My presentation will focus on the use and numbers of nonhuman primates (NHP) in research in the United States. To determine these numbers it was necessary to visit a lot of regulatory agencies—including the CDC, USDA, and NCRR/NIH (for chimpanzee data)—and Indonesia. The bottom line is that it is very difficult to quantify this in any meaningful sort of way. It seems that nobody really has the number of nonhuman primates that are used in research. In addition, different organizations and agencies count these numbers in different ways.
In some instances, it was necessary to go to the animal rights groups or to Wikipedia to find out numbers. After averaging all the numbers, the total is approximately 70,000 to 75,000 nonhuman primates each year, and that includes all types of use, whether they are being bred or held or actually used in research.
The CDC has 27 different registered NHP importers in the United States (information provided with the assistance of Bob Mullan and Gail Galland). In the early days of this field, there were over 100 importers of NHP, suggesting that greater regulation has resulted in fewer animals being imported. It is interesting to note that nearly half of these importers are commercial importers, with the rest scattered among zoos and academic institutions.
CDC has requirements for licensing a facility. All NHP are required to stay in a federally registered quarantine for at least 31 days before they can be transferred to another institution. Most institutions quarantine them for a longer period of time.
One of the confounding regulations was the requirement for records on these primates as they moved from the initial importing institution to other institutions. The importing institution has to document whether the animals are being held for scientific, exhibition, research, or educational use and that wherever they send the animals will likewise register in the same category. The intent is to keep these animals out of the pet trade. The CDC inspects facilities, reviews
import plans, monitors arriving shipments, assesses disease control measures, reviews animal health records, and investigates illness reports.
The data on NHP importation from 1994 to 2007 show a continual increase. In 2005 to 2007, the last three years for which data are available, the number rose to over 25,000 or 26,000 primates per year being brought into this country. This suggests a greater use of nonhuman primates in the United States. If the data are broken down further one finds that over 93% of imported NHP are Macaca fascicularis, or cynomolgus; 5% are M. mulatta, or rhesus, probably from China. The rest are scattered among other species.
If one looks at the data by importer, each importer brought in from one to over 10,000 animals, with the number of shipments per importer ranging from one to 70 a year. A significant statistic is the percentage of dead-on-arrivals: zero to 0.2%, which is very low. This is a huge improvement from many years ago when a 10% to 15% mortality rate was considered good. Reportable illnesses are very low now as well.
Looking at the importation data further, 60% of these animals are coming from China, followed by Vietnam and Mauritius. Over 85% of the animals are coming from three countries.
NIH (through NCRR) supports eight national primate research centers, which collectively contain almost 28,000 nonhuman primates, with the majority being rhesus monkeys. It becomes clear that it is the rhesus monkey that is used in research, not M. fascicularis (cynomolgus). Cynomolgus monkeys are used by commercial industries, pharmaceutical industries, or CROs for toxicology, efficacy, safety, and pharmacodynamics.
NCRR is moving toward the development of so-called specific pathogen–free colonies, which consist primarily of rhesus colonies, M. mulatta. Those colonies are primarily SPF-4, meaning they are free of SIV, STLB, SRV, and herpes B. There are other colonies called “superclean” that have even more viruses eliminated, such as cytomegalovirus, foamy virus, and perhaps others. At this point, roughly 5,000 rhesus monkeys in SPF-4 colonies are being produced for research by investigators throughout the country. There are plans in the national primate research centers program to create even more SPF colonies in the years to come.
The major types of research conducted at the primate centers are AIDS and other infectious diseases: these account for over 40% of research activity. Neurobiology research is also prevalent, at almost 20% of activity, and various other areas make up the rest.
Many specialized resources emerge from primate center programs. Perhaps one of the more important ones is the NHP tissue program, from which over 42,000 primate tissue samples, organs, genetic samples, cells, fluids, and more are supplied to investigators throughout the nation and internationally.
USDA annual reports provided information about how many primates were in use in registered facilities throughout the United States. There are roughly 46,000 or so NHP listed in these reports, either in column B (used for breeding) or columns C and D (used in research, in situations where there is no
pain, momentary pain, or pain alleviated with analgesia). Interestingly there were no reports of animals in column E (unrelieved pain and/or distress). This is difficult to understand since there are clearly projects going on at various facilities that fall under column E.
In summary, based on these and the previous data, an estimated 70,000 to 75,000 primates are used in research.
Now I will address the use of chimpanzees, and I would like to acknowledge the contribution of Tom Butler for the information I will share with you. The most important thing about chimps is that they share roughly 98.5% of their DNA with humans. Therein lies both the benefit and the curse of the issue of chimps in research, and there are many different thoughts about whether or not they should be used for this purpose.
Using chimps means increased expense due to the need for larger cages, larger facilities, stronger people, and more educated people. Six centers in the United States maintain chimpanzees: the Southwest Foundation for Biomedical Research (San Antonio, TX), Michale Keeling Center for Comparative Medicine (Bastrop, TX), New Iberia Research Center (Louisiana), Yerkes Primate Center (Atlanta, GA), Primate Foundation of Arizona (Mesa), and Alamogordo Primate Facility (New Mexico). All but the last two conduct research on chimpanzees.
The population of chimpanzees is continually declining.1 From the latter part of 2006 to the first part of 2007, there was a decline of nearly 100 animals or almost 10% of the chimp population. The population has been declining primarily for health-related reasons. Another consideration is that about half of the animals are owned or subsidized in part by the federal government, which traditionally does not make these chimps available for research by private industry.
A key factor is animal age. The desirable age for chimps in many research projects is roughly up to 21 years of age, which is about the time health problems begin developing, particularly cardiovascular problems. As of 2008, only about half of the US chimp population was below the age of 21, further accentuating the declining usable numbers of chimps for research. Given the current rate of decline, the number of chimps for research will be close to zero by the year 2030. While age can exclude chimpanzees from use in research, other factors to consider in choosing animals include behavioral characteristics, health status, experimental history, and current research.
So there are very few chimps available for research in the United States. Yet there are those who insist that the chimpanzee is the only animal that can be used specifically for pharmacodynamics in a way that the human is used, to test monoclonal antibodies. Many of these monoclonal antibodies cannot be tested in other warm-blooded mammals or in other NHP species because they are elimi-
1Data in this and the next paragraph are from an unpublished workshop presentation by Thomas Butler, DVM, DACLAM (currently Chair of the Board of Directors of Chimp Haven in Keithville, LA), “The Future of Chimpanzees in Biomedical Research,” on October 17, 2007.
nated very rapidly. The chimp is the only animal that processes these monoclonals in a way that they can be tested.
Vaccine development is another huge concern in considering research on chimpanzees. The chimpanzee was integral to development of the hepatitis B vaccine and is used to make improvements in it. In addition, it is the only known animal model for hepatitis C and is being used in attempts to develop a hepatitis C vaccine. However, it should be noted that in the early days of AIDS researchers thought chimps would be extremely valuable, but that was not the case.
In terms of emerging infectious diseases, many scientists feel strongly that the chimpanzee should not be allowed to disappear from the US research scene. Some argue that it would be foolhardy to let the chimp population disappear and then need the animals again for future critical research.
In 1997 the National Research Council published a report that set the stage for the future of chimps with regard to breeding (Chimpanzees in Research: Strategies for Their Ethical Care, Management, and Use). The breeding ban for chimps in federally funded facilities continues today. The report also recommended that euthanasia not be allowed for population control. Along with this, there was a suggestion that a national chimpanzee sanctuary facility be created; Chimp Haven, in Shreveport, Louisiana, took in its first animals in 2005 and now houses about 110 chimps in retirement. Although the NRC report recommended that there be 1,000 chimps available to meet current research needs, there would need to be 60 births per year. With the federal ban on breeding, there are only about 15 births per year at privately held facilities. Because the population of chimps is aging, it seems certain that the numbers will continually decline. In addition, most chimps have been used for experiments, so there will be virtually no naïve animals for future studies.
In the last part of my presentation, I would like to highlight Indonesia, a typical exporting country for NHP. Most people in the United States are unaware of Indonesia, yet it is the fourth-most populous country in the world, made up of 13,000-plus islands, and home to numerous species of nonhuman primates, including vast numbers of Macaca fascicularis. Much of the following information was obtained from the Indonesian quarantine group, which is responsible for export.
Between 2004 and 2007, there was a rapid increase in the numbers of nonhuman primates exported. Most have been going to China, which is rapidly becoming the giant in terms of NHP use as well as NHP export to the United States and other countries. In China, and to a lesser extent in Indonesia, primate centers and research centers are being built. These countries will no longer want to supply NHP to the US and other countries as they will want to develop research enterprises in their own countries. Many people from those countries have been educated in the United States and Europe and are beginning to feel ready to do research back home.
For example, the Bogor Primate Research Center in Indonesia has the capacity for many major types of research procedures, including those that involve ABSL-3 facilities. Many cutting-edge research projects are ongoing in conjunc-
tion with laboratories in that country. The center has virology labs that are as large and as well equipped as those in the United States. This is happening because the Indonesian government and others have invested money and training and now they are prepared to undertake research projects on their own. These types of activities are occurring in many other countries of origin of NHP. These countries are very capable and they are looking to increase these activities.
I will conclude by explaining why there is an increase in NHP exportation. Breeding colonies are developing everywhere in Indonesia because new laws prohibit the exportation of feral animals. Countries of origin such as Indonesia, China, India, and others will be requesting outsourcing of research from the United States.
In summary, obtaining accurate numbers of NHP used in research is difficult at best, but the trend is increasing, not decreasing. The need for research using chimpanzees remains controversial, but they will continue to be used at least in the short term.
I would like to thank Bob Mullan and Gail Galland from CDC, John Harding from NCRR and NIH, Tom Butler for giving me the chimp data, Joko Pamungkas and others from the Bogor Primate Center and the quarantine division of the Indonesian governments, and Pam Ferguson and Patti Rosendahl from Paris NHP.
In this presentation I will highlight how big the Chinese nonhuman primate (NHP) production capability is, the quality of the animals, animal welfare considerations, and how the animals are exported worldwide.
China has been a leading and major supplier country for NHP, not only to the United States but to Europe, Japan, and now to Korea. The numbers of NHP used by various countries in 2002 and estimated for 2007 are 52,000 and 59,000, respectively, by the US1; 4,000 and 5,000 by France2; 3,000 and 4,000 by the UK3; 2,000 and 3,000 by Germany4; 2,000 and 3,000 by Canada5; and 3,000 and 5,000 by Japan.6,7 China has also been exporting NHP to the Netherlands and Spain and, particularly in recent years, has been exporting macaques to Canada.
1Data extrapolated from USDA Report of Animal Welfare Act in these two years.
2The numbers of NHP used in France were extrapolated from Chinese suppliers/breeders and the report from the European Commission to the Council and the European Parliament on Statistics on the Number of Animals Used for Experimental and Other Scientific Purposes (2002). I also referred to the Ethics of Research Involving Animals, Appendix 2: Statistic-Research Involving Animals in the UK, EU, USA, and Japan.
3The numbers of NHP used in the UK were extrapolated from Home Office (2004) Statistics of Scientific Procedures on Living Animals Great Britain 2003 and from other sources, including the Ethics of Research Involving Animals, 2005.
4The numbers of NHP used in Germany were extrapolated from Chinese export sources and from the European Commission report cited above.
5The numbers of NHP used in Canada were extrapolated from Chinese export data and a personal survey of Canadian importers.
6The numbers of NHP used in Japan were extrapolated from Chinese exporters’ information as well as information provided by Japanese users and from Chinese government records. I also referred to a survey from April 2001-March 2002, performed by the Committee for Laboratory Animal Care and Use (2003).
7Author’s note: The UK, France, Germany, Canada, and Japan do not have yearly compulsory reports on the use of laboratory animals like the US. I believe that the extrapolated numbers of NHP used in 2007 in these countries are fairly close to the reality.
If one looks at the total number of the macaques imported into the US in 2006-2007, the number was higher in 2006.8 In 2007, 27,000 macaques were imported, of which cynomolgus constituted 93% and rhesus only about 5%. Based on the data from Asia that Dr. Morton presented, almost 25,000 NHP were imported from Asia, representing 93% of the total NHP imports worldwide to the United States for research use.
Specifically looking at China over the past two years, in 2006 the US imported about 11,000 cynomolgus monkeys and about 1,400 rhesus, and in 2007 about 15,000 cynomolgus and 1,350 rhesus.9 As a clarification, cynomolgus monkeys are not native to China. They have come into China from various countries in Asia. Of the cynomolgus monkeys imported in the US from Asia in 2007, 68% are from China compared to 13% from Mauritius. Imports from Cambodia, Indonesia, and the Philippines are rising.
In the past two to three years, Chinese breeders have imported a large number of cynomolgus monkeys from Cambodia. The Chinese government exerts tight control and either restricts or approves the process. For example, in order to import 2,000 or so of the animals by charter from Cambodia to China, it is necessary to acquire an import permit, which usually takes about six months. During this process, the government, both local and central, sends representatives to a region or supply farm for inspection. They check on the number and quality of the animals and whether they are wild-caught or purpose-bred animals.
In the past three years, China has increased the number of breeders. All the primate breeding facilities must obtain a license for production of the nonhuman primates through the Bureau of Wildlife Protection and Conservation in the central government. They must also be approved by a similar office in the provincial government. The government does not allow breeders to sell their animals for up to six years from the start of the facility. Many want to know how many NHP facilities there are in China. There are 32 “qualified” facilities in China, meaning that the facilities are monitored for the number of animals and their quality. Of these facilities, in 2008 23 of them were able to sell/export cynomolgus monkeys and 16 of them rhesus. Some of the facilities engage in cynomolgus monkey breeding only, some of them have both cynomolgus and rhesus, and some of them breed only rhesus monkeys.
Among the 32 NHP facilities half of them are large, meaning they have over 10,000 animals. Some of them are medium-sized or small, with 5,000 to 9,000 animals.
8The total number of the macaques imported into the US in 2006-2007 was obtained from the CDC presentation at the annual Conference of the Association of Primate Veterinarians.
9The numbers of cynomolgus macaques and of rhesus macaques were from CDC reports at APV meetings.
The number of the facilities has been increasing because there is a perceived increasing demand for macaques and particularly for cynomolgus monkeys.
China has a commercial quota system. Toward the end of each year, the Bureau of Wildlife Conservation and Protection as well as the provincial government and an ad hoc committee visit each facility and count the animals in terms of how many young, the ages of the animals, and the total number of breeders. Based on this information, they assign each facility the number of animals allowed to be commercialized (i.e., exported or used domestically in research).
From 2006 to 2008, the quota for cynomolgus production and sale increased. The quota for rhesus increased only slightly and most of the large facilities are not involved in the breeding of rhesus monkeys now.
It should also be noted that most of the breeding facilities for cynomolgus monkeys are in the southern part of China, and facilities for rhesus monkeys are mostly in the central or western part of China.
The number of breeding females in the 32 facilities was obtained from each facility as well as from government data. In 2008 there were about 62,000 breeding cynomolgus monkeys and 8,600 breeding rhesus monkeys.
About 18,000 to 19,000 macaques are exported from China each year and the mortality rate is very low, on the order of 0.06% in 2007. Transportation of NHP has not presented any problems in China. Several airlines—Air China, China Eastern, China Southern, and Hainan Airline—are willing to transport the animals from China worldwide—to the United States, Europe, Japan, and Canada. Since many of the animals come from the south of China, they are shipped from Guangzhou Airport. Animals are also shipped from Shanghai and Beijing. The major receiving airport in the US is Los Angeles, with the second being New York (JFK and Newark). Chicago and Seattle airports also receive animals. Thus, transport of nonhuman primates to worldwide places from China is not a problem.
In looking at the practices related to supply and quality of NHP in China, it usually takes about 2-3 months to obtain the commercial quota as discussed earlier. There is also a quarantine program that is very similar to that in the US for animals to be exported. The quarantine process lasts from 1½ to 2 months, during which the animals must receive three TB tests at two-week intervals and are also tested for various viral, bacterial, and parasitic diseases. China has very good diagnostic methods in terms of simian immunodeficiency virus (SIV) and B virus. However, since the US has the Virus Reference Laboratory (VRL), the viral status of animals imported into the US will be confirmed by the VRL. The animals receive vaccinations for measles and hepatitis A prior to export. Finally, each animal receives a certificate of health by the Provincial Administration of Quality Supervision, Inspection, and Quarantine (AQSIQ). Since 2007, the vast majority—about 90%—of animals are virus free. They also can be vaccinated upon request.
One issue I would like to address is the concern that China is not concerned about animal welfare. In fact, China has five regulations related to welfare of laboratory animals.10 Violation of certain of these regulations carries a penalty.11
10(1) Statute on Administration of Laboratory Animals (1988), issued by the Ministry of Science and Technology. (2) Project on Laboratory Animals during the Ninth Five Years (1997), issued by the Ministry of Science and Technology. (3) Guideline of Beijing Municipality on the Review of Welfare and Ethics of Laboratory Animals (2005), issued by Beijing Administration Office of Laboratory Animals. (4) Guideline on Humane Treatment of Laboratory Animals (2006), issued by the Ministry of Science and Technology. (5) Regulations on Punishment of Dishonorable Behavior in Science and Technology Projects (2006), issued by the Ministry of Science and Technology.
11Furthermore, most CROs conducting animal studies or testing as well as all AAALAC-accredited primate facilities have an IACUC.
By way of definition, New World monkeys and neotropical primates are the same thing and comprise primates that are indigenous to the Americas. There are four main genera of New World monkeys that are used in research: squirrel monkeys (Saimiri spp.), owl monkeys (Aotus spp.), marmosets (Callithrix jacchus), and tamarins (Saguinus mystax).
New World monkeys are used in virtually all the same ways as the Old World monkeys in biomedical research. They are used for discovery research and for preclinical research. It is essential to draw a distinction between the two [types of research], because the vast majority of primates that are imported are used for preclinical studies, safety, and efficacy studies.
Discovery research focuses on discovering something new, something previously unknown, and is typically investigator-driven research funded by the NIH. Preclinical research focuses on verifying what was learned in discovery research under the highly controlled GLP conditions that are required by the FDA or equivalent regulatory bodies in Europe. Both types of research contribute to the translational science that we talk about in the United States—from bench to bedside.
Genomic comparisons are often used as a rationale for why primates are important for certain kinds of research into human disease. New World monkeys and Old World monkeys share approximately 92% and 94%, respectively, of DNA with humans. Chimpanzees, of course, are even closer, sharing over 98%.
Among the squirrel monkeys (genus Saimiri), three species or subspecies are used in research, primarily in the United States. Saimiri sciureus sciureus is the common squirrel monkey and the one most frequently imported to the United States; S. boliviensis boliviensis, the Bolivian squirrel monkey, and S. boliviensis peruviensis, the Peruvian squirrel monkey, are also used for research. Three species of owl monkeys are used in research: Aotus nancymaae, A. vociferans, and A. azarae. Marmosets (Callithrix jacchus) are used in Europe more than in the United States, but there are several colonies around the country: one at the Wisconsin National Primate Research Center, one at the Southwest National Primate Research Center, and a small colony at the New England Primate
Research Center. Tamarins are used probably the least of the four genera, and Saguinus mystax is the most commonly used one in the United States.
Bolivian squirrel monkeys are no longer available through importation due to bans in the country of origin—Bolivia no longer exports squirrel monkeys, so we are not likely to get more of these. Bolivian squirrel monkeys have been very useful in malaria research because they are susceptible to falciparum malaria and Plasmodium vivax. They have also been used in prion disease work, the transmissible spongiform encephalopathies. Squirrel monkeys are susceptible to every prion disease with which they have been infected, including wasting disease of deer, which is a growing problem in the United States. If one looks at lesions from brains with sporadic and variant Creutzfeldt-Jakob disease (CJD), there is a little difference in the lesion. In sporadic CJD there is a generalized spongiform change. In the variant form of the disease, which is thought to have come from eating contaminated beef from animals that had bovine spongiform encephalopathy, the lesion creates a rosette around accumulations of abnormal prion protein. Squirrel monkeys develop these lesions and they look virtually identical to those seen in humans.
Owl monkeys have been used for malaria research, pathogenesis, and vaccine development work with malaria. In our colony we have seen a high prevalence of cardiomyopathies and the sequelae to chronic hypertension. These animals exhibit aortic aneurisms and hypertrophic cardiomyopathy. In the end stages of the disease they develop chronic heart failure, and instead of the heart pumping it just appears to vibrate—the contractility of the heart is virtually gone. This is, as far as we know, the only spontaneous or naturally occurring chronic severe hypertension model in a primate species.
Marmosets have been used for a number of studies of obesity, metabolic syndrome, aging, and reproductive biology. They are valued in reproductive biology because about 80% of these animals produce dizygotic twins, which share the same placenta, so there is a very high prevalence of chimerism.
Tamarins were very important in the development of hepatitis A vaccine. They are susceptible to GB virus B, which is closely related to hepatitis C virus.
In the United States there are three sources of New World monkeys. One is through importation. Second is through an agreement between the Pan American Health Organization (PAHO) and NIH through the Peruvian Primatology Project to provide small numbers of New World monkeys to NIH and through NIH to others that need them for research; not very many animals are imported that way, but it is a source. Then finally, animals can be obtained from the Center for Neotropical Primate Research and Resources at the University of Texas M.D. Anderson Cancer Center, and specifically at the Michale Keeling Center for Comparative Medicine. The Center for Neotropical Primate Research and Resources is supported through two grants from NCRR, one for squirrel monkeys and one for owl monkeys.
To obtain specific information about importation of these species, I went to the CITES1 database and looked up importation data over a 5-year period, from 2002 to 2006. Of all the species, squirrel monkeys are the most commonly imported around the world. Between 2002 and 2006 over 4,000 monkeys were exported to North America and almost 3,000 to other regions of the world.
Looking at the number of squirrel monkeys imported into the United States by year from 1997 to 2006, the highest number was in 2002 (>500); in most years it averaged between 200 and 300 a year. These numbers do not take into account animals procured from domestic colonies, and do include research colonies at institutions around the country.
Owl monkeys imported to the United States have come from two countries, Argentina (Aotus azarae) and Peru (A. vociferans and A. nancymaae). Virtually all of these animals have been used for malaria research.
Animals supplied through the Center for Neotropical Primate Research and Resources have been primarily squirrel monkeys. The owl monkey resource, for which funding began in 2004, has begun to grow, but as with all breeding resources of primates, it takes years to develop them.
To determine the types of research done in various species of NHP, I queried the CRISP database.2 Macaques are by far the most frequently used NHP, with over 650 grants/contracts in 2008 citing their use. About 40 grants this year cite the use of the squirrel monkeys, 15 cite owl monkeys, and 6 cite marmosets. One must be careful in interpreting these numbers because they only reflect NIH extramural research and do not take into account NHP used by the Department of Defense, which used many, as well as the Food and Drug Administration and the Centers for Disease Control and Prevention.
The intramural research program of NIH also uses primates, and they use more macaques than any other genus of primates. However, the NIH Office of Animal Care and Use provided information that imports through PAHO from 2002 to 2006 totaled 230 owl monkeys and 68 squirrel monkeys.
To try to bring it into perspective, New World monkeys are valuable for several key diseases. The owl monkey is an emerging model for cardiovascular disease and chronic hypertension. In the United States in 2008, an anticipated 400,000 people will die from cardiovascular disease. Looking worldwide of course it will be far, far greater.
These animals have been used extensively for malaria research. While not of foremost importance in the United States, malaria remains one of the most devastating diseases worldwide. Mortality in 2008 is expected to be almost 3 million people, and 75% of those who die from malaria are women and children.
Half of the children in Africa that die before the age of 5 die from complications from malaria.
Finally, these animals are important in studies of hepatitis B and C. Although there is now a vaccine for hepatitis B, these diseases remain a big problem. Tamarins were very important in the development of hepatitis A vaccine, and they may provide answers to some questions about hepatitis C. It is hard to determine numbers for the impact of hepatitis B and C worldwide, but it is probably on the order of 3 million or more people, and people who survive with hepatitis C for 25 years have a very, very high incidence of hepatocellular carcinoma and hepatomas. So it is also a risk factor in cancer.
I want to thank Jim Taylor, Don Bordine, and Alfie Caesar, who helped me with some numbers, as well as Larry Williams and Laura Zapalac in my department.