medical research animal experimentation

Ethics of Medical Research with Animals

U.S. Law and Animal Experimentation: A Critical Primer

Every country’s law permits medical experimentation on animals. While some countries protect particular kinds of animals from being subject to experimentation—notably great apes and endangered species—very few place concrete limitations on what researchers may cause animals to suffer, given sufficient scientific justification. What laws do, instead, is establish standards for the humane treatment and housing of animals in labs, and they encourage researchers to limit or seek alternatives to the use of animals, when doing that is consistent with the scientific goals of their research. The result, of course, is that no existing regulatory scheme is satisfactory to opponents of animal research. The law, in their view, does nothing more than make the animal research scientist into a sort of James Bond villain: superficially polite, offering fine housing and well-prepared cuisine even to those whom he intends, eventually, to kill.

Of course, the goals of animal experimentation law seem much more reasonable if one accepts that research on animals is both important for medical progress and morally permissible. On those assumptions, it makes a great deal of sense for the law to aim primarily at limiting unnecessary animal suffering even as it licenses scientifically justified experimentation. U.S. law accepts those assumptions and adopts that aim.

The system that has evolved in the United States combines elements of sometimes competing regulatory philosophies. The result is a complex, multilayered system that addresses the most important concerns, but, partly because of historical accident, also leaves some gaps. Even proponents of medical research on animals can see obvious ways in which the regulatory structure could be changed to benefit animals. Perhaps more important, though, is the fact that the existing regulatory structure, imperfect though it may be, is elastic enough to accommodate substantial changes that could reduce unnecessary animal suffering.

Multiple Regulatory Approaches

Animal welfare laws must address three main ways in which unnecessary animal suffering can occur in the context of medical experimentation. First, such suffering can occur when a given research protocol is not well justified scientifically. An experiment that was so badly designed that it could never generate any useful scientific knowledge would never warrant animal suffering. Harder cases result when the amount of suffering is ratcheted down, or the experiment’s potential to generate useful knowledge is ratcheted up. A legal regime concerned with avoiding this kind of unnecessary suffering can opt to trust in the judgment of each individual research scientist, or empower someone besides the researcher to make at least some baseline assessment of the scientific value of each new animal research protocol. It can also provide information and guidance to researchers or overseers to improve their decisions.

Second, unnecessary suffering can occur when the amount of animal suffering induced by an experiment is not strictly required to conduct the experiment—perhaps because more animals are used than are necessary; or because less sentient animals could be substituted for more sentient ones, or computer or tissue models substituted for animals entirely; or because crude experimental procedures are producing avoidable stress or pain. A legal framework seeking to avoid these kinds of unnecessary suffering will encourage or require researchers to use the three Rs: reduce (the number of animals used in experiments), replace (animals with nonanimals, higher-order animals with lower), and refine (experimental procedures causing pain or distress). [1]

Third, unnecessary suffering can occur outside the actual research protocol yet still in the research setting because of inappropriate animal handling, housing, and feeding practices. A legal regime seeking to avoid this kind of suffering will dictate humane standards for animal housing and care.

Given these goals, what sort of regulatory scheme would be best at realizing them? One can imagine a variety of available approaches, from strong, centralized state regulation and monitoring of all experimentation to a hands-off reliance on professional self-regulation among laboratory researchers. On the world stage, the United Kingdom is closest to taking the former approach, Japan to the latter. U.S. law falls somewhere in the middle, in part because U.S. law in this area is in fact the result of a gradual, decades-long merging of the government regulatory and professional self-regulatory approaches. [2]

The government regulatory approach is embodied in the sprawling, strange, and often amended Animal Welfare Act of 1966. In its original form, the AWA was designed to control pet breeding and sale practices; it was passed, in part, as a result of public outcry about the mistreatment of dogs sold to laboratories. As amended, it governs the treatment of animals in a wide range of settings, from pet shops to circuses and from zoos to laboratories. Its enforcement is delegated to the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service, whose inspectors make unannounced site visits to research facilities. Violations uncovered on such visits can result in fines and even, in extreme cases, criminal prosecution. The most common complaint about enforcement under the AWA is that it is rigid and mechanistic.

Because of its historical roots in concern for pets, the AWA’s reach is confined to warm-blooded animals, and it contains special regulations addressed to certain animal favorites: dogs, cats, rabbits, and monkeys. Its animal experimentation regulations apply to any school or research facility that purchases or transports live animals in interstate commerce or that receives federal funding. But in fact the law has never reached the bulk of warm-blooded animals actually used in research. Concern about high regulatory costs—and about possible delay in creating guidelines for other, more popular animals—led the USDA to exclude laboratory rats and mice from its oversight from as early as 1970. In spite of lobbying efforts in the 1980s by proanimal groups, a congressional amendment to the AWA in 2002 legally formalized the agency’s longtime practice, excluding rats, mice, and birds from the definition of “animal.” [3]

In general, the law and its implementing regulations have focused on setting demanding, detailed standards for animal housing and basic standards for pain control. It supports only minimal review of the scientific merit of research protocols, but it requires researchers to make efforts to “reduce, replace, and refine.”

The self-regulatory approach to animal research regulation is embodied in the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals . [4]   The Guide has existed in some version since 1963, when it was introduced as a voluntary set of professional standards for laboratory animal research. Today, the Guide’s standards are mandatory for all research facilities receiving federal funds. The Guide covers the treatment of all vertebrates, which means that, at least in federally funded research, it closes many of the gaps left open by the AWA. Not only are rats, mice, and birds covered, but also cold-blooded vertebrates like zebra fish—currently the go-to animal for laboratory studies of pain and nerve function.

The change in the Guide’s status to a rulebook has altered its content somewhat. Earlier editions’ expansive aspirational goals have given way in later editions to more readily applicable rules. There has also been considerable pressure to get the AWA’s regulatory requirements and the Guide’s standards to match, since all federally funded researchers are bound by both. Indeed, today, the two sets of standards are, if not identical, at least compatible with one another. But in general, where the AWA regulations are more rigidly prescriptive, the Guide permits lab veterinarians to use their professional judgment in applying general standards to particular species or protocols.

Clearly there is room for reform. If the AWA were amended to include rats, mice, and birds, for example, that would be a major step toward ensuring the humane treatment of all animals in public and private labs.  

Federal standards are full of specific requirements for different kinds of studies, but in general, it is fair to say that they offer the most concrete guidance on questions of animal housing and care. The regulations include detailed discussions of square footage, exercise requirements, room temperature, and more. Considerably less guidance is offered on issues of protocol evaluation and implementation of the three Rs.

Of course, this is exactly what might be expected given the incredible volume and variety of animal research in the United States. A central authority can say a lot about how to house and feed monkeys, mice, and zebra fish, and expert advice on those issues will apply to all monkeys, mice, and zebra fish in every lab, no matter what protocols they are being used for. But questions about the other possible sources of unnecessary animal suffering—the scientific justification of a given protocol, or the ways in which animal suffering connected to a given protocol might be avoided or reduced—are too numerous and varied to be answerable in advance by a central authority. With regard to those highly fact-specific questions, U.S. law relies on the expert judgment of local IACUCs.

It is no coincidence that this kind of reliance on decentralized expert committees is also the salient feature of U.S. law governing research on human subjects. The federal Common Rule, [5]  faced with a similar diversity of research protocols to evaluate, regulate, and modify, uses the same tactics as the AWA: it mandates creating research oversight committees (institutional review boards), specifies that their membership should include both relevant expertise and community representation, and empowers them to make and enforce a range of judgments about particular experimental protocols.

While the many IACUCs are expected to exercise independent judgment with regard to the scientific issues brought before them, the U.S. government does its best to inform the judgment by providing them with educational resources. The Public Health Service and the Department of Agriculture Web sites are full of guidance documents and educational resources for laboratory researchers and for IACUC members. There are documents, for example, with specific ideas about how and when to substitute lower-order animals for higher-order animals, and other documents providing up-to-date scientific news about newly developed computer models that can substitute, in some cases, for animal experimentation.

Finally, just as in the human subjects research world, federal regulations are quite commonly supplemented by private education and accreditation. Many research facilities seek accreditation by the Association for the Assessment and Accreditation of Laboratory Animal Care, a professional association of veterinarians and laboratory scientists. AAALAC provides education and does prearranged site inspections of labs once every three years. Educational and inspection standards are built largely around the requirements of the Guide , and the NIH accepts AAALAC accreditation as prima facie evidence of a facility’s compliance with the Guide’s requirements.

Toward Reform: Accountability, Uniformity, Balance

The system of decentralized oversight by local IACUCs has several obvious advantages: it permits oversight by people with knowledge of the local researchers and laboratory facilities; it allows IACUCs to develop specialized knowledge, well tailored to the research being done at their facilities; and it is likely more speedy than any alternative program of centralized governmental research oversight would be. On the other hand, the decentralization of oversight has given rise to a number of problems—which, not surprisingly, are similar to those that beset the IRB system in human subjects research.

First, there is a problem of transparency and accountability. IACUCs are for the most part fairly anonymous. Hardly anyone not directly involved in animal research knows that they exist, much less who their members are. And of course, their members are not elected or in any other way publicly accountable for the decisions they make. Most IACUC decisions do not take the form of opinions or any other form of substantive, publishable decision, but of recommendations to researchers for piecemeal alteration of protocols. A central repository of IACUC minutes, and of policies adopted by different IACUCs, might both increase accountability and stimulate new ideas by creating cross talk between IACUCs. But any such repository would have to be created with an eye toward preserving researchers’ intellectual property.

Second, decentralization almost necessarily gives rise to a lack of uniformity in decision-making and in quality of research oversight. One IACUC may conclude that a protocol involves unnecessarily harsh treatment of animals or presents an opportunity for substitution of nonanimal models; another may view the original protocol as unproblematic and requiring no amendment. A number of studies have shown that similar protocols are treated quite differently by different IACUCs. [6]  It is unclear what the implications of such findings are. Do they reveal that IACUCs have differing standards relating to animal welfare? That they judge similar protocols differently when they are presented by different researchers? Or some combination of these factors? In any case, enforced uniformity across IACUCs is a dangerous solution to propose for the problem of varying standards, in the absence of clear knowledge about whose standards are appropriate—and whose would be enforced.

A third complaint about the decentralized approach to animal-research regulation involves the perception that the U.S. government is too deferential to local IACUCs and does not take the task of auditing labs sufficiently seriously. In the early 2000s, there were some high-profile allegations made by whistleblowers from the USDA’s Animal and Plant Health Inspection Service (APHIS) that audit findings were deliberately being watered down to be less critical than the field officers originally intended them to be. [7]  U.S. audits of APHIS confirmed allegations of lax auditing in some regions of the country. [8]  The obvious reform here is to better fund and train both the regulatory overseers and those who audit their performance.

There are other important criticisms of the U.S. regulatory regime not directly connected to its choice of decentralized decision-making. First, there is the question of scientific justification for animal suffering. The AWA does not ask IACUCs to balance animal suffering against the scientific merit or promise of any given experiment. Instead, it asks IACUCs to ensure only that any given protocol has scientific merit and that any animal suffering the protocol induces is strictly necessary to that science. The result is that any study that will advance science, even in a very small way, can be used to justify tremendous amounts of animal suffering, as long as the suffering is necessary to the advance. Though they do seek to modify studies via use of the three Rs, IACUCs almost never reject protocols.

Finally, and most importantly, there is the issue of which animals are protected. As already mentioned, the hundreds of thousands of rats, mice, and birds used in private, nonfederally funded labs are not subject to any federal regulation. (Some individual states’ anticruelty statutes may apply in some cases, but there is very limited case law in the area.) Excluded, also, are cold-blooded animals. This means that there is no federal legal pressure on private firms such as drug companies to reduce or refine animal use, or to replace animals with computer or tissue models—a strategy that may be particularly feasible in studies of toxicology or drug metabolization.

Even in federally funded facilities, the living conditions of rats, mice, and birds are not subject to the USDA’s APHIS inspection; only in AAALAC-accredited facilities is there oversight beyond self-reporting, and AAALAC does scheduled inspections only once every three years. Rats and mice, it should be stressed, are the most commonly used laboratory animals. In addition, U.S. law offers no protection for invertebrate, cold-blooded animals such as cephalopods. By contrast, Europe has recently moved to protect cephalopods in light of their manifest intelligence and sentience. Nor does U.S. law prevent research on great apes, or ban (though it does regulate) the use of wild-caught animals. And the United States is one of only two governments in the world that still permits invasive research on chimpanzees, though the scope of federal funding for chimp research has recently been sharply limited. [9]  (See “Raising the Bar: The Implications of the IOM Report on the Use of Chimpanzees in Research,” in this volume.)

Clearly there is room for reform. Some needed reform involves stepping up research oversight. If the AWA were amended to include rats, mice, and birds, for example, that would be a major step toward ensuring the humane treatment of all animals in public and private labs. In addition, the inspection rate for facilities could be more frequent. Publicly funded U.S. labs are inspected by APHIS about once a year, by their own IACUCs twice a year, and by AAALAC (if they choose to be AAALAC-certified) once every three years. Compare this to the U.K. system of inspecting about once a month. Other reforms could involve improving rigid and not-terribly-useful existing regulations, like cage-size requirements currently based on animals’ body size rather than on their behavioral needs. Most significantly, the law could be reformed to permit a more explicit balancing of harms to animals (including both suffering and death) against the scientific gains at which the research aims. Empowering IACUCs to engage in such balancing is hardly radical; IRBs, for example, are already empowered to engage in such balancing in the human subjects research area, and this has not caused research to grind to a halt. Such a reform would require us to confront directly the question of how much suffering humans can impose on other species in return for small but real gains in knowledge.

Finally, a great deal can be accomplished even within an unchanged legal regime. The most urgent need is for more to be done to implement the three Rs. The familiar calls for better education about replacement techniques and more aggressive IACUC intervention on behalf of reduction and refinement are, of course, well justified. But even more dramatic reduction might be achieved if the goal of reduction were pursued not only within but also across protocols. There might be significant gains from putting animal-sharing procedures in place at the institutional level. At the moment, animals are commonly euthanized whenever the particular research project they’re involved in comes to an end, without regard to the animal’s age or health status. If a protocol involves attempts to breed, for example, mice with particular genetic traits, the pups born without those traits are routinely euthanized. If research facilities could work with researchers to use healthy animals from one study in another, rather than default to their euthanization, then fewer animals would need to be bred for suffering.

Stephen R. Latham is director of the Interdisciplinary Center for Bioethics at Yale University. He has published on a broad range of issues at the intersection of bioethics and law. He is a former board member of the American Society for Bioethics and Humanities, a former graduate fellow of Harvard’s Safra Center on Ethics, and a former research fellow of the University of Edinburgh’s Institute for Advanced Studies in Humanities. His current research includes a project funded by the Robert Wood Johnson Foundation to create a database of state statutes and cases criminalizing HIV exposure and a project on a legal framework for newborn whole-exome screening. 

• 1. The widely accepted “Three Rs” terminology was first introduced into the animal research literature in W.M.S. Russell and R.L. Burch, The Principals of Human Experimentation Technique (London: Methuen, 1959). ↵
• 2. A detailed account of the confluence of these two streams of regulation (to which my brief discussion here is heavily indebted) is provided by L. Carbone, What Animals Want: Expertise and Advocacy in Laboratory Animal Welfare Policy (Oxford, U.K.: Oxford University Press, 2004), p. 34ff. ↵
• 3. Wild-caught rats and mice are included in the regulations. For more detail, see Carbone, What Animals Want , p. 69ff. ↵
• 4. National Research Council, Guide for the Care and Use of Laboratory Animals , 8th ed.(Washington, D.C.: National Academies Press, 2011). ↵
• 5. U.S. Department of Health and Human Services, 45 CFR 46. ↵
• 6. See, for example, S. Plous and H. Herzog, “Reliability of Protocol Reviews for Animals Research,” Science 293 (2001): 608-9. ↵
• 7. See, for example, the statement of Dr. Isis Johnson-Brown, USDA whistleblower, alleging regulatory inaction on her report criticizing cage conditions at the Oregon Primate Center, at http://www.all-creatures.org/saen/articles-statementofijb.html, accessed October 2, 2012. ↵
• 8. USDA Office of Inspector General, Western Region, “Audit Report: APHIS Animal Care Program Inspection and Enforcement Activities,” Report No. 33002-3-SF, September 2005, p. i, http://www.usda.gov/oig/webdocs/33002-03-SF.pdf. ↵
• 9. See Institute of Medicine, Committee on the Use of Chimpanzees in Biomedical and Behavioral Research: Assessing the Necessity (Washington, D.C.: National Academies Press, 2011); B.M. Altevogt et al., “Guiding Limited Use of Chimpanzees in Research,” Science 335 (2012): 41-42. ↵

Ethical care for research animals

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Animal Research at Stanford University

CARING for Research Animals

Training future scientists, advancing human and animal health, stanford supports animal research, stanford university supports the conduct of biomedical research to further the understanding of the world in which we live and to apply this knowledge for the benefit of humans and animals..

Biomedical research draws on a variety of model systems to help answer questions about health and disease. Animals represent only one class of subjects for study. Human beings also are used extensively as research subjects. Alternatives to animal use, which include computer modeling, cell culture and bacterial systems, are used at Stanford whenever possible.

At Stanford, all research involving animals is subject to rigorous review by the University Administrative Panel on Laboratory Animal Care.

In addition, the federal and state governments, as well as independent accreditation organizations, work to ensure that research animals are used only when necessary and under humane conditions. Stanford is committed to conducting the highest-quality research and to providing animals used in research with the best care available.

Animal experiments have been vital in the development of many vaccines, including polio and of course, the COVID-19 vaccine, the isolation and use of insulin, the discovery of a vaccine for canine parvovirus (which causes a lethal infection in dogs) and other advances that have saved the lives of humans and animals.

Future advances in the treatment of human diseases, such as Alzheimer’s and cancer, will continue to depend on inquiries that use animals, humans and other alternatives.

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Updated April 17, 2023

Medical breakthroughs underpinned by animal research

The use of animals in biomedical research helps researchers better understand the biological processes that are central to our health. This is essential for developing safe and effective ways of preventing or treating disease.

For over a century, research using animals has advanced the scientific understanding of human health, and the impact of this research is so vast that it can be difficult to measure. However, some key recent examples of lifesaving treatments that were developed thanks to animal research are worth highlighting.

COVID-19 vaccine trials

Professor Sarah Gilbert and her team at the University of Oxford spearheaded a vaccine trial in which they used a safe version of an adenovirus. An adenovirus is a virus that can cause a common cold-like illness.

Previous work funded by the Medical Research Council (MRC) through the UK Vaccine Network used this adenovirus (known as ChAdOx1) by Professor Gilbert in the production of vaccines against the Middle East Respiratory Syndrome coronavirus.

Engineering a spike protein

The team engineered ChAdOx1 to make a specific coronavirus protein, known as the spike protein, from the SARS-CoV-2 virus. As a result, our immune system should in theory be able to recognise the spike protein as ‘foreign’ and form antibodies against it. And then attack the SARS-CoV-2 virus and stop it from causing an infection.

It is hoped that long lasting immunity can be provided through vaccination by ‘bluffing’ the body in this way, and by slipping in parts of the virus that do not harm, but induce the release of antibodies.

The vaccine testing involved animal trials in ferrets and non-human primates at the Public Health England (PHE) laboratories. The team also collaborated with researchers at the BBSRC-funded Pirbright Institute to study the effect of this vaccine in pigs.

Vaccinating millions of people worldwide

Under normal circumstances, animal work must be completed before human trials can start. But because similar vaccines have worked safely in trials for other diseases, the work was accelerated and happened in parallel. It led to the approval by the Medical and Healthcare products Regulatory Agency on 30 December 2020.

This vaccine, commonly known as the Oxford AstraZeneca vaccine, has now been administered to millions of people worldwide.

Professor Alain Townsend’s team at the MRC Human Immunology Unit worked in collaboration with:

• MRC Weatherall Institute of Molecular Medicine
• Radcliffe Department of Medicine
• University of Oxford
• the Biotechnology and Biological Sciences Research Council’s (BBSRC) Pirbright Institute.

Further vaccine development

They have shown that a new potential vaccine against COVID-19, named RBD-SpyVLP, produces a strong antibody response in mice and pigs. It provides vital information for the further development of the vaccine.

Investing in the research and development of the second generation of COVID-19 vaccines is important because they will help fill gaps in efficacy against novel variants. It also addresses issues around production and distribution such as the requirement for cold chain supply logistics.

Find out more about the Oxford-produced RBD-SpyVLP vaccine candidate .

Llama antibody has ‘significant potential’ as COVID-19 treatment

A unique antibody produced by llamas could be developed as a new frontline treatment against COVID-19 and could be taken by patients as a simple nasal spray.

The laboratory research is led by scientists at the Rosalind Franklin Institute. The research was funded by:

• Engineering and Physical Sciences Research Council (EPSRC)
• EPA Cephalosporin Fund

The research has shown that nanobodies (a smaller, simple form of antibody generated by llamas and camels) can effectively target the SARS-CoV-2 virus that causes COVID-19. It is the first step towards developing a new type of treatment against COVID-19.

Preparing for human clinical studies

The scientists are hoping to progress this work from the animal setting to prepare for clinical studies in humans.

Human antibodies have been an important treatment for serious cases during the pandemic, but typically need to be administered by infusion through a needle in hospital.

However, nanobodies have several potential advantages over human antibodies:

• they are cheaper to produce
• it is possible to deliver them directly to the airways through a nebuliser or nasal spray, so they could be self-administered at home rather than needing an injection.

This could have benefits in terms of ease of use by patients, but it also gets the treatment directly to the site of infection in the respiratory tract.

Gene therapy treatment for treating blindness

Inherited eye conditions are currently untreatable because they are caused by mutations in our DNA, which form defective copies of key genes required for normal vision. Gene therapy aims to deliver healthy copies of these defective genes directly to the retina, to correct these genetic mistakes.

MRC has been funding research into gene therapy for inherited eye diseases since 2004. Animal research in mice and dogs has been vital for establishing the necessary proof-of-concept for ocular gene therapy.

Developing a new, efficient technique

In 2011, with MRC funding, a team of scientists at the UCL Institute of Ophthalmology developed a new technique for improving the efficiency of this gene therapy. The results of which were confirmed in mouse models, a special strain of mice to study a particular human disease or condition.

Once the safety and efficacy of this approach was established in mice, the work rapidly progressed to two clinical trials. The first patients receiving this ground-breaking treatment have benefited from significant vision restoration, with more patients now in clinical trials. As well as the benefit to patients, this work is now widely regarded as a landmark for the entire gene therapy field.

Last updated: 17 August 2023

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Social Sciences | 7.25.2019

From the Archives: Animal Research

Every year, scientists use millions of animals—mostly mice and rats—in experiments. the practice provokes passionate debates over the morality and efficacy of such research—and how to make it more humane..

Click image to see full cover: The original January-February 1999 issue that this article appeared in

Read the original article as it appeared in 1999.

The volume of biomedical research, and of trials of new therapies, has increased dramatically in recent decades, fueled by advances in understanding of the genome and how to manipulate it, methods of processing huge data sets, and fundamental discoveries such as targeted and immunological approaches to attacking cancer (see “Targeting Cancer,” May-June 2018). Greater Boston, and Harvard, are major participants in academic biomedical research, in close proximity to the biotech and pharmaceutical industries, which have set up shop locally to tap into the wealth of talent and ideas. Along the way, new research techniques that are driven by more sophisticated imaging, bioinformatics (see “Toward Precision Medicine,” May-June 2015), and “organ-on-a-chip” technology have made it possible to conduct science with less reliance on various kinds of animal-testing. Given rising social concern for and interest in animal welfare (see “Are Animals ‘Things’?” March-April 2016), these converging trends make rereading this in-depth 1999 report by John F. Lauerman on the use of animals in biomedical research still timely and important. ~The Editors

“What is man without the beasts? If the beasts were gone, man would die from a great loneliness of spirit. For whatever happens to the beasts soon happens to man.”

~ Chief Seattle

Frederick Banting would never have begun his research without access to research animals. Before he had even spoken of his ideas, his first note to himself on the subject read: "Ligate the pancreatic ducts of dogs." The quiet Ontario doctor envisioned that severing the connection between the pancreas and the digestive system in a living animal would allow him to isolate the mysterious substance that would control diabetes.

During the first week in the laboratory, Banting and his assistant, Charles Best, operated on 10 dogs; all 10 died. Finally, in 1921, after months of experimentation, Banting and his colleagues isolated a material that kept a depancreatized dog named Marjorie alive for about 70 days. Exactly what information was gained from using dogs, and how many dogs were absolutely needed, is not clear. Work previous to Banting and Best’s, some of it in humans, had indicated the presence and importance of a hormone involved in glucose transport. Many more experienced scientists in the diabetes-research community believed that Marjorie had never been fully depancreatized, and thus may have never been diabetic. More likely, they said, the dog died of infection caused by her pancreatectomy. It’s possible that even the death of the famous Marjorie was unnecessary for the great discovery.

But the two Toronto researchers had isolated insulin, providing the first step toward producing it from pig and cow pancreas, available in bulk from slaughterhouses. The result—that Banting and Best "saw insulin"—appears to have justified all sacrifices. What’s the life of a dog, 10 dogs, a hundred? Before Banting and Best operated on dogs, we had no insulin; afterwards, we did.

Stories such as these are the reason our society and the vast majority of societies in the world accept the use of animals as a vital component of medical research.

Deeply entrenched traditions support the notion that animal welfare must bow to the best interests of humans. Animal domestication was among the first labor-saving devices. Humans have experimented with animal breeding, feeding, and disease control for thousands of years—not to benefit the animals themselves, but to insure that the owners obtained a maximum yield.

Today, those traditional practices have evolved into a scientific institution, the appropriateness of which is subject to perennial debate. In the United States alone, there are an estimated 17 million to 22 million animals in laboratory research facilities. To many people, animal research represents a doorway to the medical treatment of tomorrow. But to animal protectionists, and a growing number of other Americans, animal experimentation is a barbaric, outdated practice that—on the basis of a few notable past successes—has somehow retained its vestigial acceptability.

"Let’s say that it’s true, that animals were indispensable to the discovery of insulin," says Neal Barnard, M.D., of the Physicians Committee for Responsible Medicine, an animal-protection group. "That was a long time ago. I think to say, ‘It was done this way and there’s no other way it could have been done’ is a bit of a leap of faith, but let’s say that at the time there was no other way. You could also say that you couldn’t have settled the South without slavery. Would you still do it that way today? Just because something seemed necessary or acceptable at the time is not to say that we should do it in our time."

The Animal Debate

The legitimation of the animal-research debate challenges one of the most important and widely used scientific approaches to discovery about the human body and its diseases. Animal experimentation is often considered as much of a sine qua non to research as the Bunsen burner. But animal protectionists reply that the importance of animals to research is overrated, and that their pressure has exposed profligacy among experimenters.

In February 1997, a highly controversial collection of articles appeared in  Scientific American  on the subject of laboratory-animal research. The first, written by Barnard and Stephen Kaufman, M.D., of the Medical Research Modernization Committee, another protectionist group, advanced the view that data collected from animal experimentation are almost always redundant and unnecessary, frequently misleading, and by their very nature unlikely to provide reliable information about humans and their diseases. "Animal ‘models’ are, at best, analagous to human conditions," the authors wrote, "but no theory can be refuted or proved by analogy. Thus, it makes no logical sense to test a theory about humans using animals."

A rebuttal in support of animal research followed, by Jack Botting, Ph.D., former scientific adviser to the Research Defense Society in London, and Adrian Morrison, Ph.D., D.V.M., of the University of Pennsylvania School of Veterinary Medicine. Their reply cited examples of scientists from Louis Pasteur to John Gibbon, a twentieth-century pioneer in open-heart surgery, who made important breakthroughs in the treatment of human disease through animal research.

Many scientists—both supporters of animal research and advocates for its diminution—simply refused to discuss the difficult topic, recalls Madhusree Mukerjee, the editor who proposed that  Scientific American  explore the controversy and who wrote a third article, reporting on the overall state of animal research in the sciences. (Similar difficulties were encountered in researching the present article.) Mukerjee suspects that possible interviewees feared the criticism of their colleagues.

Reader response, on the other hand, was overwhelming, both pro and con. "We got a huge amount of flak for dealing with the subject at all," recalls Mukerjee. "Some of it was fairly frightening." To many animal-research supporters, it was as though the floodgates had been opened. "I am simply stunned that  Scientific American,  a paragon of promotion of scientific research, would actually offer up for debate whether animal research should occur," wrote one reader. "Please leave this question of animal research to animal-rights activists, and stop yourselves from turning into scientific wimps." "A lot of the scientific community felt [ Scientific American’ s editors] had overstepped their bounds and compromised their values by printing the Barnard-Kaufman article," says Joanne Zurlo, associate director of the Johns Hopkins School of Public Health Center for Alternatives to Animal Testing and a specialist in chemical carcinogenesis.

Those researchers who supported animal use and wrote in said the animal-protectionists’ side of the  Scientific American  debate was fraught with misstatements and scientific errors, although Mukerjee maintains that all the articles were painstakingly fact-checked. "We annoyed a lot of influential scientists," she says. "Our publication has spent more than a century describing advances in medical research, including some by fairly controversial figures. We’d never addressed the question of research on animals before, and in a sense it was a necessary thing to do. We probably lost some subscriptions because of it. But we are a bridge between the researchers who write for us and the public who read us, and we decided to let our readers decide for themselves."

Animal Welfare

Animal protectionists date their movement back to the times of Leonardo da Vinci and even Pythagoras, who are alleged to have been vegetarians. Numerous essayists and animal lovers have detailed their objections to the misuse of animals. Yet not long ago, virtually anyone who wanted to could conduct experiments on animals. In the 1960s, it was not uncommon to walk into a laboratory and find mice, dogs, cats, even monkeys, housed on the premises in whatever conditions researchers saw fit to provide. Banting himself frequently bought pound dogs and may even have caught dogs on his own; his collaborators recalled that he once arrived at the lab with a dog he had leashed with his tie.

Only in the nineteenth century did animal research begin to draw explicit objections from protectionists. A pivotal event occurred in England in 1874, when a lecturer at the University of Norwich demonstrated how to induce epileptic symptoms in a dog through the administration of absinthe. Objections were raised by students in the audience, and the dog was set free. Later, charges were filed against the lecturer under Dick Martin’s Act, an 1822 law that called for a fine of 10 shillings from anyone committing acts of cruelty against animals. Two years later, in 1876, Parliament passed the Cruelty to Animals Act, requiring a license for animal experimentation and placing restrictions on some painful forms of experimentation.

In the United States, minimal restrictions on animal experimentation prevailed until 1966, when the first federal Laboratory Animal Welfare Act (now known as the Animal Welfare Act, or AWA) was passed by Congress. In 1970 the AWA was broadened to require the use of appropriate pain-relieving drugs, and to include commercially bred and exhibited animals. Six years later, provisions were added covering animal transport and prohibiting animal-fighting contests. In 1985, Congress passed the Improved Standards for Laboratory Animals Act, which again strengthened the AWA by providing laboratory-animal-care standards, enforced by U.S. Department of Agriculture (USDA) inspectors, and also aimed to reduce unnecessarily duplicative animal-research experimentation.

In 1976, however, the AWA was amended in a rather curious way: rats, mice, birds, horses, and farm animals were specifically excluded from its purview for reasons that are not fully clear, although the USDA’s limited resources—along with political pressure from interested parties—are likely to be among them. Since rats and mice make up more than 95 percent of all research animals in this country, the amendment effectively put the vast majority of laboratory animals outside the reach of the USDA. Since then, at least one court has ruled the 1976 amendment "arbitrary and capricious."

The Mouse Warehouse

As associate professor of surgery Arthur Lage, D.V.M., walks through the doors of Harvard Medical School’s Alpert Building, people recognize him, smile, and let us pass without showing identification. He is director of the Center for Animal Resources and Comparative Medicine and the Center for Minimally Invasive Surgery at the medical school and director of the Office of Animal Resources for the Faculty of Arts and Sciences as well. We take an elevator down to a basement, where Lage swipes a card through a reader, unlocking a door to a hallway, where he speaks into a phone. A minute later, a young man clad in blue scrubs opens the door. Lage explains that he’s bringing a reporter in for a tour and that we’ll need keys to see certain rooms. The young man hands over the keys and closes the door.

At the other end of the short hallway are two doors, each leading to a sanitary changing room. When you turn the lights on in the changing rooms, the doors at either end lock automatically. After we’ve pulled blue scrubs over our clothes, Lage douses the lights and we step out of the room into another brightly lit hallway.

We’re in one of Harvard’s 16 animal facilities now, a moderately "clean" facility—meaning that it requires only minimal preparations for entry. Some laboratories would require us to remove our clothes and shower before entering; others don’t even stock scrubs. But this facility is full of mice—transgenic mice. A stray pathogen in one of the animal rooms could wipe out millions of dollars’ worth of experiments or, just as disastrous, infect a colony of mice with viruses or bacteria that might confound the results of a study.

Of course, the security isn’t intended only to repel microbes. Perhaps in frustration with perceived shortcomings in the oversight of animal experimentation, some animal-protection groups have gained a reputation for tactics that are rash and often destructive. On several occasions, animals have been "liberated" from laboratories, erasing potential results and sometimes careers. In 1989, the Animal Liberation Front took credit for the release of more than 1,200 laboratory animals, some of them infected with cryptosporidium, which can be harmful to infants and immunocompromised people. The total damage was estimated at $250,000. In 1987, a laboratory under construction at the University of California at Davis was burned; the loss was estimated at $3 million.

Although there is little evidence of violence toward animal researchers here in the United States, in Europe, where the animal- protection movement is more firmly entrenched, activists have taken aim at individuals, sometimes with disastrous results. In 1990, the infant daughter of a researcher was injured by a car bomb believed to have been set by animal protectionists. In separate, related incidents, a furrier and a breeder of cats used in experimentation were injured by letter bombs. Responsibility for the mail bombs was assumed by "The Justice Department," a militant, underground, animal-protection organization.

Even today, animal-protection groups find ways to gain access to research and testing facilities. In 1997, Michelle Rokke of People for the Ethical Treatment of Animals (PETA) infiltrated Huntingdon Life Sciences, a drug- and cosmetic-testing firm in East Millstone, New Jersey. Using a surveillance camera embedded in her eyeglasses, Rokke took hours of films that PETA claimed showed animals being slammed into cages and roughly handled. PETA president and co-founder Ingrid Newkirk said their investigation also revealed that young beagles’ legs were broken for another study at Huntingdon. Movie star Kim Basinger gave a press conference on Huntingdon’s lawn. In April 1998, the USDA fined Huntingdon $50,000 for AWA violations.

In the basement of the Alpert building, there is no evidence of such fury. Each room holds literally hundreds of mice in shoebox-sized cages, and there are so many of them it looks like a shoe warehouse. There are about 55,000 mice involved in research at Harvard at any one time, but that number is growing constantly. In 1997 it was closer to 50,000; by the end of 1998 it approached 58,000. By comparison, the numbers of other animals are almost negligible: about 1,300 rats, 145 rabbits, 115 hamsters, 70 guinea pigs, 67 primates, 35 pigs, 30 gerbils, 25 chicks, 20 dogs, 18 sheep, 6 cats, and 1 ferret. In addition, the New England Regional Primate Research Center in Southborough, Massachusetts, houses another 1,500 monkeys and other primates. Established at Harvard in 1966 with a grant from the National Institutes of Health, the NERPRC is one of seven such centers created by Congress in the early 1960s to serve as regional resources for scientists.

Surprisingly, there is no hint of animal smell within the basement facility. Temple Grandin of Colorado State University, a specialist in the behavior of captive animals, says that what mice really crave is some form of bedding—wood chips, paper, or shavings—which not all these animals have. Still, these laboratory animals, born and bred under fluorescent lights, are comfortable enough to live out lifespans they would never approach in the wild and, of course, to reproduce. And since almost all of them are involved in genetic studies, making sure they’re happy and healthy enough to reproduce is of vital importance. Keeping these buildings clean and free of infection is a triumph of research design. All the soiled animal cages are shuttled to one end of the laboratory where, before they re-enter, they pass through an enormous autoclaving machine that sanitizes the cages as well as the carts they sit on.

Amid the towers and technology of the medical area, animals one normally associates with a farm are a jarring sight. But Lage (pronounced lah-gee) led me through animal laboratories in the basement of the Seeley Mudd Building where we saw pigs, sheep, and rabbits held in small, clean pens. At one point, we watched eight sheep slated for experimental surgery frisk around a room that looked almost exactly like an office. If the straw were swept away, one could easily have moved in a desk and gone to work.

"We care for all these animals just as though they were covered by the [Animal Welfare] Act," Lage says proudly. "I think most of us believe that the act should cover rats and mice."

Although the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), like the USDA, inspects laboratory-animal facilities, including those of rats and mice, AAALAC accreditation isn’t legally required to conduct animal research. "AAALAC conducts something like a ‘peer review’ assessment," Lage says. "It’s a voluntary process, subscribed to by many, many research organizations. If you decide not to go through accreditation, you have to describe your entire program every time you apply to the government for funding for animal research."

Many laboratories and commercial drug-testing companies that receive no funding from federal sources and use only rats and mice proceed with only minimal oversight from their own institutional animal care and use committees (IACUCs). But restrictions on animal research are, if anything, increasing, not abating. Not content with the level of state and federal regulation, for example, the city of Cambridge in 1989 passed its own law creating an inspector’s office with the power to make USDA-type inspections of all research facilities housing vertebrate animals, including rats and mice.

Cambridge’s current commissioner of laboratory animals, Julie Medley, D.V.M., annually inspects 34 laboratories, makes follow-up visits to some facilities (sometimes unannounced), and reviews "hundreds and hundreds" of research protocols to ensure that all experiments meet federal standards for pain control. Investigators readily comply with Medley’s suggestions for better animal care and pain control, she says, but she perceives an undercurrent among some researchers who chafe under what they perceive as excessive government intervention in their work. "I’m sure some of the principal investigators resent these regulations," she says. "It doesn’t happen that often, but there are rare occasions when I run into resistance from an investigator."

Still, for animal protectionists, the intentions of the Animal Welfare Act, AAALAC, and state inspectors are not enough. Sandi Larson, a scientific adviser to the New England Anti-Vivisection Society, who has a master’s degree in microbiology, concedes that "not all researchers are Dr. Frankensteins. But," she adds, "they have been trained to look at animals as tools. It’s ingrained in them to shut off their compassion and act like scientists. They think there’s no room for emotions." A significant portion of the animal-protection movement believes that most experimentation on animals is without merit. If animals are different enough from humans that we can dismiss their suffering as inconsequential, isn’t it just a little too convenient that they resemble us enough to be considered a source of reliable information about human physiology?

Animal Liberation 

Peter Singer was an Oxford philosophy student who had little interest in animals, domesticated or otherwise, until he had lunch with a vegetarian friend one day and they began talking about the use and abuse of animals. Singer was quickly converted to the cause, and within a few years became its champion. One of the pivotal events in the treatment of laboratory animals in this country and throughout the world was the publication of his manifesto,  Animal Liberation,  in 1975.

Just 25 years ago, some proponents of animal experimentation still held that animals’ intellectual inferiority to humans meant that they could not be accorded the same rights as humans. Some argued that animals had no consciousness or memory, that they did not think as humans did. The quality and intensity of the pain felt by animals was still subject to debate. Singer, recently appointed DeCamp professor of bioethics at Princeton University’s Center for Human Values, refuted the assertion of animals’ inequality, pointing out that our society grants equal rights to all humans without regard to IQ or ability to function. "If the demand for equality were based on the actual equality of all human beings, we would have to stop demanding equality," he wrote. "...[T]he claim to equality does not depend on intelligence, moral capacity, physical strength, or similar matters of fact."

As for consciousness and the ability to feel pain, Singer pointed out that we have no reason to believe animals lack either one. Some of the experiments he recounts make their emotional vulnerability all too clear. In the late 1950s, for instance, psychologist Harry Harlow of the University of Wisconsin embarked on a series of experiments in which he deprived young rhesus monkeys of contact with their mothers. Young monkeys who were most completely deprived of parental contact developed very bizarre behavior, and would cling to objects that supplied the most minimal comfort, such as a scrap of terrycloth. Many of his fellow researchers considered Harlow a genius for having established the importance of interpersonal contact to normal childhood development. Singer, on the other hand, pointed out that the experiments demonstrated just how much like us monkeys really are, and he condemned the inhumanity of torturing them to obtain information that could have been elucidated in many other ways, perhaps through epidemiological studies of children who had been separated from their mothers at critical periods of development.

"You can’t have it both ways," says biochemist Karin Zupko ’77, an animal-rights advocate formerly with the New England Anti-Vivisection Society. "You can’t say that animals are different enough from people so that it’s acceptable to experiment on them, but enough like people so that the results of the experiments are valid."

Models for Medicine

Scientists, however, counter that you can, in fact, gather useful information about humans from animals that seem vastly different from us. They point to the many surgical experiments performed on pigs, dogs, and monkeys that have led to advances in transplantation, heart-valve replacement, and coronary artery bypass graft surgery.

"Research on live organisms is essential for medical advance," asserts Francis D. Moore ’35, M.D. ’39, S.D. ’82, Moseley professor and surgeon-in-chief emeritus at Harvard Medical School and Brigham and Women’s Hospital, respectively. As Moore has pointed out in testimony to the Massachusetts legislature and in his autobiographical book,  A Miracle and A Privilege,  the first successful human kidney transplant, in which Moore played a pivotal role in 1963, would not have been possible at that time without an understanding of immunology based on experiments in rats and mice. Important aspects of the surgery were developed in larger animals. "There’s no substitute for it," says Moore. "Some people say you can set up a computer program to act like a dog. Well, forget it. All animals have responses that we don’t understand, and there’s no way to set that up on a computer."

A great deal of our understanding of basic human physiology comes from experiments in large animals, like dogs and chimpanzees. Harvard physiologist Walter B. Cannon, A.B. 1896, M.D. ’00, S.D. ’37, for example, performed experiments on dogs for many years to understand the basic dynamics of digestion. Different animals may be selected for different purposes. A dog’s prostate differs from that of a human in having only two lobes, yet dogs, like humans, can develop benign prostatic hyperplasia.

"Not all animal models are ideal, but some cases are a perfect fit," says Arthur Lage. "Mice are certainly a very good model for studying human genes. Much of the genetic makeup of the mouse is very similar to that of a human; there are large regions of shared identity." That’s why, Lage explains, Harvard will probably double its use of mice over the next five years—to about 100,000 mice annually. The chief reason for this is transgenic-mouse technology—which allows the insertion and deletion of key disease genes into the mouse genome. These techniques allow researchers to study the impact of both subtle and drastic changes in the genome, and to make key predictions about how similar changes would affect humans. Mice can be bred, for example, with varying ability to express the  p53  gene, which has been implicated in a wide variety of cancers. Understanding how the activity of such genes affects cancer development promises to vastly increase our knowledge of treatment and prevention.

Philip Leder ’56, M.D. ’60, Andrus professor of genetics and head of the medical school’s department of genetics, who pioneered the technology, points out that transgenic mice have been used to test the safety and efficacy of new therapeutics; to detect biohazards; and to advance our knowledge of cancer. Yet he concedes this widely embraced methodology has yet to produce new therapies itself. "It’s impossible as yet to bring it home to lives of patients," he says, "because the development of diagnostics and therapeutics takes time."

There are many areas, however, where a direct connection between animal research and patient welfare can be argued. In the field of AIDS, for instance, research on animals has been making important contributions to the basic understanding, prevention, and treatment of this life-threatening disease.

In 1981, Norman Letvin ’71, M.D. ’75, received a call that would change his life. It concerned an epidemic of mysterious deaths, all caused by unusual pathogens and cancers, such as pneumocystis carinii pneumonia, cytomegalovirus, and rare lymphomas. But the patients suffering from these infections were not humans, but laboratory monkeys.

We now recognize these so-called "opportunistic infections" as signals of the presence of the human immunodeficiency virus (HIV) that causes AIDS. But at that time, the disease was just being recognized in humans, the term "AIDS" itself was unknown, and the cause of all these infections was still a frightening mystery.

Letvin, now professor of medicine at Harvard, says HIV probably began as a relatively harmless virus that infected some species of African monkeys. When it crossed species lines, it did so in several directions, spreading simultaneously into both human and additional non-human primate populations. In these new populations, the infection had much more serious consequences than in the African monkeys: it was lethal. But to Letvin, the realization that a parallel syndrome was occurring in man and monkeys was a tremendous opportunity.

"A great deal of effort has been expended on trying to find rodent and rabbit models for studying HIV infections, but they have not proven terribly useful," Letvin notes. "The only way we can see what happens in the first few minutes, hours, and days after infections—questions that are essential to answer in order to develop an HIV vaccine—is by working in animal models. We are forced to work in these models if we want to answer these questions." (The number of monkeys needed for such an experiment, he hastens to point out, is relatively small: usually about six.)

In Letvin’s experiments, monkeys are inoculated with candidate vaccines against HIV. After a brief period during which the vaccine draws a response from the host monkeys’ immune system, the animals are inoculated with a strain of immunodeficiency virus that brings on an AIDS-like disease. Periodic blood samples are taken to monitor their white blood cell counts and viral replication. An experimental model that causes the monkeys to get sick is more informative, Letvin explains, because even if the vaccine doesn’t prevent infection, it may slow the course of the disease enough to be useful.

"There’s little question that exciting animal data is a major drive for the initiation of human studies," Letvin says. "It’s not a gatekeeper, but an important piece of a complex puzzle we use to determine whether to go forward with the long march into humans. There are hundreds of approaches one could take. If a strategy does look promising, an animal trial makes it easier to determine whether it’s worth spending millions of dollars to measure its safety and efficacy in humans."

Letvin points out that an AIDS vaccine would save millions of human lives, particularly in populations where expensive treatment is not available. Thus the use of animals in research on diseases such as AIDS seems fated to continue for years to come. If the past is any indication, it will probably yield a rich crop of new medical information.

Perhaps the more accurate question then—under the circumstances—is, how much do we care about animal suffering? Is it worthwhile to consider that issue in our quest for better treatment for diseases?

The Three R’s

Since Peter Singer formulated his ideas, the animal-protection movement has gone from a series of staccato eruptions to a steady influence on the course of medical research. Everyone involved in the animal-research debate admits that the situation has changed considerably during the last 25 years. Ernie Prentice, a nationally recognized expert in the regulation and ethics of animal research and a member of the institutional animal care and use committee at the University of Nebraska Medical Center, can remember a time when animals were routinely subjected to painful measures without pain control. In one well-publicized experiment, pigs were burned without anesthetic; in another long-running research project, monkeys were subjected to traumatic blows to the head without analgesics. Animals progressed to the end stages of artificially induced malignancies, renal failure, and heart disease, all without any form of pain control.

"Those kinds of projects would not be permitted now. They would be unacceptable for at least two reasons," says Prentice. "One is that we now have regulations that clearly ban this kind of experimentation, and those regulations are adequately enforced to make sure that they’re followed. At the same time, there is heightened ethical sensitivity among both researchers and IACUCs. If you had sat in on a meeting of an IACUC in 1985 and were able to compare the level of discussion back then with what goes on today, you would see a tremendous difference."

Increasingly, members of the protection community are taking legal steps to gain input into animal-treatment guidelines, and have found more conventional ways to exert pressure. Marc Jurnove, a member of the Animal Legal Defense Fund (ALDF), is suing the USDA for "aesthetic and recreational injuries" that he suffered when seeing the living conditions of chimpanzees and apes at a Long Island zoo. Jurnove charged that the USDA failed to adopt and enforce adequate standards for the animals’ well-being, as is required by the AWA. This past September, the U.S. Court of Appeals for the District of Columbia Circuit, the nation’s most influential circuit court, upheld Jurnove’s right to sue. Recently, the ALDF also led animal-rights groups in successfully suing the National Academy of Sciences for access to records and to committee meetings pertaining to a guide on the care and use of laboratory animals.

Some major funding organizations have also embraced the animal-rights movement. The Doris Duke Charitable Foundation, with assets of $1.25 billion, is one of the 25 wealthiest philanthropies in the country. Although it funds medical research, one of its restrictions is that animals not be used as subjects. This creates a sticky situation for the board, which hopes to fund research on AIDS, cancer, heart disease, and sickle-cell anemia, areas heavily dependent on animal research in the past.

But the effort to occupy a middle ground, supporting the principles of reduction, replacement, and refinement of animal research while acknowledging its necessity, has been extremely frustrating.

Several research institutions have established centers of animal-rights advocacy. The Center for Animals and Public Policy at Tufts University and the Center for Alternatives to Animal Testing at Johns Hopkins University, for example, have tried to establish liaisons with both protectionists and researchers. "I wasn’t running around throwing bombs," says Andrew Rowan, Ph.D., former director of the Tufts Center and now senior vice president of the Humane Society of the United States. "I was engaging colleagues in scientific debate without being obstreperous. People were shouting past each other." Veterinarian Peter Theran, vice president of the health and hospitals division of the Massachusetts Society for the Prevention of Cruelty to Animals and director of the MSPCA’s Center for Laboratory Animal Welfare, says that his group has had to walk a fine line. "We try to maintain a rapport with both sides," he stresses. "I have to say that we often don’t agree with some of the more aggressive groups, like PETA. But there’s a tendency to paint the animal-welfare community with a broad brush. And that makes dialogue extremely difficult."

"When you say you’re for animal welfare, you’re perceived as rabid," says Joanne Zurlo of the Johns Hopkins center. "At the same time, we can’t deal with groups like PETA because they believe in abolition of animal use. When we organized the first World Congress on Alternatives and Animal Use in the Life Sciences in 1993, we invited representatives from every organization to sit at the table. PETA would not join. Even the American AntiVivisection Society sent a representative, but members of the hard-line groups who were picketing outside hounded her and called her a murderer."

Human Lives, Humane Experiments

The growth of the animal-protection debate has been fraught with acrimony. The results, however, go beyond the additional credibility that has been afforded animal protectionists. Scientists, too, find that they can be more open about the feelings they have or may have had for the creatures in their care, and are more free to explore alternative methods of experimentation.

"All of us, whether we’re doing research on animals or not, recognize that this is something that is not optimal," Andrew Rowan says. "If society didn’t feel that we needed the information, we wouldn’t do research on animals. But society feels we do, and so do scientists. There’s a tension between our concern about causing pain and distress and killing animals and our need for new knowledge. No one would say that the animals in research benefit from it, and in a world that was perfect we wouldn’t be doing this. We’re engaged in encouraging people to make animal welfare a higher priority without compromising their ability to gather information."

Neal Barnard, of the Physicians Committee for Responsible Medicine, argues that the route away from animal research should carry us toward population-based efforts like the Framingham Heart Study, in which heart researchers have closely followed the health habits and outcomes of 5,000 adults for just over 50 years. That study was a key factor in galvanizing current national efforts to lower cholesterol, combat hypertension, and encourage proper diet and exercise to reduce mortality from heart disease.

"Those areas where we struggle the most, clinically, are those where we haven’t exploited good clinical research and are relying on animal models," Barnard says. "Look at cardiac defects. We don’t know how they’re caused because no one has done the equivalent of the Framingham study for heart defects, even though it’s quite feasible. The Centers for Disease Control and organizations study these congenital abnormalities only in a very haphazard way.

"Of course," he continues, "there have been some brilliant exceptions, such as the research on neural tube defects. It was found through observation of humans that these defects were associated with deficiencies in folic acid, and that by taking vitamin supplements you can reduce the risk. The same with fetal alcohol syndrome: the breakthroughs came in studying humans, not animals."

Politics frequently obscures our view of research bias, Barnard says. He has called for a Framingham-style study of the health implications of cow’s milk consumption, which has been implicated in some studies as a possible cause of Type 1 diabetes in children. Barnard believes that the political strength of the dairy industry has kept such a study from becoming a reality even though some 700,000 Americans suffer from Type 1 diabetes.

Even within the scientific community, there is an increasing willingness to admit that current research methods can be improved upon. A wide variety of in vitro tests have been proposed (among them, the use of human tissue culture and in vitro cell-culture assays), as well as increased reliance on computer modeling and the creative application of human epidemiological studies. Both government and industry experts agree that if new techniques eliminate or reduce the use of animals, so much the better. "[T]he current rodent bioassay for assessing carcinogenicity costs $1 million to $3 million and requires at least 3 years to complete," reads the summary of a January 1997 meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals. The main topic of the meeting was the development of alternatives to animal research, and the report continues, "More efficient testing methods may reduce the time required to bring new products to the marketplace and increase the amount of useful information that can be obtained."

Most researchers recognize that the humane treatment of animals isn’t only compassionate—it’s also good science. Imagine trying to measure the effect of blood-pressure medication on a dog that hasn’t been walked in days. We now know that animals’ feelings, behavior, and emotions have a profound effect on their physiological functioning—as is the case with humans. Consequently, after strong initial opposition to the Animal Welfare Act, most researchers have come to support it.

The Humane Society of the United States represents one example of how animal protectionists can set reasonably limited goals that promote animal welfare in ways that better serve both animals and humans. "We’ve contacted animal care and use committees and asked them to work with us to identify techniques that cause pain and distress and figure out ways to share ways to eliminate that in research," says HSUS’s Andrew Rowan. "Some of the committees are rather suspicious; they see a hidden attempt to stop all animal research. The response has been slight so far. But we think that most researchers are bright people and will understand that our primary goal is just to eliminate animal suffering wherever possible."

Norman Letvin, who frequently debates animal protectionists, knows that there are many who would like to end the practice of animal research for good. Although he is ready and willing to discuss the morality and ethics of his work, he thinks that calling an end to the practice would hurt society enormously.

"It is very easy to take an absolutist position and say it is wrong to cause the death of another living animal," Letvin says. "The difficulty in what [researchers] do comes in saying, ‘I understand that what I’m doing is causing the death of a limited number of animals, but I’m making a judgment that the information gained from this limited, focused experiment will yield results that will justify doing the study.’ Many humans infected with viruses or suffering from cancer or heart disease enter into studies that allow the development of new therapeutics. Every day, thousands of humans say, ‘It is worth it for me to be involved in those studies because, even though I probably won’t benefit, others will.’ In the end, the decisions I’m making with respect to experimental animals are not dissimilar."

As we walked to a new facility on Longwood Avenue, Arthur Lage reminded me that it was the former site of Angell Memorial Animal Hospital, which has since moved to Huntington Avenue in Jamaica Plain. He points out where horses were tethered in the courtyard as they waited to be seen by a veterinarian. He indicates a barely visible tower protruding from the rear roof where distempered dogs were once quarantined. "It was hard work," he recalls, somewhat wistfully, of the internship and residency he served at Angell. "But it was rewarding. You might sit up all night with a sick dog or cat, trying to save its life."

Today, Lage cannot devote as much time to saving animals’ lives. Instead, as he says, he’s helping save human lives through animal research, while ensuring that animals are used humanely. Embodied in his work are many of the contradictions that many of us feel when we consider the millions of animals—from mice to monkeys—that annually give their lives for human health. The use of animals in research will not end today, nor tomorrow, but opinions on the matter appear to be evolving, perhaps toward a better life for animals in the laboratory, and toward better science.

John Lauerman used to write the magazine’s " Harvard Health " column. He is coauthor of a book on diabetes and, with Thomas Perls, M.P.H. ’93, M.D., and Margery H. Silver, Ed.D. ’82, of  Living to 100,  forthcoming from Basic Books in March.

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Replacing Animal Testing with Stem Cell-Organoids : Advantages and Limitations

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• Published: 19 April 2024

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• Guiyoung Park 1 ,
• Yeri Alice Rim 2 , 3 , 4 ,
• Yeowon Sohn 5 ,
• Yoojun Nam   ORCID: orcid.org/0000-0003-4583-3455 5 , 6 &
• Ji Hyeon Ju 2 , 3 , 4 , 6  

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Various groups including animal protection organizations, medical organizations, research centers, and even federal agencies such as the U.S. Food and Drug Administration, are working to minimize animal use in scientific experiments. This movement primarily stems from animal welfare and ethical concerns. However, recent advances in technology and new studies in medicine have contributed to an increase in animal experiments throughout the years. With the rapid increase in animal testing, concerns arise including ethical issues, high cost, complex procedures, and potential inaccuracies.

Alternative solutions have recently been investigated to address the problems of animal testing. Some of these technologies are related to stem cell technologies, such as organ-on-a-chip, organoids, and induced pluripotent stem cell models. The aim of the review is to focus on stem cell related methodologies, such as organoids, that can serve as an alternative to animal testing and discuss its advantages and limitations, alongside regulatory considerations.

Although stem cell related methodologies has shortcomings, it has potential to replace animal testing. Achieving this requires further research on stem cells, with potential societal and technological benefits.

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Use of Large Animal Models for Regenerative Medicine

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Introduction

Historically, animal models have contributed substantially to the advancement and study of vaccines, surgical techniques, and various scientific experiments [ 1 ]. However, owing to the problems associated with animal testing, researchers are now questioning whether animal models and tests are the best options for these procedures. Growing animal testing is ethically concerning amid scientific evolution. According to the Humane Society International Organization, more than 100 million animals are killed annually worldwide for scientific purposes (Humane Society International). The animals used vary depending on their traits and include rats, mice, rabbits, dogs, cats, guinea pigs, zebrafish, swine [ 2 , 3 ].

In December 2022, the U.S. Food and Drug Administration (FDA) announced animal testing is no longer mandatory safety approval of products [ 4 ]. However, products that are used on the human body still require safety testing. In other words, testing for toxicity, compatibility, and safety is compulsory for products; however, animal testing is unnecessary for conducting these tests. In response, research facilities and companies have introduced alternatives such as computer simulations and in silico models. Stem cell therapy has gained popularity throughout the medical field, and various studies are underway to gain deeper knowledge [ 5 ]. With the emergence of this stem cell-based test, alternative methods have also arisen, potentially offering to become a replacement for animal testing.

When comparing test options, alternatives offer more beneficial attributes than animal testing. Non-animal tests are cost-effective, less time-consuming, and simpler procedures than animal tests [ 6 ]. However, most research institutions use animal models. This is because animal testing has been a longstanding experimental approach for decades [ 7 , 8 ]. Efforts are being made to replace animal testing with the use of human cells, as animal testing results often exhibit interspecies differences with humans, thus lacking the ability to reliably predict clinical outcomes. Application of advancing stem cell technology continue, but completely replacing animal experimentation poses significant challenges. Therefore, it is important to conduct further studies to advance the science of alternative testing methods. This review aimed to summarize the use of stem cell technology as an alternative to animal testing and discuss its advantages and limitations.

Current State of Animal Testing

Uses of animal testing.

Animal testing has been used for decades, and in the 21st century, the number of tests has increased considerably [ 2 ]. With approximately 100 million animals used for testing annually worldwide, science has been rapidly evolving. The primary function of animal testing is to test drugs, their toxicity, and their compatibility with the human body to ensure safe use. Hence, pre-launch testing is crucial. Companies and research facilities must subject their products to clinical trials before introducing them to potential customers.

Neurological disorder such as Parkinson’s and Alzheimer’s have also been modeled in animals to understand their mechanisms and to determine suitable treatments [ 9 , 10 , 11 ]. For instance, in the case of Parkinson’s disease, various animal models have been employed, including Caenorhabditis elegans, Zebrafish, and mice. Additionally, genetically modified mice carrying mutations associated with proteins like α-synuclein, Parkin, Pink1, and LRRK2, as well as mice induced with α-Synuclein Pre-Formed Fibril (PFF), are utilized to assess dopaminergic neuronal loss and investigate changes in α-synuclein aggregation. In Alzheimer’s disease, transgenic mice carrying mutations associated with familial Alzheimer’s disease (FAD), such as the 5xFAD model, are commonly used. These models allow for the evaluation of amyloid beta reduction through histological methods and the assessment of drug efficacy using behavioral tests like the Maze, providing insights into underlying disease mechanisms. Animals utilized as disease models contribute significantly to our comprehensive understanding of the mechanisms behind various illnesses, facilitating our grasp of these conditions. Research conducted using these animal disease models has indeed contributed to the discovery and development of treatments. However, it’s scientifically crucial to acknowledge that these animal models often present disparities in lifespans compared to humans and may not entirely mirror the intricate etiology of human diseases. Additionally, while animal experimentation is utilized for various conditions such as cancer, diabetes mellitus, and traumatic brain injury, it’s constrained by its inability to fully capture the nuances of the human immune system and intricate disease mechanisms (Table  1 ).

In addition to modeling diseases, animals are also used to test cosmetics or healing rates of products. In the cosmetics industry, animals are typically used to test skin or eye irritation to assess the safety of these products in humans [ 17 , 18 ]. The Draize test, developed in 1944 to test for such hazards in rabbits [ 19 ], is used to test products such as drugs and balms for wound healing. It involves creating wounds on animals to gauge recovery rates [ 16 ].

Related laws, Guidelines, and Principles

As of 2023, current regulations state that the FDA no longer deems animal tests necessary for evaluating product safety [ 4 ]. This enables companies and research facilities to explore possible non-animal testing when obtaining product approval. Additionally, out of 195 countries worldwide, only 42 have laws or regulations limiting animal testing for products (The Humane Society). Animal testing laws have been implemented by banning animal testing or limiting its use during testing. Europe completely banned cosmetics tested on animal testing in 2013 [ 3 , 20 , 21 ]. This demonstrates a push to limit animal testing; however, the movement remains ineffective because of the absence of laws against animal testing in most countries.

Guidelines for animal experimentation and clinical trials for drug development and safety testing have varied procedures among companies and researchers up to now. So, the Guidance for Industry for Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals from the Center for Drug Evaluation and Research provides guidelines for the safety assessment of products compiled from regulatory standards of several countries. According to these guidelines, preclinical trial researchers should consider factors such as animal species, age, delivery method (dosage, administration, treatment regimen, etc.), and test material stability [ 22 ] (Fig. 1 ).

( A ) Procedure of new drug approval as stated by the Food and Drug Administration (FDA). In the preclinical research stage, small, medium, and large animals are usually used for testing new drugs. ( B ) iPSCs that can replacing animal testing. PBMCs or fibroblasts are reprogrammed to iPSCs and subsequently differentiated into target modeling cells such as neurons, cardiomyocytes, and hepatocytes. ( C ) iPSC-derived 3D organoids enable in vitro efficacy and safety testing. Organ-on-a-chip embedded with organoids used in in vitro tests, created using BioRender

The FDA has also provided a drug development process that includes these steps. The first step in drug development is discovering and researching a new drug (discovery and development stage). The second stage is preclinical research, in which drugs have to undergo a series of animal tests (or alternative tests, if possible) for safety. The FDA strongly suggests that animal preclinical trials follow Good Laboratory Practice (GLP). The main elements of GLP are as follows [ 23 ]: appropriate use of qualified personnel, quality assurance, appropriate use of facility and care for animals, proper operating procedures for animals used in trial, individual animal data collection and evaluation, testing product properly handled and analyzed, study proceeds with an approved protocol, data should be collected as outlined in the protocol, and full report prepared after procedures.

To enhance clinical translation, reproducibility issues in preclinical trials, such as biased allocation, insufficient controls, and lack of interdisciplinary, uncharacterized, or poorly characterized supplies [ 24 ]. The third step involves clinical testing on humans to assess safety and efficacy. The fourth and fifth stages comprise FDA post-market safety monitoring for all approved drugs [ 25 ].

Guidelines also suggest the 3R (replacement, reduction, and refinement) principle, which recommends that scientists follow certain criteria during clinical trials. Replacement involves using other testing methods other than animal testing [ 26 ]. In computer models, tissues, or stem cell research, if alternatives to animal testing exist, researchers should prioritize their use. Reduction involves minimizing the number of animal tests [ 26 ]. Questioning the necessity of animal tests during a particular part of our research and reducing their numbers imbues the concept with meaning. Refinement focuses on minimizing stress and providing the best care to animals [ 26 ], including providing proper food, entertainment, and clean well-maintained shelters.

As International efforts for animal replacement methods, research and development into alternative testing methods is already underway in both Europe and the United States, with each regulatory body establishing its own initiatives. In Europe, the European Center for the Validation of Alternative Methods (ECVAM) was founded in 1992, and since 2013, the sale of cosmetics containing ingredients tested on animals has been completely banned. Moreover, there are plans to expand the scope to include medical devices, health supplements, and pharmaceuticals in the future. In the United States, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) was established in 2000. The objective is to reduce animal testing by 2025 and eliminate mammalian animal testing entirely by 2035 through innovative advancements in alternative testing methodologies. In 2022, amendments to the Food, Drug, and Cosmetic Act in the United States removed mandatory animal testing requirements in the drug development stage and presented alternative testing methods as viable non-clinical trial options.

Problems/limitations of Animal Testing

A pressing issue with animal testing is the ethical concerns stemming from it. Most studies have demonstrated that these models undergo invasive procedures that often result in pain or even death. Research indicates that animals share pain and emotional capacity with humans [ 27 ]. Thus, sacrificing them for research can appear cruel. Advocates call for equitable treatment, opposing animal testing as inhumane and cruel. Such ethical issues has always followed animal testing and are ongoing [ 28 ].

Moreover, some studies have indicated that animal testing is not an accurate model for medicines or substances, highlighting the need for accurate and efficient testing alternatives that are similar humans. The complexity of human disease mechanisms raises doubts whether animal models can accurately replicate them.

Physiological differences between animals and humans mean a product safe for animals may not guarantee human safety [ 29 ]. Interspecies differences have led to poor results in correlating animal testing with human outcomes, consequently causing several clinical trial failures [ 30 ]. Between 2010 and 2017, clinical trials for drugs had a greater chance of failing phase І, owing to safety and efficacy [ 31 ]. In addition, even if a product passes phase І there is still a 90% rate of failure while undergoing the necessary procedures [ 32 , 33 ]. Prolonged use of animal testing can ultimately endanger humans, as some drugs and products approved through trials were later deemed harmful. Concerns such as high cost and long laborious procedures will be discussed below.

Benefits of Replacing Animal Testing

The main benefits of replacing animal tests with alternatives are as follows: cost-effective, time efficient, less complex testing procedures, and societal benefits.

Stem cell modeling is less expensive than animal testing. The Draize test mentioned before costs approximately $1,800, whereas non-animal testing methods cost considerably less [ 6 ]. Affordable procedures offer renewed chances for past costly research to emerge. A decrease in the cost of procedures would facilitate new drug development, making opportunities for new technologies easier.

Animal testing requires prior preparation that is often complex and time consuming. Several guidelines of various organizations worldwide follow certain principles and procedures. For animal testing, factors such as providing clean and well-maintained shelters, food, necessary supplies for survival, and entertainment are laborious [ 26 ]. Alternatives are time-efficient and less laborious, simpler protocols, and fewer supplies to maintain procedures.

Alternatives to Animal Testing Related to Stem Cells

Organoids are organ-like structures derived from self-organizing stem cells in 3D cell cultures. They exhibit organ-specific characteristics and originate from stem cells undergoing self-organization [ 34 , 35 ]. . They are beneficial over previous 2D cell culture, as they can show near-physiological cellular composition and actions [ 36 ]. Organoids are typically established from embryonic stem cells (ESCs), human pluripotent stem cells (PSCs), and adult stem cells [ 37 , 38 , 39 ]. The potential of organoids as alternatives stems from their correlation with patient reactions to products such as drugs, indicating that they are a promising for rare diseases where clinical trials are impractical [ 39 ]. Organoids have a wide range of applications and are suitable for studies of infectious diseases, hereditary diseases, and toxicity, and can provide personalized medicine for individual patients [ 38 ].

Recent studies have shown that PSC organoids can form complex brain organoids that are useful for modeling traumatic brain injury [ 15 ]. Organoids derived from PSCs are of various types, including stomach, lung, liver, kidney, cerebral, and thyroid, and can contribute to organ failure or dysfunction. Cancer organoids are cultured from thin tumor sections, which are efficient for studying cancer syndromes [ 34 ]. Organoid studies on Alzheimer’s disease highlight the possibility of using familial or sporadic Alzheimer’s disease induced pluripotent stem cells (iPSCs) to model brain activity [ 40 ]. Thyroid follicles derived from hESCs have the potential to be used as organoids to treat hypothyroidism [ 41 ] (Table  2 ). Technology development of 3D bioprinting organoids is underway, promising better productivity. Bioprinting for organoids includes inkjet-based bioprinting, laser-assisted bioprinting, extrusion-based bioprinting, and photo-curing bioprinting [ 42 ]. Ongoing studies are also exploring 3D printing technology using organoids, offering the possibility of creating organs for patient-tailored services and toxicology research.

However, organoids still possess limitations that render them unsuitable tools to replace animal testing. Organoids lack of vasculature structure affects growth and maturation, leading to differences in behavior compared to the original tissue [ 59 ]. This may result in only partial replication, leading to an incomplete disease model [ 38 ]. Moreover, the complexity and heterogeneity of certain organs, such as the brain or immune system, pose challenges for complete replication in organoid models. This inability to replicate such complexity can affect the translatability of findings from organoid studies to clinical applications. Research and experiments involving organoids often require lengthy culture protocols, which can vary depending on the type of organoid being cultivated. In some extreme cases, organoid culture may extend for months or even years, as seen in examples such as intestinal organoids(8 weeks or more), retinal organoids(6 ~ 39 weeks or more), brain organoids(12 weeks or more), and liver organoids(4 ~ 8 weeks or more) [ 60 , 61 , 62 , 63 , 64 ]. Even after going through the lengthy process, there are sometimes a lack of established organoids in sufficient numbers. This limited availability of organoids can hinder the procedure of functional testing, which can lead to insufficient research outcomes. Organoids also lack the intricate network of connections that can be seen in living organisms. Inter-organ communication is crucial when checking metabolic health, and with organoids lacking such an important factor, it is difficult to create treatments for any abnormalities regarding infection and diseases. Organoids also lack a diverse set of cell types, structural organization, and physiological functions in comparison to functioning organs, which limits the ability to accurately replicate disease processes and responses to treatment [ 59 ]. When compared to animal models, organoids fall behind, as animal models offer a broader view of processes for diseases, immune responses, and systemic effects of treatments. Another noteworthy concern arises from the fact that current production technology for organoids under GMP (Good Manufacturing Practice) standards has yet to be established.

Quality Control of Organoid

For organoids to serve as suitable models for diseases or experimental purposes, quality control (QC) is essential. Accuracy and consistency in production lead to more precise results, ensuring better therapeutic treatments or modeling. If quality control for organoids isn’t established sufficiently, problems such as inconsistent test results, misinterpretation of existing data, wastage of valuable resources, reproducibility issues, unreliable models, and ethical concerns regarding biomedical studies could arise.

Organoid structures and functions can be assessed through multiple methods. Structural assessment of organoids can be performed using bright-field imaging for both quantitative and qualitative research. Additionally, methods such as immunofluorescent staining, transmission electron microscopy, and scanning electron microscopy are also utilized [ 65 , 66 ]. The functionality of organoids can be assessed through qPCR and single-cell or bulk cell RNA sequencing, which provide quantitation of marker gene expression, revealing cell identity and composition [ 67 ]. Assay methods like ELISA and colorimetric assays are useful for secretome quantification while Luciferase essays help measure enzyme activity [ 65 , 68 ]. Staining methods such as Glycosaminoglycan (GAG) staining(specifically for synovial mesenchymal stromal cell (SMSC) organoids), immunofluorescence staining, and Alizarin red staining mainly help with visualizing components within the organoid [ 65 , 68 , 69 ]. There are also more direct methods like implantation to test the in vivo functions of organoids [ 65 , 70 ] (Table  3 ).

Extracellular microenvironment, which contain such things as soluble bioactive molecules, extracellular matrix, and biofluid flow, contributes to the growth rate and formation of organoids. Given the variation in extracellular microenvironments across different types of organoids, it is imperative to modulate the extracellular microenvironment accordingly for each organoid type. This ensures the production of organoids with consistent quality across different production batches [ 71 ].

Regulations/Applications Regarding Organoids from the FDA

While there aren’t any specific regulations regarding organoids from the FDA(Food and Drug Administrations) as of in the recent years, there are two categories of applications that include framework for cell related therapies, which include organoids. There are two applications, Biologics License Application (BLA) and the Investigational New Drug (IND) Application. The BLA, as stated in the official website of FDA, is a request for permission to introduce and deliver for a biologic product(vaccines, somatic cells, gene therapy, tissues, recombinant therapeutic proteins, organoids, etc.) into interstate commerce. Requirements for a BLA includes applicant information, product/manufacturing information, pre-clinical studies, clinical studies, and labeling. The IND application is a request for authorization to administer an investigation drug or biological product to humans. IND had three types: Investigator IND, Emergency Use IND, and Treatment IND which could fall into two categories being commercial or non-commercial. The IND application must contain the following broad areas of information: Animal Pharmacology and Toxicology studies, Manufacturing Information, Clinical protocols and Investigator Information.

When examining the current ongoing clinical trials( ClinicalTrials.gov ) in the application of organoids, it can be noted that they are being utilized in refractory cancers, osteosarcoma, high-grade glioma, advanced breast cancer, and colorectal cancer. This pertains to the utilization of the organoid platform to investigate the sensitivity to various drugs (chemotherapy, hormonal therapy, targeted therapy) by exposing them to each individual agent (or combination of agents). It is anticipated and ongoing to aid in clinical decisions regarding the optimal treatment option for each patient.

Organ-on-a-chip

Organoid chips(OoC) can be regarded as the outcome of merging biology and microtechnology, serving as microfluidic cell culture devices [ 72 , 73 ]. OoC has the ability to mimic the cellular environment, which leads to an examination of their effects on cell communication with more accessibility and ease. The chips are generally designed by collecting cells (primary cells, transformed cell lines, human ESC, or iPSCs) using equipment with pumps(that enable fluid flow), incubators, sensors, and microscopes to monitor and examine the cells in the system [ 49 , 74 ] (Fig.  1 ). Depending on the type or cell or method cells can be aggregated in matrix or matrixless conditions [ 75 ].

Various types of human organ chips, including the liver, heart, eyes, kidneys, bones, intestines, and skin, are used to simulate the breathing motion. Single-organ chips such as liver-on-a-chip and lung-on-a-chip are useful for observing individual chemical reactions [ 53 ]. There are also multiple organ-on-chip, which are organ-chips connected to a vast system [ 76 ]. The main purpose of multi-organ-on-chips is to simulate the entire body, recognizing that a single organ does not represent the entire human system. Using multiple organ-on-chips connected to one system allows the analysis of how various organs communicate with each other.

The U.S. Food and Drug Administration (FDA) and the U.S. National Institutes of Health (NIH) have provided project support for tissue chips for drug screening, including lung-on-a-chip. Additionally, efforts are being made globally to advance the utilization of organoid chips, such as the establishment of the European Organ-on-Chip Society in Europe.

A limitation of OoCs is their complex experimental setup [ 77 ], which can be avoided with clear guidelines or protocols. Cell medium changes also raise concerns about chip environments [ 77 ]. There is also the issue of using animal models to validate OoC systems initially [ 78 ]. To address this, OoC experts recommend forming well-established collaborations with developers, toxicologists, and pharmaceutical companies to explore alternative solutions.

iPSCs(Induced Pluripotent stem Cells)

iPSCs are a recent development in the field of disease modeling. Having traits such as self-renewal and pluripotency, iPSCs can transform into various cells within the human body (Fig.  1 ); thus, reprogramming patient cells creates personalized medicine for specific diseases [ 79 , 80 ]. The ability to produce a large batch of iPSCs with only a small number of patient samples is important [ 81 , 82 ]. The objectives of iPSC models closely align with the 3R principle [ 83 ]. Replacing animal models in research while adhering to reduction and refinement principles is expected to be advantageous.

iPSCs are research to find cures for various diseases and are used as broad disease models (Table  2 ). For example, iPSCs from patients with Parkinson’s disease differentiate into midbrain dopaminergic neurons (DAns) in the substantia nigra pars compacta (SNpc), which can be used to model Parkinson’s disease on a cellular basis [ 43 , 44 , 45 ]. For cardiac diseases, which include a decrease in cardiomyocytes that leads to scar formation and ultimately heart function failure, there are existing studies that explore iPSCs for novel therapeutic cures [ 84 ]. iPSC-derived progenitors such as human HCN4 + and human ESC derived ROR2+, CD13+, KDR+, PDGFRα + cells later generate cardiomyocytes [ 47 ]. For cancer modeling using iPSCs, reprogrammed tumor specimens or iPSCs with premalignant or early genetic lesions can show the stages of cancer [ 49 ]. iPSCs from patients that are healthy and those with Alzheimer’s disease differentiate into the main brain cells, modeling the human brain with a functional blood barrier. Further research could drive drug discovery [ 9 ]. Studies of organ failure or dysfunction have shown that human iPSCs are useful. Research on lung regeneration has shown that endogenous and exogenous stem cells mediate therapeutic results [ 50 ]. Another study focused on the use of liver hepatoblasts, which could help alleviate hepatotoxicity through liver development and hepatic differentiation [ 85 ].

However, iPSCs are still in a relatively early developmental phase and have several limitations. Concerns for researchers regarding iPSCs is in vitro culture adaptation and tumorigenicity, the inability to completely reflect in vivo 3D environments, and the variation of differentiated cells depending on the protocol [ 86 , 87 ]. Quality control of differentiated cells and influencing factors are crucial for iPSC researchers, impacting their applicability as medical models or treatments.

Figure 2 Human diagram showing multiple stem cell-related technologies that can be applied to various human organs.

A BioRender diagram depicts diverse stem cell technologies for human organs

Limitations

Stem cell-related methodologies, such as organoids, are a very new technology in the field of animal alternative testing. In the early developmental stage, alternative stem cell models and technologies still require a few years of testing. Animal testing is still used today, owing to its historical role in safety and efficacy assessment. New alternatives have been presented; however, the uncertainty of these methods have caused most researchers to adhere to old protocols. In cases of complex diseases arising from various factors such as cardiovascular, neurodegenerative, and infertility, complete replacement by animal alternative testing methods may still be impractical. In such instances, it is crucial to concurrently employ animal experimentation alongside alternative testing methods utilizing organoids or stem cells to bolster data reliability. As a component of these endeavors, numerous researchers have undertaken disease modeling, such as stroke, utilizing brain organoids and cardiac organoids in in vitro experiments. The solution involves focusing on alternative testing methods [ 88 ]. By transforming old methods and creating alternatives, this shift could be the norm. There has already been a move toward that goal, as the FDA has established a cross-agency working group (The Alternative Methods Working Group) to promote various alternative methods, such as in vivo, in vitro, in silico , or system toxicology modeling [ 89 ]. In the 2021, FDA report titled “Advancing Regulatory Science at FDA,” the most prioritized area is identified as “Advancing Novel Technologies to Improve Predictivity of Non-clinical Studies and Replace, Reduce, and Refine Reliance on Animal Testing.”

Given ongoing research in alternative stem cell-related methods, this appears promising to replace animal testing. These alternatives offer advantages for scientists and the public. However, it is important to acknowledge that iPSCs, organoids, and OoCs each have distinct strengths and limitations. With continued advancements and studies to further understand these issues, these limitations can be avoided.

Data Availability

All data pertaining to this manuscript are included within the article.

Abbreviations

Food and Drug Administration

organ-on-chip

induced pluripotent stem cell

pluripotent stem cell

Embryonic stem cell

Center for Drug Evaluation and Research, GLP, Good Laboratory Practice

Dopaminergic neurons

Substantia Nigra pars compacta

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This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI22C1314).

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VA continues ‘approved’ experiments on dogs, cats and monkeys after Congress orders an end to live-animal tests by 2026

A hospital staff member took this 2019 photo of a cat used in experiments at the Louis Stokes Cleveland VA Medical Center. Tests involved placing electrodes in the cat’s bladder and using a device to stimulate urination. The VA in 2024 is seeking to implant wires and sensors in the legs and backs of cats in a separate experiment to test an implant for translating signals from a prosthesis to the nervous system. (White Coat Waste Project)

WASHINGTON — Researchers from the Department of Veterans Affairs will implant pacemakers in the hearts of 54 dogs that will be euthanized at the end of the tests and surgically embed wires and sensors into the backs and legs of cats in separate experiments that the VA plans to conduct on live animals in 2024, according to agency documents.

Though the Department of Veterans Affairs is under order by Congress to phase out live animal experiments using cats, dogs and primates “with limited exceptions” by 2026, the agency continues to support live-animal research at VA facilities across the U.S., according to the VA.

More than 62,000 cats and dogs are in U.S. labs for live animal experiments run by government agencies, colleges and universities, and private companies, according to the Humane Society of the United States, a nonprofit organization that focuses on the welfare of animals.

The VA has been phasing out live animal testing on dogs, cats and primates since 2018.

Terrence Hayes, the VA press secretary, said the agency is assessing a new congressional directive adopted in March to eliminate the live-animal tests “with limited exceptions” within two years.

“VA is reviewing the recently signed fiscal year 2024 appropriations law to ensure any implementation of the new provisions fully meet congressional intent, including using of funding, program requirements and reporting to our congressional partners,” he said.

A provision requiring the VA to end live animal research is part of the Consolidated Appropriations Act, signed into law March 9. The legislation requires the VA to provide a plan for ending the tests within 90 days of the bill’s enactment.

In 2024, the VA’s list of “approved research” on live animals includes two separate experiments using dogs at the Richmond VA Medical Center in Virginia.

The experiments involve implanting pacemakers in the hearts of dogs to induce extra heartbeats that disrupt the regular heart rhythm, causing a sensation of fluttering in the chest. The purpose is to measure deteriorating heart muscle and heart failure caused by the extra heart beats.

The dogs will undergo open heart surgery to implant a pacemaker device and a radio telemetry system. Catheters also will be positioned on the heart surface, according to the project description.

Fifty-four dogs will be used in the experiment, after which “most will be euthanized,” according to VA documents.

Dogs not euthanized will be granted a four-week recovery at which time the pacemakers will be disabled, and the animals further studied. Those dogs also will be euthanized at the conclusion of the tests.

The White Coat Waste Project, a nonprofit watchdog group, said records obtained by the organization under the Freedom of Information Act show no dogs are currently confined at the Richmond VA Medical Center or being used there for heart experiments.

A separate VA experiment using cats is approved for 2024 for the Louis Stokes VA Medical Center in Cleveland for testing the durability of implanted medical devices to stimulate nerve sensation in patients who have undergone amputations.

“New prosthetic technology for amputees can restore natural sensations,” according to the project proposal published by the National Institutes of Health.

Funding through September for the experiments is about $270,000, according to information the VA published on its website.

The experiments involve surgically embedding wires and sensors into the legs and backs of cats, according to documents obtained by the White Coat Waste Project.

The procedures risk paralysis and death in the cats, which is counter to directives by Congress for restricting these types of tests, said Justin Goodman, senior vice president of White Coat Waste Project.

The experiment is to test a miniaturized implant that translates electrical signals from a prosthesis to the nervous system, which could allow veterans who lost a limb to achieve a better sense of balance and motion in digits and joints, Hayes said.

He described the experiments as safe and said the cats will be placed into adoptive homes at the conclusion of the research in six months.

The VA also has approval in 2024 to continue experiments on dozens of rhesus macaque monkeys for measuring treatment outcomes for spinal cord injuries.

The experiment at the VA San Diego Health Care System involves damaging a monkey’s spinal cord in surgical procedures.

The monkey then undergoes “multiple major survival surgeries” along with stem cell therapy to address injuries and observe recoveries.

“Each of these surgeries will add to the body of knowledge we can gain about recovery from spinal cord injury,” according to the project description on the VA website.

“This research is to explore the possibility that neural stem cells can be used to help bridge the damaged tissue and restore communication across the site of the injury,” according to the project description.

The experiment identifies the use of restraint chairs for behavioral testing to force monkeys recovering from spinal cord injuries to use the hand with limited use to perform tasks. The monkeys also are expected to walk on treadmills and retrieve food to improve function, according to the project description.

The experiments using the monkeys are being conducted in conjunction with other agencies including University of California-Davis, which has one of the biggest primate laboratories in the country, Goodman said.

Animals are purchased from breeders licensed to sell dogs, cats, primates and other animals to laboratories for use in live-animal experiments.

Goodman said his organization objects to the VA using taxpayer dollars to purchase animals and submit them to painful experiments. He said animals often are euthanized and dissected at the end of the research, as part of the study.

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• VA ordered to end experiments on dogs, cats and primates by 2026

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• Published: 15 March 2024

Metagenomic insights into the wastewater resistome before and after purification at large‑scale wastewater treatment plants in the Moscow city

• Shahjahon Begmatov 1 ,
• Alexey V. Beletsky 1 ,
• Alexander G. Dorofeev 2 ,
• Nikolai V. Pimenov 2 ,
• Andrey V. Mardanov 1 &
• Nikolai V. Ravin 1  

Scientific Reports volume  14 , Article number:  6349 ( 2024 ) Cite this article

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• Antimicrobials
• Microbial communities
• Industrial microbiology

Wastewater treatment plants (WWTPs) are considered to be hotspots for the spread of antibiotic resistance genes (ARGs). We performed a metagenomic analysis of the raw wastewater, activated sludge and treated wastewater from two large WWTPs responsible for the treatment of urban wastewater in Moscow, Russia. In untreated wastewater, several hundred ARGs that could confer resistance to most commonly used classes of antibiotics were found. WWTPs employed a nitrification/denitrification or an anaerobic/anoxic/oxic process and enabled efficient removal of organic matter, nitrogen and phosphorus, as well as fecal microbiota. The resistome constituted about 0.05% of the whole metagenome, and after water treatment its share decreased by 3–4 times. The resistomes were dominated by ARGs encoding resistance to beta-lactams, macrolides, aminoglycosides, tetracyclines, quaternary ammonium compounds, and sulfonamides. ARGs for macrolides and tetracyclines were removed more efficiently than beta-lactamases, especially ampC , the most abundant ARG in the treated effluent. The removal efficiency of particular ARGs was impacted by the treatment technology. Metagenome-assembled genomes of multidrug-resistant strains were assembled both for the influent and the treated effluent. Ccomparison of resistomes from WWTPs in Moscow and around the world suggested that the abundance and content of ARGs depend on social, economic, medical, and environmental factors.

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Introduction.

The spread of antimicrobial resistance (AMR) in the environmental microbiome has become one of the frequently noted problems in recent years, along with global climate change, food security and other technological challenges. Numerous studies show that from year to year, in addition to increasing the cost of hospitalization and treatment of patients infected with multidrug-resistant bacteria, the number of deaths of such patients is growing 1 , 2 . Understanding the mechanisms underlying the emergence, selection and dissemination of AMR, and antibiotic resistance genes (ARGs), is required for the development of sustainable strategies to control and minimize this threat. The dissemination of antibiotic resistant bacteria (ARB) and ARGs occurs differently and this process is more active in urban territories rather than in rural ones. The rate of spread of ARGs and ARB in urban areas is obviously determined by the high population density and, as a rule, wastewater which flows from these areas contains both ARG and ARB. Most antibiotics used in medicine, agriculture and the food industry, as well as resistant bacteria, end up in wastewater. Wastewater treatment plants (WWTPs) therefore could provide a comprehensive overview of ARG abundance, diversity and genomic backgrounds in particular region 3 . Moreover, wastewater and WWTPs are places where ARGs and ARB are particularly abundant and are often considered “hotspots” for the formation of strains with multiple resistance and one of the main sources of the spread of AMR in the environment 4 .

Despite numerous studies on the role of WWTPs in resistome diversity and dissemination, each new study is, in terms of time and geography, unique, as many urban areas and countries have not yet been studied. In addition, some studies are dedicated to explore only one component of the wastewater treatment system, such as wastewater, activated sludge or treated effluent, and there is a lack of research that would give a comprehensive view of the diversity and change in the composition of the resistome at different stages of water cleaning, from wastewater to treated effluent, released into the environment.

Usually, wastewater treatment in large facilities takes place in three stages. The first stage includes physical methods of water cleaning, the second stage is microbiological treatment in bioreactors with activated sludge (AS), and the third stage is the final treatment of water and its disinfection. At the second stage, than could be performed using several technologies, microorganisms of AS are used to remove organic matter, ammonium, and (in more complex processes) phosphorus 5 . At this stage, the removal of microorganisms present in the wastewater, including ARB, occurs due to their adsorption on AS particles, which are removed along with excess AS. The efficiency of this process differs for various bacteria and depends on the purification technology used. Therefore, purification technologies directly affect the removal of particular ARGs and ARB, however, this issue was poorly studied 6 .

ARGs representing all known resistance mechanisms have been found in WWTP environments 7 . ARGs for beta-lactams, macrolides, quinolones, tetracyclines, sulfonamides, trimethoprim, and multidrug efflux pump genes have been found in the incoming wastewater, AS, and treated effluent in various countries 7 , 8 . Recently, Munk and coauthors (2022) using metagenomics methods characterized resistomes of 757 urban wastewater samples from 243 cities in 101 countries covering 7 major geographical regions. They reported regional patterns in wastewater resistomes that differed between subsets corresponding to drug classes and were partly driven by taxonomic variation 3 . Although this study did not analyzes the composition of the wastewater resistome after treatment, there are numerous evidences that the prevalence of ARB and ARG in rivers may increase downstream from the point of discharge of treated wastewater into them 9 , 10 . In a study of WWTPs in Germany, 123 types of clinically significant antibiotic resistance genes were found in treated wastewater discharged into water bodies 11 . An analysis of the presence of 30 ARGs at different stages of wastewater treatment at WWTPs in Northern China showed that the content of most ARGs in the treated effluent was lower compared to the influent entering the treatment, although an increase in the abundance of some ARGs and their release into the environment was also observed 12 . A metagenomic analysis of WWTP in Hong Kong revealed seasonal changes in the content of several types of ARG and its decrease in the treated effluent 13 , 14 . Most ARGs were reduced by more than 98% in the treated effluent compared to the wastewater entering the treatment 14 . Some other studies have also reported a decrease in ARGs after wastewater treatment 15 , 16 , 17 . However, in other studies, no changes in the ARG content or even an increase were observed 17 , 18 , 19 . Although there are numerous studies of resistomes in WWTP-related environments the distribution of samples was geographically biased and covered mostly North America, Western Europe, Eastern Asia (mostly China), Australasia, and few places in South America/Caribbean and Sub-Saharan Africa 3 .

In order to expand the geographical coverage and our knowledge about global resistome abundance and diversity, we analyzed resistomes of wastewater before and after treatment at large-scale WWTPs in the city of Moscow (Russia). Although Moscow WWTPs are among the largest in the world and may play an important role in the spread of antibiotic resistance, the resistomes of municipal wastewaters in Moscow have not previously been studied by modern molecular genetic methods. Previously we performed 16S rRNA gene profiling of AS microbial communities at large-scale WWTPs responsible for the treatment of municipal wastewater ion Moscow 5 . Comparison of microbial communities of AS samples from WWTPs in Moscow and worldwide revealed that Moscow samples clustered together indicating the importance of influent characteristics, related to local social and environmental factors, for wastewater microbiomes 5 . For example, due to the relatively low cost of water for household consumption, wastewater in Moscow has a relatively low content of organic matter. Apparently the presence of ARB and ARGs in communal wastewater depends on the frequency of antibiotic use and the range of drugs used. These factors differ in different countries and cities. Therefore, the characterization of the resistome and the role of Moscow WWTPs in the dispersion of ARGs is an important goal. Of particular interest is also the assessment of the impact of wastewater treatment technology on the composition of the resistome and the degree of ARG removal.

Here we present the first metagenomic overview of the composition of resistome of influent wastewater, AS and treated effluent released into the environment at two Moscow WWTPs employing different treatment technologies.

Characteristics of WWTPs and water chemistry

The Lyuberetskiy WWTP complex of JSC “Mosvodokanal” carry out the treatment of wastewater in the city of Moscow with a capacity of about 2 million m 3 per day. This complex consists of several wastewater treatment units (hereafter referred to as WWTPs). They purify the same inflow wastewater but otherwise are independent installations between which there is no transfer and mixing of AS. Two WWTPs implementing different technologies for wastewater treatment were chosen as the objects of study. The first one (LOS) is operated using anaerobic/anoxic/oxic process, also known as the University of Cape Town (UCT) technology. There the sludge mixture first enters the anaerobic zone, where phosphate-accumulating microorganisms (PAO) consume easily degradable organics, then to the anoxic zone, where denitrification and accumulation of phosphates by denitrifying PAO occur, and finally to the aerobic zone, where organic matter and ammonium are oxidized while PAO accumulate large quantities of polyphosphate. The second WWTP (NLOS2) uses a simpler nitrification–denitrification technology (N-DN). In the aerobic zones organics and ammonium are oxidized, while in the anoxic zone nitrate is reduced to gaseous nitrogen. This treatment technology removes organic matter and nitrogen, but was not specially aimed to remove phosphates. The production capacity of LOS is approximately 2 times more than that of NLOS2; there are no other important differences between these WWTPs besides treatment technology.

Sampling and chemical analysis

Wastewater and AS samples were collected in September 2022 and kindly provided by “Mosvodokanal” JSC. The temperature of water samples was about 24 °C. Samples of AS from bioreactors of two WWTPs were taken in 50 ml Falcon tubes (BD Biosciences). Wastewater samples (influent and effluents from two WWTPs) were taken in 5 L plastic bottles. The cells were collected by centrifugation at 3000 g for 20 min at 4 °C.

Wastewater quality values, namely, biochemical oxygen demand (five days incubation) (BOD 5 ), chemical oxygen demand (COD), total suspended solids (TSS), sludge volume index (SVI), ammonium nitrogen (N-NH 4 ), nitrate nitrogen (N-NO 3 ), nitrite nitrogen (N-NO 2 ) and phosphorus (P-PO 4 ) in the influent and effluents of two WWTPs were measured by the specialized laboratory “MSULab” according to the Federal inspection of environmental management’s protocols for chemical analyses of water.

DNA isolation, 16S rRNA gene sequencing and analysis

Total genomic DNA was isolated using a Power Soil DNA isolation kit (Qiagen, Germany). DNA for each sample was isolated in four parallel replicates, which were then pooled. PCR amplification of 16S rRNA gene fragments comprising the V3–V4 variable regions was performed using the universal primers 341F (5′-CCTAYG GGDBGCWSCAG) and 806R (5′-GGA CTA CNVGGG THTCTAAT) 20 . The obtained PCR fragments were bar-coded and sequenced on Illumina MiSeq (2 × 300 nt reads). Pairwise overlapping reads were merged using FLASH v.1.2.11 21 . All sequences were clustered into operational taxonomic units (OTUs) at 97% identity using the USEARCH v.11 program 22 . Low quality reads were removed prior to clustering, chimeric sequences and singletons were removed during clustering by the USEARCH algorithms. To calculate OTU abundances, all reads obtained for a given sample were mapped to OTU sequences at a 97% global identity threshold by USEARCH. The taxonomic assignment of OTUs was performed by searching against the SILVA v.138 rRNA sequence database using the VSEARCH v. 2.14.1 algorithm 23 .

The diversity indices at a 97% OTU cut-off level were calculated using USEARCH v.11 22 . To avoid sequencing depth bias, the numbers of reads for each sample were randomly sub-sampled to the size of the smallest set.

Sequencing of metagenomic DNA, contigs assembly and binning of MAGs

Metagenomic DNA was sequenced using the Illumina HiSeq2500 platform according to the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA). The sequencing of a paired-end (2 × 150 bp) NEBNext Ultra II DNA Library prep kit (NEB) generated from 145 to 257 million read pairs per sample. Adapter removal and trimming of low-quality sequences (Q < 30) were performed using Cutadapt v.3.4 24 and Sickle v.1.33 ( https://github.com/najoshi/sickle ), respectively. The resulting Illumina reads were de novo assembled into contigs using SPAdes v.3.15.4 in metagenomic mode 25 .

The obtained contigs were binned into metagenome-assembled genomes (MAGs) using 3 different programs: MetaBAT v.2.2.15 26 , MaxBin v.2.2.7 27 and CONCOCT v.1.1.0 28 . The results of the three binning programs were merged into an optimized set of MAGs using DAS Tool v.1.1.4 29 . The completeness of the MAGs and their possible contamination (redundancy) were estimated using CheckM v.1.1.3 30 with lineage-specific marker genes. The assembled MAGs were taxonomically classified using the Genome Taxonomy Database Toolkit (GTDB-Tk) v.2.0.0 31 and Genome Taxonomy database (GTDB) 32 .

ARG identification

Open reading frames (ORFs) were predicted in assembled contigs using Prodigal v.2.6.3 33 . ARGs were predicted using the NCBI AMRFinderPlus v.3.11.4 ( https://github.com/ncbi/amr/wiki ) command line tool and its associated database 34 . The predicted protein sequences of all ORFs were analyzed in this tool with parameter “-p”.

Efficiency of wastewater treatment

Two wastewater treatment technologies were used in the investigated WWTPs,—nitrification/denitrification at NLOS2 and more advanced anaerobic/anoxic/oxic UCT process at LOS. LOS removed more than 99.5% of organic matter (according to the BOD5 data) and more than 99.9% of ammonium while the performance of NLOS2 was poorer (Table 1 ). Particularly noticeable differences were observed in nitrate and nitrite concentrations in the effluents suggesting the lower efficiency of denitrification in the NLOS2. Interestingly, although the NLOS2 unit was not designed to remove phosphorus, the concentration of phosphates in the treated effluent at this WWTP is only slightly higher than at LOS. The treated influent at LOS contains fewer solids consistently with lower SVI. Overall, the technology used at LOS plant is more efficient.

Microbiomes of the influent wastewater, activated sludge and treated effluent

The 16S rRNA gene profiling of microbial communities revealed 1013 species-level OTUs (97% identity) in the influent and 1.2–1.7 times more OTUs in the AS and treated effluent samples (Supplemental Table S1 ). The Shannon diversity indices correlated with the number of detected OTUs and increased in the series “influent” – “activated sludge” – “effluent” at each WWTP (Supplemental Table S2 ).

Analysis of the microbiome of wastewater supplied for biological treatment showed that that the most numerous phyla in the microbial community were Firmicutes (28.4% of all 16S rRNA gene sequences), Campylobacterota (28.0%), Proteobacteria (20.9%), and Bacteroidota (10.5%) (Fig.  1 ). These were mainly representatives of the fecal microbiota, which are often found in wastewater. The phylum Firmicutes was dominated by Streptococcaceae (9.7%, mostly S treptococcus sp.), Lachnospiraceae (5.9%), Ruminococcaceae (3.0%), Carnobacteriaceae (1.7%), Peptostreptococcaceae (1.6%) and Veillonellaceae (1.4%). Most of Campylobacterota belonged to the family Arcobacteraceae (26.8%) of the genera Arcobacter (19.9%), Pseudarcobacter (2.5%) and uncultured lineage (4.3%), as well as by sulfur-oxidizing Sulfurospirillum (1.0%). Among the Proteobacteria the most abundant genera were Acinetobacter (7.8%) , Aeromonas (1.8%) and Pseudomonas (1.1%). Most of the identified Bacteroidota were typical fecal contaminants such as members of the genera Bacteroides (2.6%), Macellibacteroides (1.5%), Prevotella (1.4%), and Cloacibacterium (1.2%).

Microbial community composition in the influent, AS and treated effluent samples according to 16S rRNA gene profiling. The composition is displayed at the phylum level. INFL, influent wastewater; AS-LOS, AS at LOS plant; CW-LOS, treated effluent at LOS plant; AS-NLOS2, AS at NLOS2 plant; CW-NLOS2, treated effluent at NLOS2 plant.

Activated sludge of WWTP bioreactors is a complex microbial community consisting of physiologically and phylogenetically heterogeneous groups of microorganisms involved in the removal of major contaminants from wastewater. The composition of AS microbiomes was very different from the microbiome of incoming wastewater (Fig.  1 ). The phyla Campylobacterota (less than 0.5%) and Firmicutes (2–4%) were much less abundant in AS microbiomes. Proteobacteria was the dominant group in the microbiomes of AS (23–40%), but its composition differed from the microbiome of influent wastewater: instead of the fecal microflora (Enterobacterales and others) the AS community harbored lineages involved in the purification processes ( Competibacteraceae , Rhodocyclaceae , Nitrosomonadaceae , etc.). Likewise, Bacteroidota were among the most numerous phyla in AS microbiomes at both LOS (6.5%) and NLOS2 (14.1%), but instead of Bacteroidales mostly comprised Chitinophagales and Sphingobacteriales typical for AS communities. The numerous groups of AS community also included Chloroflexi (22% and 10% in LOS and NLOS2, respectively), Patescibacteria (1.8% and 9.9%), Nanoarchaeota (4.3% and 9.1%), Nitrospirota (3.9% and 7.3%), Verrucomicrobiota and Myxococcota (about 4% in both WWTPs). Bacteria that play an important role in the removal of nitrogen ( Nitrospira and Nitrosomonas ) and phosphorus ( Dechloromonas ), as well as glycogen-accumulating Ca . Competibacter, have been found in large numbers. The abundance of these functional groups is consistent with the high efficiency of nitrogen and phosphorus removal.

The main source of microorganisms in treated effluent is the AS, from which they are washed out; bacteria from the influent water may also be present in minor amounts. Therefore, as expected, the microbiome composition of treated wastewater was similar to that of activated sludge. Consistently, compositions of microbiomes of treated effluent were similar to that of AS samples. However, some differences were observed, in particular, the microbiomes of the treated effluent contained many Cyanobacteria (7.74% and 3.49% for LOS and NLOS2, respectively) which were found in minor amounts both in the influent water and in the ASs (< 0.5%). Probably, these light-dependent bacteria proliferate in the final clarifier and then can be easily washed out with the effluent.

Diversity of resistomes

The results of metagenomic analysis of incoming wastewater revealed 544 ARGs in the assembled contigs, classified into 33 AMR gene families (Table 2 and Supplemental Table S3 ). Among the most numerous were classes A, C, D and metallo- beta-lactamases, rifampin ADP-ribosyltransferase, Erm 23S ribosomal RNA methyltransferase, aminoglycoside nucleotidyl-, acetyl- and phospho-transferases, the ABC-F type ribosomal protection proteins, chloramphenicol acetyltransferase, trimethoprim-resistant dihydrofolate reductase, quaternary ammonium compound efflux SMR transporters, lincosamide nucleotidyltransferases, tetracycline efflux MFS transporters and tetracycline resistance ribosomal protection proteins (Table 2 ). These genes may enable antibiotic inactivation (373 genes), target protection (85 genes), efflux (44 genes) and target replacement (25 genes).

The abovementioned genes confer resistance to most of commonly used drugs: beta-lactams (198 genes), macrolides (74 genes), rifamycin (60 genes), aminoglycosides (51 genes), tetracycline (27 genes), phenicols (27 genes), diaminopyrimidines (19 genes), quaternary ammonium compounds (16 genes), glycopeptides (15 genes), lincosamide (13 genes), fosfomycine (12 genes) and drugs of 11 others classes (Fig.  2 ).

ARGs identified in wastewater and AS samples categorized by drug classes.

About twice less ARGs were identified in AS samples from both WWTPs. Like in the influent, beta-lactamases of classes A, D, and metallo-beta-lactamases were the most numerous, while only a few genes for class C enzymes were found (Table 2 ). Other families of ARGs, numerous in the influent, were also numerous in AS microbiomes. A notable difference between the resistomes of the AS samples is the greater number of rifampin-ADP-ribosyltransferase genes ( arr ) in NLOS2 compared to LOS (63 vs 33). The largest number of arr genes was assigned to Bacteroidota, and the lower relative abundance of this phylum in AS at LOS likely explains these differences. Like in the wastewater, resistance to beta-lactams, macrolides, rifamycin, aminoglycosides, and tetracyclines was the most common (Fig.  2 ). On the contrary, genes for some drug classes were underrepresented in AS resistomes, especially for diaminopyrimidines (3 and 2 genes for LOS and NLOS2, respectively) and glycopeptide antibiotics (2 and 0 genes).

The results of metagenomic analysis of treated effluent showed that the diversity of these resistomes was only slightly higher than that of the corresponding AS samples. This result was expected since the main source of microorganisms in the effluent is activated sludge, from which they are partially washed. However, resistomes of treated effluent at both WWTPs contains about twice more class A beta-lactamase genes than AS samples suggesting less efficient absorption of their host bacteria at AS particles (Table 2 ).

Quantitative analysis of antibiotic resistance genes of WWTP

The results described above provide information on the diversity of resistance genes, but not on their abundance in the metagenomes, which depends on the abundance of corresponding bacterial hosts. To quantify the shares of individual ARGs in the metagenome and resistome, the amounts of metagenomic reads mapped to the corresponding ARGs in contigs were determined. In total, the resistome accounted for about 0.05% of the metagenome of wastewater supplied for treatment, while the shares of resistomes in the metagenomes of AS and treated effluent samples were 0.02% and 0.014% at the LOS and NLOS2 WWTPs, respectively.

Quantitative analysis of the content of individual ARGs in metagenomes showed that the structure of the influent resistome was very different from that of AS and treated effluent. The relative content of ARGs accounting for more than 1% in at least one analyzed resistome is shown in Fig.  3 . The LOS and NLOS2 WWTPs differed significantly from each other, and the differences between the AS and effluent resistomes at each WWTP were much less pronounced.

The relative abundancies of particular ARGs in the resistomes. Only ARGs with shares greater than 1% in at least one sample are shown, all other ARGs are shown as “others”.

The resistome of the influent was not only the most diverse, but also the most even in composition. The shares of none of the ARGs exceeded 5% of the resistome, and the 23 most common ARGs accounted for a half of the resistome. The most abundant ten ARGs were qacE, sul1, ampC, blaOXA, msr(E), erm(B), mph(E), tet(C), aph(3'')-Ib and aph(6)-Id, conferring resistance to antiseptics, sulfonamides, beta-lactams, macrolides, aminoglycosides (streptomycin), and tetracyclines.

AS and treated effluent at LOS plant was strongly dominated by a single AGR type, class C beta-lactamase ampC , accounting for about 45% of their resistomes. This gene was also the most abundant one in the resistomes of AS and effluent at NLOS2 (14.8% and 18.2%, respectively). Apparently it originates from the influent wastewater supplied for treatment where its share in the resistome was 3.2%. AmpC β-lactamases are considered clinically important cephalosporinases encoded on the chromosomes and plasmids of various bacteria (especially Enterobacteriaceae ), where they mediate resistance to cephalothin, cefazolin, cefoxitin and most penicillins 35 . Close homologues of this gene, with a nucleotide sequence identity of 99.8–100%, have been found in plasmids and chromosomes of various Proteobacteria ( Thauera, Sphingobium, Aeromonas etc.). Since in all samples ampC was found in short contigs with very high coverage, it is likely widespread in the genomes of various bacteria in different genetic contexts.

The second most abundant ARG in the resistomes of AS samples was sulfonamide-resistant dihydropteroate synthase ( sul1 ). It accounted for 4–5% of AS and treated effluent resistomes in LOS and for about 11% in NLOS2, while its share in the influent water resistome was about 5%. The sul1 gene is usually found in class 1 integrons being linked to other resistance genes, including qacE 36 . Consistently, sul1 and qacE were found in one contig assembled for the influent water samples and assigned to Gammaproteobacteria. Another sulfonamide-resistance gene, sul2 , was also numerous, accounting for about 2% of the resistomes in the influent and LOS samples, and for about 4% in the AS and water treated at NLOS2.

Since ARGs entering the activated sludge and then into the treated effluent originate mostly from wastewater supplied for treatment, the absolute majority of ARGs present in the influent in significant amounts (more than 0.2% resistome) in were also found in AS and effluent samples. The only exception macrolide 2′-phosphotransferase gene mph(B) accounting for 0.51% in the influent resistome. Likewise, all ARGs accounting for more than 0.2% of resistomes in the treated effluent were present also in the influent.

Potential multidrug resistant strains

One of the most important public health problems is the spread of multidrug resistant pathogens (MDR), which refers to resistance to at least one agent in three or more chemical classes of antibiotic (e.g. a beta-lactam, an aminoglycoside, a macrolide) 37 . Such strains can arrive with wastewater entering the treatment, and also form in AS communities. AS are dense and highly competitive microbial communities, which, along with the presence of sublethal concentrations of antibiotics and other toxicants in wastewater, creates ideal conditions not only for the selection of resistant strains, but also for the formation of multiple resistance through horizontal gene transfer 4 . To identify MDR bacteria, we binned metagenomic contigs into metagenome-assembled genomes (MAGs) and looked for MAGs comprising several ARGs. Only MAGs with more than 70% completeness and less than 15% contamination were selected for analysis: 117, 56, 72, 94 and 121 for influent, AS of LOS, effluent of LOS, AS of NLOS2 and effluent of NLOS2, respectively. Five MAGs of MDR bacteria were identified in the metagenome of the influent, one—in AS of LOS, two—in the LOS effluent and one in the NLOS2 effluent (Table 3 ). These MAGs were assigned to unclassified genus-level lineages of Ruminococcaceae and Cyclobacteriaceae, Phocaeicola vulgatus, Streptococcus parasuis, Ancrocorticia sp., Enterococcus sp., Bacillus cereus and Undibacterium sp.

Disscussion

We characterized the composition of microbial communities and the resistomes of influent wastewater, activated sludge and treated effluent from two WWTPs in city of Moscow, where various biological water treatment technologies are used. Among the predominant bacteria in the influent wastewater we found mainly fecal contaminants of the genera Collinsella , Bacteroides , Prevotella , Arcobacter , Arcobacteraceae , Blautia , Faecalibacterium, Streptococcus , Acinetobacter , Aeromonas and Veillonella 38 , 39 , 40 , 41 , 42 , 43 . Previously, we performed 16S rRNA gene profiling of wastewater before and after treatment at one WWTP (LOS) and revealed that all abovementioned potential pathogens were efficiently removed and their relative abundance in the water microbiome decreased by 50‒100 times 44 . Similar pattern of removal of potential pathogenic bacteria was observed here for NLOS2 where another water treatment technology is used.

An important indicator of the dissemination of ARG is the proportion of the resistome in the entire metagenome before and after wastewater treatment. In the influent, the resistome accounted for about 0.05% of the metagenome, which corresponds to approximately two ARGs per bacterial genome. Approximately the same values are typical for most countries 3 . After treatment, the fraction of the resistome in the wastewater metagenomes decreases, but, surprisingly, only by 2–4 times. However, since the total concentration of microorganisms in treated effluent is approximately two orders of magnitude lower than in raw wastewater, it is likely that the total abundance of ARGs in the treated effluent is significantly reduced.

Apparently, fecal contaminants effectively removed during treatment are not the only carriers of ARG in wastewater, which are also found in bacteria characteristic of activated sludge and thus appearing in the effluents. Unfortunately, due to the high diversity of microbiomes and the tendency of ARG to be present in multiple copies in different genomic environments, most of the contigs containing ARG turned out to be short, which did not allow to define their taxonomic affiliation.

The resistome of influent water includes 26 ARGs, the share of which is more than 1%. Among of them the prevalence of ampC, aadA, qacE, bla, qacF and qacL is specific for Moscow WWTPs, since these genes were not among the 50 most common ARGs according to the results of a worldwide analysis of wastewater resistomes in large cities 3 . Different ARGs were most “evenly” represented in the influent wastewater while in the AS and treated effluent, a clear selection of particular types of ARGs was observed, which obviously reflects a change in the composition of microbiomes. A vivid example is the increase in the proportion of ampC in the resistomes, especially at LOS.

The discovered ARGs can confer resistance to most classes of antibiotics and among the resistomes of the studied WWTPs in the city of Moscow, genes conferring resistance to beta-lactam antibiotics were the most common, they accounted for about 26% of the resistome in the water supplied for treatment (Fig.  4 ). Similar values have been observed for wastewater in some other countries, particularly in Eastern Europe and Brazil, where 20 to 25% of reads were assigned to ARGs conferring resistance to beta-lactams 3 . According to data for 2021, beta lactams accounted for about 40% of the total antibiotic consumption in Russia in the medical sector 45 .

The relative abundancies of ARGs in the resistomes categorized by drug classes.

Like in most wastewater resistomes in different countries, ARGs conferring resistance to macrolides, aminoglycosides and tetracycline were also among the most abundant in wastewater from Moscow (Fig.  4 ). Resistance to macrolides, rather than beta-lactams, was most common in wastewater from most countries in Europe and North America, while in Moscow ARGs to macrolide were the second most common. Macrolides and tetracyclines are also widely used in medicine in Russia (20% and 5% of total antibiotic consumption in 2021, respectively). On the contrary, medical consumption of aminoglycosides in Russia is rather low (< 1% of the total), therefore, the high abundance of relevant ARGs was unexpected. The opposite pattern was observed for quinolones, which make up about 22% of the antibiotics used in medicine, but their ARGs accounted for only about 1% of the resistome. However the main mechanisms of resistance to quinolones, mutations in the target enzymes, DNA gyrase and DNA topoisomerase IV, and increased drug efflux 46 , were not addressed in our study.

A peculiar feature of Moscow wastewater resistome was the high content of resistance genes to sulfonamides (about 9%), which were not among the major genes in wastewater resistomes worldwide 3 . Sulfonamides are synthetic antimicrobial agents that currently have limited use in the human medicine, alone or mainly in combination with trimethoprim (a dihydrofolate reductase inhibitor), in the treatment of uncomplicated respiratory, urinary tract and chlamydia infections 7 , 47 . Different sulfonamide ARGs ( sul1, sul2 and sul3 ) were detected in the wastewater in the some countries, including Denmark, Canada, Spain and China, applying culture dependent, independent and qPCR methods 7 . The opposite picture was observed for streptogramin resistance genes, which were among the ARGs in the majority of resistomes worldwide, but in Moscow wastewater they accounted for less than 1%. This is probably due to the limited use of this drug in Russia.

Another distinguishing feature of the resistome of wastewater in Moscow is the high content of ARGs conferring resistance to quaternary ammonium compounds (QAC), about 9%. It can be explained by the frequent use of these antiseptics in medicine. QACs are active ingredients in more than 200 disinfectants currently recommended for inactivation the SARS-CoV-2 (COVID-19) virus 48 . A recent study showed that the number of QACs used to inactivate the virus in public facilities, hospitals and households increased during the COVID-19 pandemic 49 . Indeed, the results of a study dedicated to the study of wastewater resistome worldwide 3 did not reveal the presence of QAC ARGs in the wastewater, since the samples for this study were collected before the pandemic.

An important issue is the extent to which different water treatment technologies remove ARGs. The effective removal of ARG was primary due to a decrease in the concentration of microorganisms in treated effluent, since the share of resistome in the metagenome after treatment decreased by only 2.6 –3.7 times and the NLOS2 plant appeared to be more effective in this respect. However, compared to LOS, treated effluent at NLOS2 contains approximately twice as much suspended solids, probably due to poorer settling characteristics of the sludge indicated by the higher SVI. Therefore, the overall efficiency of removing ARGs from wastewater at two WWTPs may be similar.

Considering the relative abundances of ARGs in the resistomes, genes conferring resistance to macrolides and tetracyclines were removed more efficiently than beta lactamases, especially ampC , and rifampin ADP-ribosyltransferase genes. The low efficiency of removal of the ampC gene and the increase in its abundance in the resistome after wastewater treatment were previously reported for WWTPs in Germany 50 . Efficient removal of ARGs to macrolides ( ermB, ermF, mph(A), mef(A) ) and tetracyclines ( tet(A), tet(C), tet(Q), tet(W) ) has been reported in a number of studies worldwide 51 . ARGs enabling resistance to sulfonamides, tetracyclines and chloramphenicol were more efficiently removed at LOS than at NLOS2, while the opposite was observed for beta lactamases (Fig.  4 ). The later became the most abundant class of ARGs in the treated effluent.

Metagenomic analysis not only identified resistance genes, but also revealed probable MDR strains based on the analysis of assembled MAGs. We identified 9 such strains in both influent, AS and treated effluent. The real number of MDR strains is probably higher, since only a small fraction of all metagenomic contigs was included in the assembled high quality MAGs.

Phocaeicola vulgatus , (formerly Bacteroides vulgatus ), is a mutualistic anaerobic bacteria commonly found in the human gut microbiome and frequently involved in human infections. The results of whole genome analysis showed presence of blaTEM-1 and blaCMY-2 ARGs, which confers resistant to beta-lactams 52 , 53 . P. vulgatus was also identified as potential host for the transmission of tetracycline ARGs 54 . Streptococcus parasuis is an important zoonotic pathogen that causes primarily meningitis, sepsis, endocarditis, arthritis, and pneumonia in both pigs and humans 55 . A variety of MDR strains of this bacterium have been described. For instance, S. parasuis strain H35 was isolated from a lung sample of a pig in China; several ARGs, including optrA , catQ , erm(B), lsa(E), msr(D), mef(A), mdt(A), tet(M), lnu(B), aadE and two copies of aacA-aphD , were found in the chromosome and cfr(D) was detected on plasmid pH35-cfrD 56 . MDR strain of Bacillus cereus was identified in the effluent water microbiome. This bacterium is known as human pathogen and a common cause of food poisoning with toxin-producing property 57 . Bacillus cereus was isolated from drinking water treatment plant in China and antimicrobial susceptibility testing revealed that it was resistant to cefoxitin, penicillin tetracycline 58 , macrolide-lincosamide-streptogramin (MLSB), aminoglycoside and tetracycline antibiotics 59 . Assembled MAG B.cereus from effluent water contained ARGs conferring to macrolides, beta-lactams, fosfomycin and streptogramin and may be considered as MDR strain. Genomes of members of the genera Streptococcus (AS of LOS) and Enterococcus (influent), not identified at the species level, were found to contain multiple ARGs. Most of species of these genera are opportunistic and true pathogens known for their drug resistance 60 , 61 . One MAG from the influent water metagenome was assigned to uncultured lineage of the family Ruminococcaceae. Members of this family are typical non-pathogenic gut inhabitants, although genomes of some strains could harbor ARGs 62 .

Three MAGs retrieved from influent wastewater microbiome ( Ancrocorticia ) and treated effluent water ( Cyclobacteriaceae and Undibacterium ) were found to contain several ARGs. However, we found no evidences about pathogenic and MDR strains in these taxa. It is possible that these environmental bacteria acquired ARGs via horizontal gene from outside their lineages. WWTPs are an ideal environment for horizontal gene transfer (HGT), since when bacteria are exposed to strong selective pressures, such as the presence of antimicrobials, the horizontal acquisition of ARGs enables genetic diversification and create the potential for rapid gains in fitness 63 .

Conclusions

Metagenome sequencing of the raw wastewater, activated sludge and treated wastewater at two large WWTPs of the Moscow city revealed several hundreds of ARGs that could confer resistance to most commonly used classes of antibiotics.

Resistome accounted for about 0.05% of the wastewater metagenome and after wastewater treatment its share decreased by 3–4 times.

The resistomes were dominated by ARGs encoding resistance to beta-lactams, macrolides, aminoglycosides, tetracycline, QAC, and sulfonamides. A peculiar feature of Moscow wastewater resistome was the high content of ARGs to sulfonamides and limited occurrence of resistance to streptogramins.

ARGs for macrolides and tetracyclines were removed more efficiently than ARGs for beta-lactamases.

A comparison of wastewater resistomes from Moscow and around the world suggested that the abundance and content of ARG in wastewater depend on social, medical, and environmental factors.

Data availability

The raw data generated from 16S rRNA gene sequencing and metagenome sequencing have been deposited in the NCBI Sequence Read Archive (SRA) and are available via the BioProject PRJNA945245.

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Acknowledgements

This work was partly supported by the Russian Science Foundation (Project 22-74-00022 to S.B.).

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Shahjahon Begmatov, Alexey V. Beletsky, Andrey V. Mardanov & Nikolai V. Ravin

Winogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Prosp, bld. 33‑2, Moscow, Russia, 119071

Alexander G. Dorofeev & Nikolai V. Pimenov

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S.B. and N.V.R. designed and supervised the research project; A.G.D. collected the samples and analysed chemical composition of wastewater; A.V.M. performed 16S rRNA gene profiling and metagenome sequencing; S.B., A.V.B., N.V.P., and N.V.R. analysed the sequencing data; S.B. and N.V.R. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Shahjahon Begmatov or Nikolai V. Ravin .

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Begmatov, S., Beletsky, A.V., Dorofeev, A.G. et al. Metagenomic insights into the wastewater resistome before and after purification at large‑scale wastewater treatment plants in the Moscow city. Sci Rep 14 , 6349 (2024). https://doi.org/10.1038/s41598-024-56870-0

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H5N1 bird flu: What threat does it pose?

Dr. Rajesh Gulati at UC Riverside answers some common questions about the virus and its spread

The World Health Organization has raised concerns about the spread of H5N1 bird flu , the disease caused by infection with avian (bird) influenza (flu) Type A viruses. Currently, H5N1 bird flu is causing outbreaks in U.S. poultry and dairy cows.

Dr. Rajesh Gulati , interim chair of internal medicine at the University of California, Riverside  School of Medicine , answers some questions about H5N1 bird flu. Gulati is the associate dean of graduate medical education at UCR and a practicing hospitalist at Riverside Community Hospital.

Q: How is this new virus strain behaving differently than strains in earlier outbreaks?

This new strain of avian influenza (H5N1) is different from earlier strains because it has adapted and changed. It has used new genetic material from wild bird genes and infected more wild bird species than previous strains have. It has also been affecting mammals — wild ones, such as bears and foxes, but also domesticated ones, like dairy cattle and cats. We can see this new virus strain is also sticking around longer than previous outbreaks. 

Q: How does the virus spread? How might it spread to humans?

The virus initially caused outbreaks in North American poultry species but has now been detected in many types of mammals as well as a few humans. It is spread through droplets or dust from infected animals, which a human can inhale or transfer from hands to the eyes/nose/mouth. We are not sure about the spread from person to person, but viruses are able to adapt and mutate rapidly. It is important to refer to the Center for Disease Control and Prevention for their research and recommendations.

Q: What precautions should people take?

Many precautions need to be taken by those in the dairy industry in direct contact with possibly infected animals, or those who often interact with wild birds. These individuals should be using PPE (such as respirators), creating isolated areas, and monitoring exposures. The virus is affecting dairy cattle; we want to make sure we are not consuming raw milk products, which contain many more dangerous microbes than possibly just H5N1 and are only drinking pasteurized milk. 

In addition, for those of us with pet cats or dogs, it’s important to limit their interactions with wild birds to avoid any spread of the virus to our homes. We should also try to limit our interactions with wild birds, including their feathers, feces, or nests.  

In general, one of the best ways to protect against viruses is frequent hand washing with soap and water and to avoid touching your hands to your face/mouth/nose/eyes if they are contaminated in any way. 

Q: How can the spread be contained?

The current methods of control for H5N1 will likely be quarantining and culling (selective slaughter) of infected animals. If infection is suspected, it’s important to trace it back to the source and see the interactions along the way. If there is a need for a vaccine, COVID has shown us that our research centers can get them made (relatively) quickly and administered to prevent an outbreak from becoming catastrophic. For now, the virus appears to be contained to some domesticated mammals and the individuals who were interacting with them on a frequent basis. 

Q: The virus has jumped to livestock and wild animals. How might this pose a danger to humans?

The lives of humans and the animals we consume and acquire products from are tightly linked. We interact frequently with domestic animals for farming, trade, and food production, which increases the opportunity for zoonotic transmission — infections that are spread between people and animals. It has the possibility of seriously disrupting our food supply and leading to significant economic losses. 

Due to the rapid mutation, this virus can spread across species. It can lead to strains that are more virulent, that is, extremely severe or harmful, or even adapt itself to person-to-person transmission. This could lead to widespread outbreaks or even a global pandemic.  

Header image credit: wildpixel/iStock/Getty Images Plus.

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Animal Testing and Medicine

Rachel hajar.

Department of Cardiology and Cardiothoracic Surgery, Hamad Medical Corporation, Doha, Qatar

“The greatness of a nation and its moral progress can be judged by the way its animals are treated.” - Mahatma Gandhi

Animals have been used repeatedly throughout the history of biomedical research. Early Greek physician-scientists, such as Aristotle, (384 – 322 BC) and Erasistratus, (304 – 258 BC), performed experiments on living animals. Likewise, Galen (129 – 199 / 217 AD), a Greek physician who practiced in Rome and was a giant in the history of medicine, conducted animal experiments to advance the understanding of anatomy, physiology, pathology, and pharmacology. Ibn Zuhr (Avenzoar), an Arab physician in twelfth century Moorish Spain, introduced animal testing as an experimental method for testing surgical procedures before applying them to human patients.

In recent years, the practice of using animals for biomedical research has come under severe criticism by animal protection and animal rights groups. Laws have been passed in several countries to make the practice more ‘humane’. Debates on the ethics of animal testing have raged since the seventeenth century. Theodore Roosevelt in the nineteenth century stated, “Common sense without conscience may lead to crime, but conscience without common sense may lead to folly, which is the handmaiden of crime.”

Those against, contend that the benefit to humans does not justify the harm to animals. Many people also believe that animals are inferior to humans and very different from them, hence results from animals cannot be applied to humans. Those in favor of animal testing argue that experiments on animals are necessary to advance medical and biological knowledge. Claude Bernard, known as the father of physiology, stated that “experiments on animals are entirely conclusive for the toxicology and hygiene of man. The effects of these substances are the same on man as on animals, save for differences in degree”. Bernard established animal experimentation as part of the standard scientific method.

Drug testing using animals became important in the twentieth century. In 1937, a pharmaceutical company in the USA created a preparation of sulfanilamide, using diethylene glycol (DEG) as a solvent, and called the preparation ‘Elixir Sulfanilamide’. DEG was poisonous to humans, but the company's chief pharmacist and chemist was not aware of this. He simply added raspberry flavoring to the sulfa drug, which he had dissolved in DEG, and the company marketed the product. The preparation led to mass poisoning causing the deaths of more than a hundred people. No animal testing was done. The public outcry caused by this incident and other similar disasters led to the passing of the 1938 Federal Food, Drug, and Cosmetic Act requiring safety testing of drugs on animals before they could be marketed.

Another tragic drug fiasco occurred in the late 1950s and early 1960s with thalidomide. It was found to act as an effective tranquilizer and painkiller and was proclaimed a ‘wonder drug’ for insomnia, coughs, colds, and headaches. It was found to have an inhibitory effect on morning sickness, and hence, thousands of pregnant women took the drug to relieve their symptoms. Consequently, more than 10,000 children in 46 countries were born with malformations or missing limbs (phocomelia, from the Greek meaning ‘limb’). The drug was withdrawn in 1961 and 1968 after a long campaign.

The above-mentioned incidents and others illustrate the harm to humans from the use of substances that have not been first tested on animals and underline the importance of animal experimentation to avert or prevent human tragedy. The practice of using animals in biomedical research has led to significant advances in the treatment of various diseases.

Issues such as ‘cruelty’ to animals and the humane treatment of animals are valid concerns, and hence, the use of animals in experimentation is greatly regulated. This has led to the 3Rs campaign, which advocates the search (1) for the replacement of animals with non-living models; (2) reduction in the use of animals; and (3) refinement of animal use practices. However, total elimination of animal testing will significantly set back the development of essential medical devices, medicines, and treatment. By employing the 3Rs when continuing to use animals for scientific research, the scientific community can affirm its moral conscience as well as uphold its obligation to humanity to further the advancement of science for civilization and humanity.

Stop COVID Cohort: An Observational Study of 3480 Patients Admitted to the Sechenov University Hospital Network in Moscow City for Suspected Coronavirus Disease 2019 (COVID-19) Infection

Collaborators.

• Sechenov StopCOVID Research Team : Anna Berbenyuk ,  Polina Bobkova ,  Semyon Bordyugov ,  Aleksandra Borisenko ,  Ekaterina Bugaiskaya ,  Olesya Druzhkova ,  Dmitry Eliseev ,  Yasmin El-Taravi ,  Natalia Gorbova ,  Elizaveta Gribaleva ,  Rina Grigoryan ,  Shabnam Ibragimova ,  Khadizhat Kabieva ,  Alena Khrapkova ,  Natalia Kogut ,  Karina Kovygina ,  Margaret Kvaratskheliya ,  Maria Lobova ,  Anna Lunicheva ,  Anastasia Maystrenko ,  Daria Nikolaeva ,  Anna Pavlenko ,  Olga Perekosova ,  Olga Romanova ,  Olga Sokova ,  Veronika Solovieva ,  Olga Spasskaya ,  Ekaterina Spiridonova ,  Olga Sukhodolskaya ,  Shakir Suleimanov ,  Nailya Urmantaeva ,  Olga Usalka ,  Margarita Zaikina ,  Anastasia Zorina ,  Nadezhda Khitrina

Affiliations

• 1 Department of Pediatrics and Pediatric Infectious Diseases, Institute of Child's Health, Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
• 2 Inflammation, Repair, and Development Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, United Kingdom.
• 3 Soloviev Research and Clinical Center for Neuropsychiatry, Moscow, Russia.
• 4 School of Physics, Astronomy, and Mathematics, University of Hertfordshire, Hatfield, United Kingdom.
• 5 Biobank, Institute for Regenerative Medicine, Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
• 6 Institute for Regenerative Medicine, Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
• 7 Chemistry Department, Lomonosov Moscow State University, Moscow, Russia.
• 8 Department of Polymers and Composites, N. N. Semenov Institute of Chemical Physics, Moscow, Russia.
• 9 Department of Clinical and Experimental Medicine, Section of Pediatrics, University of Pisa, Pisa, Italy.
• 10 Institute of Social Medicine and Health Systems Research, Faculty of Medicine, Otto von Guericke University Magdeburg, Magdeburg, Germany.
• 11 Institute for Urology and Reproductive Health, Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
• 12 Department of Intensive Care, Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
• 13 Clinic of Pulmonology, Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
• 14 Department of Internal Medicine No. 1, Institute of Clinical Medicine, Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
• 15 Department of Forensic Medicine, Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
• 16 Department of Statistics, University of Oxford, Oxford, United Kingdom.
• 17 Medical Research Council Population Health Research Unit, Nuffield Department of Population Health, University of Oxford, Oxford, United Kingdom.
• 18 Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
• 19 Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Oxford, United Kingdom.
• 20 Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
• PMID: 33035307
• PMCID: PMC7665333
• DOI: 10.1093/cid/ciaa1535

Background: The epidemiology, clinical course, and outcomes of patients with coronavirus disease 2019 (COVID-19) in the Russian population are unknown. Information on the differences between laboratory-confirmed and clinically diagnosed COVID-19 in real-life settings is lacking.

Methods: We extracted data from the medical records of adult patients who were consecutively admitted for suspected COVID-19 infection in Moscow between 8 April and 28 May 2020.

Results: Of the 4261 patients hospitalized for suspected COVID-19, outcomes were available for 3480 patients (median age, 56 years; interquartile range, 45-66). The most common comorbidities were hypertension, obesity, chronic cardiovascular disease, and diabetes. Half of the patients (n = 1728) had a positive reverse transcriptase-polymerase chain reaction (RT-PCR), while 1748 had a negative RT-PCR but had clinical symptoms and characteristic computed tomography signs suggestive of COVID-19. No significant differences in frequency of symptoms, laboratory test results, and risk factors for in-hospital mortality were found between those exclusively clinically diagnosed or with positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT-PCR. In a multivariable logistic regression model the following were associated with in-hospital mortality: older age (per 1-year increase; odds ratio, 1.05; 95% confidence interval, 1.03-1.06), male sex (1.71; 1.24-2.37), chronic kidney disease (2.99; 1.89-4.64), diabetes (2.1; 1.46-2.99), chronic cardiovascular disease (1.78; 1.24-2.57), and dementia (2.73; 1.34-5.47).

Conclusions: Age, male sex, and chronic comorbidities were risk factors for in-hospital mortality. The combination of clinical features was sufficient to diagnose COVID-19 infection, indicating that laboratory testing is not critical in real-life clinical practice.

Keywords: COVID-19; Russia; SARS-CoV-2; cohort; mortality risk factors.

© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: [email protected].

Publication types

• Observational Study
• Research Support, Non-U.S. Gov't
• Hospitalization
• Middle Aged

Grants and funding

• 20-04-60063/Russian Foundation for Basic Research