Opportunistic infections: why worry? ILAR 39 (4): 264.
There have been considerable improvements in the microbiological quality of research animals. These changes were spurred in the early 1980s by the use of new diagnostic methods that revealed a spectrum of infectious agents indigenous to most rodents from commercial and research colonies. Part of the challenge in improving the microbial quality of research animals required design of new biocontainment/bioexclusion systems that were effective but easier to use than the flexible film isolator. The current problem is how to maintain the pathogen-free status of rodents that arrive at facilities housing animals under both conventional and specific pathogen-free conditions. This problem is further constrained by budgetary issues and burgeoning animal populations. Serological diagnostic improvements of the 1980s are now being followed by a molecular revolution. Polymerase chain reaction (PCR), although expensive, is rapid and may be designed as a "universal" test or an agent-specific test. PCR can reveal agents that cannot be cultured by conventional means. One example would be Helicobacter species, which have fastidious in vitro growth requirements and are difficult to culture. Helicobacter hepaticus and Helicobacter bilis have been shown to induce inflammatory bowel disease both spontaneously and experimentally in immunocompromised and genetically altered rodents. Opportunistic infections are often thought of as those that are superimposed on an immunocompromised host by virtue of genetic makeup or chemical induction. However, the immune systems of rodents infected with some of the more common pathogens can be functionally altered to such an extent that they become unacceptable subjects for immunological assays and/or more susceptible to the deleterious effects of other agents. Laboratory rodents have become world travelers and stresses associated with transportation may have amplified effects in animals that already have impaired immune function.
A broad definition of opportunistic is "denoting an organism capable of causing disease only in a host whose resistance is lowered, for example, by other diseases or drugs." Factors rendering rodents less resistant include not only those drugs or infections that may alter immune function but also manipulations researchers impose on the animal genome.
Genetically altered rodents may have unanticipated phenotypes that include clinical disease manifestations induced by organisms heretofore unrecognized or thought to be commensals.
1. What are the three steps of PCR?
2. Which of the following test(s) use red blood cells?
a. Complement fixation
b. Hemagglutination inhibition assay
3. Hairless and rhino mice have which cell abnormalities?
a. T cell
b. B cell
c. NK cell
d. PMN cell
4. What are the two major defects of nude homozygous mice?
5. What mating scheme is used for the production of nude mice and why?
1. Denature, anneal, and extend (elongate).
2. A and B.
4. Failure of hair growth, dysgenesis of thymic epithelium.
5. Male homozygotes mated with female heterozygotes; female homozygotes have poor lactating ability.
Risks of infection among laboratory rats and mice at major
biomedical research institutions. ILAR 39 (4): 266.
A survey was done among the top 102 institutions that received funds from NIH in 1996. The survey asked about measures to monitor animals and animal products obtained from external sources. Virtually all respondents had some type of animal quarantine and testing in place, but few had testing programs for cell lines, immune sera, transplantable tumors, or other animal products.
Financial support for developing such programs was reported as not often being available. Among the respondents, 70% or the mouse colonies and 60% of the rat colonies are maintained under SPF conditions. Surveillance testing for most institutions was done quarterly (3-4 times/year), mainly for resident colonies and rodents obtained form noncommerical sources (other research institutions). Serology was the primary diagnostic screening used to detect viruses, whereas culture, serology and microscopy were used to detect bacterial and parasitic infections. Few institutions used molecular test for diagnosis of disease. Approximately half of the respondents recovered cost for health monitoring through per diem charges, and half charged investigators directly or supported surveillance with institutional funds. Many institutions had some capacity for on-site diagnostic lab support but most lab work was performed buy a pathologist. Although most institutions use SPF technology in mouse and rat colonies, problems with infectious agents are still experienced. In SPF mouse colonies, cornaviruses, parvoviruses, ecto/endoparasites were reported to be present in 10-30% of these colonies. Helicobacter infections were reported to be present in 10% of the colonies. Since most institutions don't test for Helicobacter, the numbers of cases are more likely higher than reported. Cornaviruses were present in more than 70% of the institutions, pinworms about 70%, ectoparatisties 40%, TMEV more than 30%, and five other viral and bacterial agents at 10-30%. Surprisingly serological evidence of ectromelia and LCM viruses were reported for mice. SPF rat colonies in the institutions surveyed reported that the prevalence of cornaviruses, parvoviruses, PVM, Sendai virus, CAR bacillus, and M. pulmonis ranged from 20-40% and approached 70% for pinworms. Survey respondents were asked to summarize major concerns about preventative health at their institutions and factors that lead to the presence of infection. One factor was inadequate financing for health care, therefore inadequate compliance by investigators. Federal funds given to investigators does not include funding for animal health. The investigators or the institution must assume the cost. Facility shortcomings which includes inadequate separation of microbiologically incompatible animals, inadequate quarantine/space, diagnostic equipment, and staffing problems can contribute to the risk of infection as well. This survey showed that problems with infectious agents as well as other aspects that contribute to the success of an animal health program transcends most institutional borders.
Microbiological assessment of laboratory rats and mice. ILAR
39 (4): 272.
A health surveillance program is used to detect any pathogen from a specific profile of infectious agents. If an agent is detected in a sample groups, the larger population in a room or unit should be considered infected with that agent. In determining the agent profiles used to determine the health status of incoming rodents in order to prevent disease introduction and to monitor the health status of animals arleady housed in a facility, is is important to consider the interaction of specific microorganisms in light of the resident populations' specific rodent strains. The authors recommend the following as an example of a comprehensive rodent health surveillance program: 1. Appropriately anesthetize the animal to collect the blood sample 2. Record body weights fro each animal 3. Examine the carcass, including the pelage and skin, grossly for dermatophytes, ectoparasites and other causes of dermatosis and alopecia 4. Sample nasoturbinates by a wash or swab for bacteria and mycoplasma 5. Obtain oropharyngeal sample for bacterial culture 6. Check tympanic bullae 7. Reflect the skinfrom the ventral midline and grossly examine organs in the thoracic and abdominal cavities for lesions/abnormalities. Remove lungs for histo and/or DNA extraction for pathogen determiniations 8. Examine abdominal rogans grossly for abnormalities/lesions. Remove sections from ileum, liver and kidney for histo. 9. Perform wet mounts on intestinal scrapings, intestinal/cecal contents 10. Resect the urocyst to check for T. cassicauda 11. Resect the appendix to check for parasites 12. Collect fecal samples for culture, DNA extraction and parasitology Serological detection of infectious agents can be done using several methods, including ELISA, IFA, HAI. Interpretation of a positive serological result requires looking as several different aspects of the results, which include: 1. has adequate sampling been performed 2. testing has been performed on more than a few animals 3. a high frequency of of positives has been detected. The authors recommend retesting in 2-3 weeks to see if there is an increase in incidence of positive titers. The authors conclude that it is impractical for them to define a specific list of organisms to test for, as well as the frequency of testing as this will vary with each facility and its needs. For sampling, sentinels should be housed in a manner that maximizes their exposure to the organisms infecting the animals being monitored. Generally, infectious agents are transmitted most efficiently via animal contact. Fomite transmission can be achieved using dirty bedding testing, but not all organisms are transmitted via soiled bedding, such as CAR bacillus. Immunocompetent animals should be used for serological testing and that they should be in the colony for at least 1 month before testing to ensure that titers are present for detection. Animals should be sampled over multiple animals for path, histo and microbiology because some diseases are age dependent. In conclusion, the frequency of testing, selecting which infectious agents to test for, as well as the type of quality control system to use must be determined based on factors such as what are the acceptable risks researchers are will to work with, the types of facilities available, the housing methods used(conventional vs. microisolator lids vs. ventilated racks) as well as the source of animals housed.
1. TP ________ X 100 = TP +FN
b. Specific gravity
2. TN ______ X100 = TN + FP
a. Specific gravity
1. d 2. b.
Current strategies for controlling/eliminating opportunistic
microorganisms. ILAR 39 (4): 291.
"The research benefits of controlling or eliminating ... (opportunistic) microorganisms must be balanced against the control measures' cost, complexity, and probability of success." This paper outlines one approach for determining the relative risk associated with each organism and then developing an appropriate control strategy. The first step is to perform a RISK ASSESSMENT - the process of analyzing the nature and relative importance of a microorganism to a research program. The following factors to be considered in a risk assessment were briefly discussed: Prevalence of the organism; its Species Specificity; Research/ Disease Effects; Transmissibility of Microorganisms; Zoonotic Potential, Manipulation and Access (how many people have access to the animals and what do they do to them?); Movement/Transportation of the animals; risk of exposure from Husbandry Supplies, Research Equipment, and Other Materials; and Concurrent Use of Hazardous Agents. The risk assessment defines which organisms you wish to control. The second step then is to develop CONTROL STRATEGIES FOR MICROORGANISMS. Although a complete control strategy covers the whole program of acquiring, housing, transporting, and utilizing animals, this paper primarily addresses only the BIOEXCLUSION system or the facilities, equipment, and procedures used to exclude microbes from groups of animals within the institution. Some available tools for bioexclusion that were described include Barrier Facilities/Rooms, Isolators, Microisolators, Cubicles, Ventilated Cabinets, and Mass Air Displacement/ Laminar Flow Rooms. Other components of the bioexclusion program that were discussed include Assessment of Animal Health Status (both before and after they enter the facility), Personnel Procedures (What organisms are people bringing into the facility?), Clothing, Training, Changing the Health Status of Animals (rederivation, medication), and Disaster Plan (what do you do when you find an infected animal?).
Reflections on future needs in research with animals. ILAR
39 (4): 306.
"The purpose of the article is to describe the animal-related resources needed as we enter the 21st century to continue the progress of biomedical research. A discussion of future animal care needs is appropriate in this ILAR Journal issue devoted to opportunistic infections because many genetically manipulated rodent--the tools of the future--will be exquisitely sensitive to opportunistic infections. The authors postulate that major needs will exist for animals, staffing, and infrastructure to facilitate scientific progress between the time of writing and 2010".
Animal-Based Research Trends:
-continued need of investigators to breed their own rodents in-house
-emergence of zebrafish as a key vertebrae model
-increasingly sophisticated roll of the veterinarian as a member of the research team
Genetically Manipulated Animals (transgenic, knock-out, knock-in rodents)
-dominant trend in animal-based research will involve genetically manipulated animals
-these rodents require high-quality housing conditions and intensive health monitoring beyond the requirements of less susceptible rodents.
In-house animal breeding
- needed do to production of genetically manipulated animals to study multigene effects
- common rodent pathogens will continue to threaten animal health
Training and Education
- maintain healthy animals in a constant environment to generate valid data
- distinguish nongenetic factors from genetic effects
- supervise quarantine and rederivation services
- develop skills in investigative phenotyping of new genetically manipulated animals
- must distinguish phenotypic effects of genetic alterations from "background" changes
- support rodent quality assurance testing
- develop techniques of complex breeding methods to study multigene interactions
- improve animal care by early detection of clinical problems
- properly utilize barrier techniques and infection control concepts
- comprehend the essential role played by animal-based research
- provide adequate protective housing,
- provide phenotyping and diagnostic imaging support
- preservation of unique germplasm
- rederivation of infected/infested animals
- miniaturized telemetry and drug delivery systems,
- provide for regional monoclonal production facilities
- specialized resources for studying nonhuman primate models
Trained veterinarians, scientists, animal care staff, and the general public are central to continued growth in biomedical knowledge. Their efforts must be supported by modernized infrastructures- animal housing, facilities, transgenic rodent resources, miniaturized equipment, antibody and primate resources- to continue to improve health care and gain biomedical knowledge in the 21st century utilizing animal models.
Future Directions in Rodent Pathogen Control. ILAR 30 (4):
Weisbroth roughly divided the last 100 yr of research involving laboratory animals into 3 periods. During the first period of domestication (1880-1950), many species were brought into laboratories and the range and prevalence of pathogens was decreased. The second period of gnotobiotic derivation (1960-1985) was marked by the development of organized laboratory animal science and medicine. During this period, cesarean derivation was developed and greatly facilitated the reduction and elimination of several pathogens. The third period (1980-present) has been the period of eradication of the indigenous murine viruses. This has been achieved through serologic testing for antibodies to specific pathogens and subsequent elimination or cesarean rederivation of antibody-positive colonies.
Microbes have been designated as pathogens, opportunists or commensals. This designation may need to be revised given the recent evidence that some commensals may have subtle effects on the host. (i.e. MAD-2; Syphacia obvelata).
Pathogens from the past cannot be totally eliminated and will continue to appear due to contamination by wild rodents. Additional effects of currently known organisms will be reported in the future as new techniques are discovered to study them. Microbes will continue to change as new pathogens are discovered and reported.
The author encourages investigators to consider microbial status in all experimentation, and especially in studies examining cellular or subcellular mechanisms. He also promotes the development of a data base of information related to microbial effects on host physiology which should be updated regularly and accessible electronically.
Opportunistic infections in research rodents: the challenges
are great and the hour is late. ILAR 39 (4): 316.
This is an editorial addressing the problem of dwindling resources in the face of increasing need for laboratory animal facilities. The author points out that the anticipated need for mice is increasing, largely due to the availability of transgenic and knockout technology. At the same time, available space for mice is decreasing, and many institutions do not have the facilites/infrastructure available to house more animals. To further complcate matters, accurate phenotyping of genetically engineered mice requires well-defined SPF status, and many facilities do not have the resources to obtain and maintain SPF rodents. A change in accounting methodology required by the federal government has shifted animal care costs from institutions (overhead) to direct costs for the investigators, making research with animals almost prohibitively expensive. Finally, a shift in funding priorities at the NCRR has dramatically decreased the clinical training available for lab animal veterinarians; this plus the traditionally small number of veterinary students entering the comparative medicine field is creating a shortage of qualified laboratory animal veterinarians.
Deer mice as laboratory animals. ILAR 39 (4): 322.
The deer mouse (Peromyscus maniculatus) is being used increasingly toxicological and epidemiological research, as well as in ecological, behavioral and genetic studies. The wild deer mouse is among the most abundant small mammals in North America and they are ranged as far north as Alaska and as far south as central Mexico. In 1962, the deer mouse colony at the University of South Carolina was established. There is now more than 30 distinct "wild-type" and mutant genetic stocks, which form the nucleus of the Peromyscus Genetic Stock center. Deer mice can be utilized to monitor environmental pollution using exposed wild animals compared with laboratory-bred controls. A genetically variant Peromyscus outbred stock was a source of the alcohol dehydrogenase "null" variant extensively employed in ethanol metabolism research. Two major disadvantages of the deer mouse as laboratory animals are the unavailability of highly inbred, genetically homogeneous strains and difficulty in handling due to their quickness and jumping ability. The inbred animals tend to have reduced fertility and viability.
Peromyscus have been implicated in 2 human diseases of current interest: 1. Deer mice (P. maniculatus) are carriers of the pathogen producing the recent hantaviral pulmonary syndrome (Four Corners disease) outbreak in the southwestern United States. 2. The deer mouse and the congeneric white-footed mouse (P. leucopus) are known hosts for the larval stage of the tick (Ixod which transmits the Lyme disease spirochaet (Borrelia). Wild peromyscus mice may also carry ehrlichiosis. Deer mice and other Peromyscus have long been considered ideal for evolutionary research and for biological rhythms research.
Peromyscus are sexually mature by 50 days of age and the estrous cycle of the deer mouse is 5 days and gestation is 22 days, except in lactating mothers for which it is delayed between 4-5 days. Breeding is optimized when animals are continuously retained in breeding pairs. Copulatory plugs are not conspicuous in Peromyscus and are not a reliable indication of mating. Litters should be removed from breeding cages at 25 days. Peromyscus colonies are subject toinspection by the US Department of Agriculture at regular intervals.
1. Name two diseases that Peromyscus may carry that are a public health concern?
2. True/False: Since Peromyscus are mice they do not have to be inspected by the USDA.
1. Hantavirus pulmonary syndrome and Borrelia burgdorferi (Lyme diseae).