Animal Welfare Information Center Newsletter, Summer 1994, Vol. 5, no. 2

The Cotton Rat in Biomedical Research

by
Gregory A. Prince
Vice President and Director of Research, Virion Systems, Inc., Rockville, MD

[Introduction]
Natural History
The Cotton Rat as a Model of Human Disease
Care and Handling
Experimental Manipulation

For over a half century the cotton rat has served as a model for a remarkably extensive list of human and rodent pathogens. Currently its use is most important in studies of human respiratory syncytial virus, human adenoviruses, human parainfluenza virus type 3, and Litomosoides carinii (causative agent of cotton rat filariasis). In addition, it is being used in studies of the pineal gland, as a model for environmental toxicology, and as a primary animal model of human genetic therapy using the adenovirus vector. In spite of its past and present importance as well as its future potential, the model has been underutilized due to three shortcomings. First, until recently there has been no commercial source of cotton rats. Second, the lack of an inbred cotton rat has precluded important immunologic studies. Third, there have been no commercially available immunologic reagents directed against cotton rat tissues. The first two of these limitations have been overcome, as an inbred cotton rat (Sigmodon hispidus), developed in collaboration with Dr. Carl T. Hansen at the National Institutes of Health (NIH), is now available commercially through Virion Systems, Inc. The third limitation, the lack of immunologic reagents, is currently being addressed through a program in our laboratories, supported by a grant from the National Center for Research Resources, NIH, which will eventually result in the development of a broad library of reagents against cotton rat immunoglobulins, cellular antigens, and cytokines

Detail of drawing by J.J. Audubon

Natural History

The cotton rat is a New World rodent, whose distribution extends from the Southern United States through Mexico, Central America, Colombia and Venezuela. In many of these regions it is the most common feral mammal, forming an important link in the food chain as well as posing occasional problems to agriculture. Seven species of cotton rats have been identified: Sigmodon hispidus, S. alleni, S. arizonae, S. fulviventer, S. leucotis, S. mascotensis, and S. ochrognathis. S. hispidus has the largest geographical distribution, extending from southern Virginia to Florida, then westward through southern Arizona, and most of Mexico, Central America, Colombia and Venezuela. It is also the ancestor of the other six species. Although there is little phenotypic difference between these species, there is striking karyotypic diversity, ranging from 22 chromosomes for S. arizonae to 52 for S. hispidus.

The Cotton Rat as a Model of Human Disease

Polio

The widespread use of the cotton rat in biomedical research began over a half century ago in the midst of an outbreak of one of the most feared infectious diseases of this century, poliomyelitis. In the latter half of the 1930's an epidemic of polio swept across the country. Although polio virus was known to cause paralytic disease in monkeys, economic and logistical problems led investigators to search for a smaller animal model. Dr. Charles Armstrong, working at the NIH in 1937, dispatched several of his workers throughout the Southern United States with instructions to live-trap and bring back to NIH any small mammals which might be adaptable to use in a laboratory setting. Many species were obtained in this manner, and all were inoculated with polio virus obtained from a fatal case in Michigan. Among the animals was one cotton rat, S. hispidus, and it, alone, developed paralytic disease. Following the publication of Armstrong's report in 1939, the cotton rat quickly became a widely used model for polio. By 1941 one laboratory, supported by a grant from the National Foundation for Infantile Paralysis (subsequently known as the March of Dimes), developed a breeding colony of over 1000 animals, and made these animals available to other laboratories at a cost of $0.50 each. Although the inbred mouse eventually superseded the cotton rat as a model for polio, several important advances in polio research occurred as a result of the cotton rat, detailed in over 30 published papers extending from 1939 to 1979.

Typhus

During World War II, British troops in Southeast Asia were being decimated by endemic ("scrub") typhus. A paper published in 1937 describing the susceptibility of the cotton rat to the causative agent led to a major initiative in Great Britain to develop a scrub typhus vaccine using the cotton rat. The classified project, code-named "Operation Tyburn" and directed by the Wellcome Foundation, involved the construction of an extensive animal housing facility in Sussex, the transport of cotton rats across the Atlantic in American bombers, and a large-scale breeding program designed to produce in excess of 10,000 animals per month. Although the end of the war brought Operation Tyburn to an abrupt halt, and the full utility of the vaccine was never determined, over 300,000 doses of vaccine were prepared in the facility.

Filariasis

Near the end of World War II, a report was published describing filariasis in the cotton rat, caused by an endogenous pathogen, Litomosoides carinii. Between 1945 and 1993 over 200 scientific papers were published describing studies of cotton rat filariasis, far more than for any other laboratory use of the cotton rat. This model contributed greatly to an understanding of the pathogenesis and immunology of filariasis and served as a primary means of testing chemotherapeutic agents.

Respiratory Syncytial Virus

Although the cotton rat is indigenous to the New World, its utility as a model for polio, typhus, and filariasis led to the establishment of breeding colonies in several European countries. In 1970, a team of Soviet scientists reported the susceptibility of the cotton rat to pulmonary infection by respiratory syncytial virus (RSV), the primary cause of infant pneumonia throughout the world. While earlier efforts in other laboratories had shown that other mammalian species were susceptible to nasal RSV infection, this group was the first to describe a small-animal model of pulmonary RSV disease. In the quarter century since this discovery, the most important contribution of the cotton rat to biomedical research has been as a model of RSV disease. Recently concluded clinical studies, which were based upon data generated in the cotton rat model, showed that human IgG with high neutralizing activity against RSV can prevent serious RSV disease in infants and children at highest risk for life-threatening disease. Further clinical trials, also based upon data generated from the cotton rat model, will determine if the same preparation will be effective in treating RSV pneumonia in infants. In addition to these studies of passive prophylaxis and therapy, ongoing studies in several laboratories to develop an RSV vaccine are relying heavily upon the cotton rat model to demonstrate both efficacy and safety.

Adenoviruses and Genetic Therapy

In 1984, Pacini and co-workers published a paper describing the pathogenesis of human adenoviruses in the cotton rat. Several subsequent papers have verified the utility of the cotton rat for the study of adenovirus pathogenesis, and have laid the groundwork for understanding the genetic basis of viral disease.

Since the cotton rat is the only small animal known to be susceptible to human adenoviruses, and since the disease produced in cotton rats is remarkably similar to that seen in humans, much of the recent work exploring genetic therapy (which employs a human adenovirus as a vector for the replacement gene) has been done in the cotton rat. The demonstration of in vivo expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the cotton rat correlated well with the subsequent pattern of expression in the cells of cystic fibrosis patients treated (in vitro) with the adenovirus-CFTR vector.

Care and Handling

Virion Systems, Inc., maintains two breeding colonies of cotton rats: inbred S. hispidus and outbred S. fulviventer. (The latter is in the 13th generation of an inbreeding program which, if successful, will yield an inbred strain of S. fulviventer within 3 years.) The two species are maintained and handled in the same manner. We have found that large polycarbonate rat cages with locking wire lids are the best form of housing. Up to five adult animals may be housed in one cage. Dietary requirements for breeding and maintenance are satisfied by standard rodent chow (4 percent ideal protein (Ed. note: commercially available rodent diets, prepared from natural products, are approximately 20 percent crude protein)) and water, with no requirements for supplements. We have tried several types of bedding, including shredded hardwood and hardwood chips, but prefer Care Fresh (Absorption Corp., Bellingham, WA), which consists of "100 percent reclaimed paper mill byproducts." We have found that this produces less dust than the other types of bedding (and cotton rats, being more active than Mus and Rattus, generate more dust), and serves as an adequate nesting material. Bedding should be changed twice weekly.

Handling cotton rats may prove unsettling to people used to dealing with Mus and Rattus. Cotton rats are not aggressive, although they are commonly mislabeled as such. They will, however, attempt to bite when they are picked up. Furthermore, since they move very fast, and can jump vertically over 12 inches, they pose a challenge to the novice handler. We recommend that the handler use common garden-type leather gloves. Initially, one may wish to place the cage to be changed in a deep sink to minimize the possibility of an animal escaping. In cages containing more than one animal, it is best to slide the cage top enough to reach in with the hand, but not to remove the lid, as this allows for multiple escapes. With a bit of experience and improved reflexes, most handlers will be able to change cages on a countertop.

Animals for breeding setups should be paired at the time of weaning (3-4 weeks of age), as pairing at older ages results in increased fighting. The pair should not be separated when a litter is born, as this increases the likelihood that the female will kill the male upon reintroduction. The gestation is 27 days; inasmuch as the female goes into estrous at the time a litter is born, many breeding pairs will produce a litter each month, with an average of five pups per litter. Unlike some other rodent species (hamsters and some mouse strains, for instance), the cotton rat rarely turns on her infants due to handling. Indeed, a mother will readily accept foster infants, even if of a different age than her own litter.

Experimental Manipulation

Cotton rats have been subjected to a wide range of experimental and surgical protocols and have proven to be a remarkably adaptable animal. The following tips may prove useful:

Anesthesia: For short-term anesthesia, we prefer methoxyflurane (Penthrane, Metofane). We use a glass instrument jar with gauze containing a small amount of anesthetic. Anesthesia is usually induced within 1 minute, and the animals remain anesthetized for about 1 minute following removal from the jar. This is adequate time for intranasal inoculation, various forms of injection, and bleeding from the retroorbital venous plexus. Neither ether nor isoflurane is acceptable, as induction times are very short and deaths are frequent.

Longer term anesthesia may be performed in two ways. For surgical procedures, induction with methoxyflurane may be followed with closely monitored maintenance using a 50 ml centrifuge tube containing methoxyflurane-soaked gauze, the opening of which is positioned near the nose of the animal. We have maintained anesthesia in this manner for up to 30 minutes with no apparent ill effects. For protocols involving administration of substances via aerosol, an alternate method is to use a mixture of ketamine HCl (25 mg/kg) and acepromazine maleate (2.5 mg/kg) given intramuscularly.

Intravenous injections: Unlike Mus and Rattus, cotton rats do not have an accessible tail vein. Substances which must be injected directly into the blood stream may be introduced via intracardiac injection into anesthetized animals. A needle (22 gauge or smaller) is introduced at a low angle slightly below the sternum and to the cardiac side of the midline, and inserted about 1.5 cm (in an adult animal). Aspiration is necessary to verify entry into the right ventricle, upon which the material may be injected. With practice, this method is as quick as tail-vein injection, and mortality in our hands is less than 1 percent.

Retroorbital bleeding: Blood samples of up to 500 ul (100 gm animal) may be obtained from anesthetized animals by inserting a Pasteur pipet about 1-2 mm, applying light pressure to the pipet at the same time it is twisted slightly to penetrate the venous plexus. While the pipet is being held with one hand, slight pressure should be applied to the jugular vein by placing the opposite thumb lightly on the neck of the animal. This will greatly increase the flow of blood into the pipet. Daily blood samples may be drawn in this manner without apparent ill effect.

Euthanasia: We have found that carbon dioxide intoxication is the preferred method of sacrificing animals, and is a method consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

Virion Systems, Inc., can provide cotton rats of any age, although we usually ship animals at 4-6 weeks of age. Orders for up to 100 animals can generally be filled within 1-2 weeks. Although we plan to develop a large array of immunologic reagents over the next 3 years, we currently have only two reagents: polyclonal rabbit antisera against cotton rat IgG and against the C3 component of complement. Both will soon be available in purified form (IgG), with or without fluorescein isothiocyanate (FITC) labeling. We are preparing a book on the cotton rat, which will serve as a reference manual with an extensive bibliography (over 1,000 published references). However, publication of the book is still several years away, and in the meantime, we provide information from our bibliographic files on an informal basis. Further information is available from us by telephone (301-309-1844) or FAX (301-309-0471).

This article appeared in the Animal Welfare Information Center Newsletter, Volume 5, Number 2, Summer 1994

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