Presented by Steven H. Weisbroth, DVM
Platform Presentation at the Tribranch, AALAS Annual Meeting
Adams Mark Hotel, Philadelphia, PA, June 3-5, 2002
First of all, I want to thank the organizers of this year's Scientific Program for the Tribranch Meeting for giving me this bully pulpit from which to discuss emerging diseases of laboratory rodents. I believe that my experience at a diagnostic laboratory has given me a unique perspective from which to observe the changing patterns of rodent and lagomorph diseases over the years. They really have changed and it is past time for us to recognize that they have. I would like to introduce a discussion of emerging diseases by first reviewing how the patterns of disease agent encounters have changed over the years- in order to appreciate where you are at present, it's necessary to know where you came from.
You are all familiar, of course, with the basic precepts of microbiologic assessment of the health status of laboratory animals. Development of this data on a repetitive schedule forms the objective basis on which to: 1) establish and/or reconfirm the ongoing microbiologic status of commercial and institutional rodent breeding colonies, 2) to develop institutional procurement standards for supplier eligibility based on animal health criteria, and 3) to continuously monitor the health status of institutional research animal resident colonies, including recent arrivals undergoing equilibration or quarantine prior to release for use, those currently involved in research protocols and those coming off study. The strategy of health surveillance is to detect by examination of one or more sample groups the presence of any pathogen from a specific profile of infectious agents. In an agent is detected in the sample group, even in a single individual, the inference can be made that the larger group, represented by the sample, is contaminated, as well. Of equal importance is the inability to detect any of a specific profile of agents under circumstances controlled for adequacy of the sample and detection technology. By this process the designated production or research unit may be demonstrated as free of the agents on the list.
Comprehensive rodent health surveillance programs are oriented to the systematic diagnostic examination of sample groups of animals against a predetermined list of pathogenic agents. In developing the lists of agents of concern, it is first necessary to understand that each of the laboratory species is host to an etiologic spectrum composed of arthropod ectoparasites, helminth and protozoan endoparasites, bacteria, viruses, rickettsial, and fungal forms typically associated by common diagnostic experience as indigenous to that species. For testing purposes the etiologic classes are organized into panels of the more common indigenous agents. In Table 1 I have summarized a comprehensive profile that, with minor exceptions, is widely used by rodent producers and users in the United States and which forms the basis for the FELASA panels used in Europe, as well. The profile forms the objective basis for a spectrum of agents that high quality research rodents are expected to be free of and is widely used as a procurement specification for that purpose.
Over the years, the principle effect of disease control, eradication, and exclusion programs has been to systematically reduce both the range of diversity and the frequency of encountering the agents of concern on this list. With the rodents, the following stages (summarized in Table 2) in the process can be recognized:
As rodents were domesticated and brought into the laboratory, they brought with them the entire panoply of infectious agents associated with their wild counterparts. Under the improved conditions of sanitation, nutrition, interdiction of life cycles, and maintenance of breeding colonies within stable, indoor environments, a number of infectious conditions became progressively infrequent or simply disappeared form laboratory environments. Such agents as the rickettsias, numerous helminths, bacterial forms such as Leptospira, the rat bite fever agents Spirillum minus, and Streptobacillus moniliformis and even the Salmonella species serve as examples of agents that disappeared or which became notable by their infrequency under husbandry conditions that applied during the first half of the 20th Century.
That is not to say that the general health of laboratory rodents or standards for the care of these animals in the 1950s and 1960s was all that good. Biomedical experimentation expanded greatly in the decades following World War II and with it a burgeoning if the utilization of laboratory rodents. The ravages of the primary mycoplasmal, bacterial, and viral pathogens imposed serious limitations on the successful conduct of research and testing programs reliant on the use of laboratory rodents. At a certain point in the 1960s it became clear that none of the standard veterinary medical approaches of improved husbandry and sanitation, vaccination, or antibiotic chemotherapy could effectively address the relentless effects of intercurrent disease on the research process. Surely, Mycoplasma pulmonis, the agent of respiratory mycoplasmosis was the agent that motivated the turn to a different approach to control or eradicate these diseases. The different approach was to prevent disease by excluding it. Henry Foster of Charles River Laboratories and C. N. Wentworth Cummings of Carworth Farms were among the first to perceive that the principles of gnotobiology, originated to explore the dimensions of germfree biology, could also be applied to large scale production of laboratory rodents from which the ineradicable diseases of the parents could be excluded by cesarean derivation.
The process is initiated by hysterectomy of the gravid uterus from the donor parent which is passed, by antiseptic immersion, into an isolator with sterile interior, life support systems and foster females with which to rear the neonates in the resected uterus. At some point the germfree neonates are associated with the 4-6 microbial forms necessary for normative intestinal physiology. These “associated” rodents, termed “gnotobiotes”, are retained in isolators as nucleus colonies used to produce progeny destined for transfer to large barrier production colonies. The offspring from within the barrier are offered for sale or for institutional use. In principle, such barriers may be continued in use indefinitely, unless routine testing indicates a “break” or penetration of the barrier by an unacceptable microbial form.
As the managers of commercial and institutional breeding colonies became adept with the principles and practice of gnotobiotic derivation, the process was adopted as the standard throughout the field and such terms as “pathogen free” and “specific pathogen free”, or “SPF&rduo; came into common parlance to describe the status of such animals. The process was remarkably effective as a means of producing rodents free of, at the time, common primary mycoplasmal, bacterial, and parasitic diseases, but less so with the murine viruses. At the time, little was known about the viral status of SPF animals, but more importantly, since there was little clinical perception of infection, there was correspondingly little concern about their presence in rodent hosts.
It was only after the aggregate burden of the primary parasitic, bacterial, and mycoplasmal agents had been substantially reduced that the more subtle morphologic and physiologic effects of the rodent viruses could be recognized. While, on the one hand, it is true that same derivation process that excludes parasitic, mycoplasmal, and bacterial agents also effectively excludes viruses, on the other, it is equally true that the Laboratory Animal Science establishment - the breeders, facility managers, and scientific users – in the decades from 1960 – 1980, knew about and tolerated viral enzootics out of indifference or uncertainty as to their health significance, or were simply unaware of their presence in production barriers and user facilities. In the absence of clinical signs and lesions, the effects of the murine viruses on their hosts could not be appreciated, or in most cases, even recognized. That state of affairs could not long last as a growing swell of research reports and conferences began defining the deleterious effects of the murine viruses. This concern ushers in the next stage, the unfinished business of the murine viruses.
The murine viruses, as a group, have only limited potential for serious clinical manifestation. In fact, most of the murine viruses were discovered or initially encountered as contaminants of research involving some transplantable neoplasm, tissue culture, or biologic derivative from infected, but clinically silent rodent hosts. Mouse pox (ectromelia) is practically the singular example of a virus with high potential for morbidity and mortality in immunocompetent mouse stocks. Several of the others, e.g. lymphocytic choriomeningitis virus (LCM), sialodacryoadenitis virus (SDAV), Sendai virus (SEN), mouse hepatitis virus (MHV), and Kilham's rat virus (KRV) serve as examples of agents that are ordinarily asymptomatic or with only moderate potential for pathogenicity in normative rodent stocks, but which have been indicted as agents of disease, in conventional terms, by some niche conferred by age group, genetic status or by rodents rendered inmmunodeficient by some heritable or transgenic process. Recent years have seen a great expansion of institutional holdings of immunodeficient, mutant, and transgenic stocks that have acted as flash points for clinical episodes in rodents having decreased resistance to agents that are ordinarily clinically silent in their normative rodent counterparts. Agents that include several of the Helicobacter species, the hyperkeratosis agent, Corynebacterium bovis, Pneumocystis carinii, and several of the murine viruses (MHV, MVM, PVM) are all extensively documented as clinically important disease hazards of this higher risk population. More important, by virtue of their clinical silence in immunocompetent rodent stocks are the many murine viruses that introduce some variability of cellular metabolism or reflexive cellular response to infection that interferes with the research process. Most of the murine viruses act as examples of this phenomenon.
By the early 1980s immense pressure from the research community, similar to that in the 1950s, began to develop as a result of too frequent viral complication of research in the areas of molecular biology and biotechnical research and product development. For about the last 15 years or so, an unofficial, but nonetheless consensually accepted international effort has been made to implement a virus free standard for the use of laboratory animals. It is important to understand that virtually every breeder and biomedical research institution in the country has had to come to grips with the scientific community's collective resolution to conduct research with virus free rodents. This effort has involved commercial and institutional rodent suppliers, the animal care unit administrators who procure and maintain rodent stocks, and the scientific users who conduct and report research results. In short, the entire chain that deals with the supply, care, and utilization of laboratory rodents.
Facilitating the process has been the wide availability of testing reagents to enable large scale health surveillance programs. This strategy, i.e., systematic and repetitive testing for the indirect serologic indicators of infection (antibodies) in sera from colony residents or sentinels has been universally adopted as standard practice to monitor viral contamination status of rodent colonies. The effort has been quite successful when viewed from a larger perspective. Over the last 10-15 years both the diversity of rodent viruses encountered and the frequency with which such infections are detected have declined markedly. We are, at present, in the terminal, mopping up stages of this international effort. It is also true that progress in viral eradication has not occurred without periodic shutdowns at user facilities and individually wrenching dislocations in research programs. Every animal care program manager has had to learn the techniques for prevention, control, and eradication of murine virus infections and how to deal constructively with the political balancing act required to achieve the support and compliance of institutional animal users. Most well managed animal care programs operate at present for long periods of time without serologic evidence of viral exposures and without detection of other indigenous agents by routine health surveillance. Laboratory animal specialists can take deserved pride in having guided the research animal effort in this country to the present level of quality. So, is the war over? Have the good guys won? Are the diagnostic labs to become like the Maytag repairman waiting for the phone to ring? Yes and no, but not really.
Perversely, the diseases keep coming. What makes them different is that they either don't appear in Table 1 as amongst the agents defined by history and experience as indigenous to the particular rodent hosts, or they appear to biologically differ sufficiently as to suggest that something new and different is at hand. I am suggesting use of the term “post-indigenous” as a descriptor for an unfolding cluster of seemingly new conditions diagnostically taking shape as time goes on. They are summarized in Table 3.
Although admittedly based on circumstantial criteria, these conditions seem to have several common threads that have suggested themselves to me from my perspective at a diagnostic laboratory:
If not from other rodents, where might such infections originate? This is an open question, at present, but I believe Occam's razor suggests the answer. Occam's razor states that when attempting the solution to a problem with several alternative explanations, the simplest explanation is usually correct. In my opinion, the source of most of the infections in this cluster is human and reflects the relatively open and unrestricted exposure of barrier-produced laboratory rodents to their human contacts; i.e. to the animal care personnel and investigators they come in contact with after arrival at the user institution. Of course, whether infection occurs depends on the chance concurrence of human virus shedder and proximity of susceptible rodent host, but the institutional carelessness typically exercised in rodent maintenance programs almost ensures a steady flow of virus infections- and that is what we see. As a whole, we have grossly underestimated and ignored the potential for communicability of human agents to laboratory animals. This casualness is one of the last unexamined and essentially unregulated aspects of the research animal environment. Surely it is an area we are going to have to look at more closely directly in the context of animal disease control. The rationale for doing so can be illustrated by the following points:
There appears little doubt that, at present, Laboratory Animal Science is witnessing a major restructuring of the lists of indigenous pathogens of laboratory rodents. Contemporary commercial and institutional rodent supplier stocks have little, if any, realistic potential as reservoirs for these agents. The truly indigenous pathogens, those listed in the comprehensive profile in Table 1, have largely been eradicated, even at user institutions. The process has been driven by constant resupply from disease free production sources and the highly structured and microbially limited environments permitted by good laboratory animal management practice. Circumstances seem to suggest, with some exceptions, that the reservoirs for the cluster of agents listed here as “emergent” are probably humans coming in contact with the more microbially limited rodent hosts. If so, and if we mean to further limit and reduce “rodent” disease as we currently experience it, we are going to have to more carefully standardize and regulate such contacts more rigorously than we do at present.
(b) Rat Panel
|PVM||Pneumonia Virus of Mice||PVM|
|REO-3||Respiratory Enteric Orphan III||REO-3|
|Sialodacryoadenitis Virus/Rat Coronavirus)||SDAV/RCV|
|Kilham's Rat Virus/Rat Parvovirus||KRV/RPV|
|MVM||Minute Virus of Mice|
|MHV||Mouse Hepatitis Virus (coronavirus)|
|EDIM||Epizootic Diarrhea of Infant Mice|
|MCMV||Mouse Cytomegalovirus (Salivary Gland Virus)|
|LDHV||Lactic Dehydrogenase Elevating Virus|
|III||Eradication of the Indigenous Murine Viruses||1980-1996|
|IV||Post-Indigenous Disease||1996-the Present|