutations likely to cause disease.

One essential component of working with fish is setting up matings between them. This is essential if you want to obtain embryos for studying them, or when crossing different genetically modified lines and many other procedures. Fish are placed in small mating tanks in the late afternoon before the actual mating, as zebrafish begin to mate right after sunrise in the wild. These mating tanks have a removable insert between the fish and the floor of the tank, so the fish cannot consume their own eggs, which they would otherwise do.

It is also possible to tilt the separation between the floor of the tank and the fish to further stimulate the fish, as they prefer shallow water for egg laying. If you need the embryos at a specific stage you can use tanks with a separator between the male and female, so you can control the time of the mating. An occasion when you would need to do this is when using the gene editing technique CRISPR to modify zebrafish genes, a process which requires injections of the Cas9 enzyme and appropriate guide RNAs during the first stage of development. Matings can be done in small groups or in pairs. It is very important to be able to correctly identify the sex of the animals – not only do you obviously need a male and a female to have a successful mating, but you also need to know this when you combine different transgenic lines. Here you would take a male from one line and a female from another, so you can put them back in the correct tank after the mating, as it is otherwise nearly impossible to identify individual fish.

Zebrafish mating tank with removable separation before and after assembly.

Zebrafish mating tank with removable separation before and after assembly.

Once the eggs have been laid and fertilized, you can collect them in a sieve, and place them in petri dishes containing water with some salts and minerals essential for development. Embryos are then raised at 28.5°C. Here at the Sanger, the zebrafish larvae are placed in nursery tanks when they are five days old and the yolk that feeds them during early development has run out. These fry reach sexual maturity within three months, from which point on they are considered adults and housed in the main facility. Zebrafish in the lab can live about two to three years, but usually we use younger fish for breeding, as they  lay more eggs.

In summary, a lot of work needs to be done before any actual research can be carried out. Moreover, a lot of effort is put into ensuring the health and welfare of all laboratory animals. The next time you read about some exciting new discovery made using animal research, try to picture how much effort was needed before any actual science was done!

Jan Botthof

Cotton Rats, Calves and Clinical Trials: New RSV vaccine shows great promise.

Respiratory syncytial virus (RSV) affects almost two-thirds of babies in their first year of life, and is a leading cause of bronchiolitis and severe respiratory disease in infants, young children, immunocompromised individuals, and the elderly throughout the world. It is a major cause of hospital admission for infants, and results in up to 200,000 deaths per year in children under the age of 5 years worldwide. Development of an effective vaccine is a public health priority, but has proven difficult, in part due to fact that RSV infection cannot be easily studied in the standard mouse and rat species that are most commonly used in laboratory research.

Oxford University researchers have announced the successful completion of the first trial of a vaccine against RSV in adult humans, which indicated that the vaccine was safe and could induce a robust immune response (though this Phase 1 study did not evaluate its ability to protect against RSV).

In winter RSV accounts for more than 10% of UK infant hospitan admissions.

In winter RSV accounts for more than 10% of UK infant hospital admissions.

In two papers published back-to-back in the journal Science Translational Medicine this week, the University of Oxford team and their colleagues at The Pirbright Institute and the Italian biotechnology firm Okairos (now Reithera Srl) report on the successful Phase 1 clinical trial in adult human volunteers, and the animal studies that led to the trial (1,2).

The basis for their vaccine was a vector derived from the chimpanzee adenovirus PanAd3, which was modified to express several highly conserved human RSV (HRSV) proteins, which they had shown could provide a good level of protection against RSV infection in cotton rats, which are one of the less commonly used laboratory animals, but a very useful model of viral infection of the respiratory system. The cotton rat immune system more closely resembles that of humans at a genetic level than that of more commonly used laboratory mouse and rat species, and because of this similarity cotton rats have been successfully used to evaluate novel therapies for RSV prior to clinical trials, including predicting the efficacy of these drugs in children. As we discussed in an earlier post on the development of gene therapy for Hemophilia B, choosing the right adenoviral vector for the task is critical, and the chimpanzee adenovirus was chosen because there is no preexisting immunity to it in the human population that would compromise its effectiveness as a vaccine vector.

Because studies with other virus-vectored vaccines had shown that heterologous prime/boost with Chimpanzee adenovirus based vector followed several weeks later by a modified vaccinia Ankara (MVA) – a vector used in several experimental vaccines, including HIV vaccines – generates a stronger  immune response than with chimpanzee adenoviral vector alone, the researchers next examined this strategy in cotton rats, demonstrating that intranasal prime/boost immunization was effective in protecting against infection, and did not lead to adverse effects.

The cotton rat - a valuable model for studying lung disease. Image: J.N. Stuart

The cotton rat – a valuable model for studying lung disease. Image: J.N. Stuart

While the cotton rat is a valuable model for the study of respiratory infections, it does not demonstrate all the clinical features of RSV infection, the infection tends to be less severe and to be cleared more quickly in cotton rats than in human infants, and  the extent to which vaccine efficacy in the cotton rat model of HRSV can predict efficacy in humans  is unclear. The scientists therefor evaluated the prime/boost vaccine in a more stringent model, calves, which are the natural hosts of the bovine form of RSV – called bovine RSV (BRSV). The disease course and epidemiology of BRSV infection in calves is very similar to that of HRSV in children, and the very high degree of similarity in sequences of the BRSV proteins to their HRSV equivalents used to develop the vaccine  suggested that the calf would be a valuable preclinical animal model to evaluate the safety and efficacy of their prime/boost vaccine strategies (2).

The results of this evaluation indicated that the different vaccination strategies they assessed were safe and did not cause any adverse immune responses, and showed that heterologous vaccination strategies that used an intranasal administration of the  PanAd3-based vector followed by intramuscular administration of the MVA-vased vector provided superior protection against BRSV infection compared to the PanAd3-based vector alone, or repeated doses of the PanAD3-based vector. As the authors described in the article reporting on the successful phase 1 trial (1) noted in their introduction, these studies paved the way for the evaluation of this vaccine strategy in 42 human volunteers:

In developing this approach toward an RSV vaccine in humans, homologous and heterologous combinations of PanAd3-RSV, including IN vaccination route, and MVA-RSV were tested in preclinical models. The genetic vaccines elicited RSV-specific neutralizing antibodies and T cell immunity in nonhuman primates and protective efficacy in challenge experiments in rodents with human RSV and in young seronegative calves with bovine RSV (32, 33). Of critical importance in both rodent and bovine challenge models was the absence of immunopathology associated with ERD after vaccination, with the calf model acting as a translational model for the development of a vaccine for the pediatric population. All regimens fully protected the lower respiratory tract from bovine RSV infection in the calf, and heterologous combinations resulted in sterilizing immunity in both upper and lower respiratory tracts (33).

The demonstration that the prime/boost vaccine strategy developed by the Oxford University team can safely induce a strong immune response in adult humans, and protect against RSV infection in both the cotton rat and calf models, is very promising, and pave the way for further clinical trials.  Professor Andrew Pollard of the Oxford Vaccine Group, who lead the clinical trial, is keen to now move the development of this much needed vaccine forward:

Both components of the vaccine were found to be safe and to create an immune response.

‘While I am delighted with these results, this was just a first trial. We need this vaccine for children and the elderly and that is where the efforts in vaccine development will now focus.’

Paul Browne

  1. Christopher A. Green et al. “Chimpanzee adenovirus– and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults” Science Translational Medicine, Vol 7, Issue 300, 300ra126, 12 August 2015 Link
  2. Taylor G. et al.”Efficacy of a virus-vectored vaccine against human and bovine respiratory syncytial virus infections” Science Translational Medicine, Vol 7, Issue 300, 300ra127, 12 August 2015 Link

Canada Releases 2012 Animal Use Statistics

Earlier this month the Canadian Council on Animal Care (CCAC) released its report on the number of animals used in Canada for scientific purposes. The CCAC is an independent oversight body that oversees the ethical use of animals in research. They also develop guidelines and promote training programs to ensure that all individuals involved in animal research or welfare are properly trained before being allowed to work with the animals. The CCAC reports that in 2012, 2,889,009 animals were used for research, teaching and testing in Canada. This is down 444,680 animals, from 3,333,689 animals that were used in 2011. These numbers include all vertebrates and Cephalapods, but do not include invertebrates like fruit flies or nematode worms. Animals can be used in more than one protocol provided these additional protocols do not result in pain.

2012 Canadian Animal research and testing Graph

Mice (43.2%), fish (28.8%), rats (7.8%) and birds (6.6%) were the most common species, together accounting for 86% of animals used. These numbers represent a shift in the type of animal used, as fish have been the animal most frequently used by Canadian institutions for the past three years. The majority of animals (61%) were used in studies of a fundamental nature/basic research, representing 1,815,083 animals. There has been significant changes to the reporting methodology utilized to analyze the current data and the CCAC made the following statement with respect to the 2012, report:

“Due to these differences in data management and reporting, it is not possible to make accurate comparisons with CCAC PAU and CI data from previous years.”

2012 Canadian Research and Testing Table

More information about animal research in Canada can be found within the Speaking of Research Media Briefing Notes for Canada.