key: cord-022255-5s2l1kua
authors: Kunstyr, Ivo; Nicklas, Werner
title: Control of SPF Conditions: FELASA Standards
date: 2007-09-02
journal: The Laboratory Rat
DOI: 10.1016/b978-012426400-7.50047-9
sha: 
doc_id: 22255
cord_uid: 5s2l1kua

Only experimental animals of a good microbiological quality will give any kind of guarantee of an experiment undisturbed by health hazards. It is for this reason that so-called (specific pathogen free) SPF animals are used for animal experiments. Certain requirements are necessary to maintain the desired SPF organism. Physical barriers together with appropriate operating methods aim at preventing contamination with pathogens and penetration by wild rodents. As a consequence, barrier units are not easily accessible for personnel, which is sometimes considered a disadvantage by experimenters. Finally, monitoring programs help to detect and control potential sources of contamination and may therefore be of crucial importance for the management of a facility housing animals of a good microbiological quality. The main purpose of health monitoring is to detect or prevent infections, which might influence physiological characteristics of animals or their health. Appropriate health monitoring helps to avoid imprecise results and allows all the experiments necessary to be carried out with a minimum number of animal. It is found that sufficient number of animals have to be monitored to obtain relevant information on a given population. It is important that the monitoring must be performed on a regular basis to detect unwanted microorganisms in good time. The recommended frequency is every 12 week.

Only experimental animals of a good microbiological quality will give any kind of guarantee of an experiment undisturbed by health hazards. It is for this reason that so-called 'SPF' (or specific pathogen free) animals are used for animal experiments.

Here we focus on 'SPF' rats, although experimental rats of conventional and possibly even germ-free hygienic status are also used in research and testing.

Most infectious agents can severely influence experimental results. Therefore the detection and subsequent elimination of infectious agents is essential if improved and more reliable results from animal experiments are to be obtained. At the same time, the use of such animals reduces the number of animals needed and therefore makes an important contribution to animal welfare.

The term 'SPF' means that the absence of individually listed microorganisms has been demonstrated for a population by regular monitoring of a sufficient number of animals at appropriate ages by appropriate and accepted methods. 'SPF' animals originate from germ-free animals. These are usually associated with a defined microflora and subsequently lose their gnotobiotic status by contact with environmental and human microorganisms. Such animals are bred and housed under conditions that prevent the introduction of unwanted microorganisms, i.e. organisms that have the potential to induce disease in animals (or humans) or which are known to influence the physiological properties of their host and thus the outcome of experiments (Table 8 .1). 'SPF' animals are morphologically and physiologically 'normal', well suited for modelling the situation of a human population.

It has to be stressed that most infections in experimental rodents are subclinical. The absence of clinical manifestations therefore has very limited diagnostic value. However, modifications of research results due to natural infections often occur in tlhe absence of clinical disease. Such modifications may be devastating for experiments because they often remain undetected (Table 8 .2).

The types of interference of an agent with exper!imental results may be diverse. As an example, a detailed list of the potential influences of Kilham rat virus (KRV), a frequently occurring rat pathogen, on research results is given in Table 8 .3 (see also Mossmann et al., 1998) . More information about the considerable effects on research due to infectious agents can be found in various review articles (Bhatt et al., 1986; Lussier, 1988; National Research Council, 1991; Hansen, 1994; Mossmann et al., 1998; Baker, 1998; Nicklas et a/., 1999) .

Most infectious diseases are multifactorial. An infectious agent alone or in insufficient quantities is usually not able to elicit the disease. Support by other factors is necessary. Some factors that can lead to an overt disease are listed in Table 8 

Certain requirements are necessary to maintain the desired hygienic quality. Physical barriers together with appropriate operating methods aim at preventing contamination with pathogens and penetration by wild rodents. As a consequence, barrier units are not easily accessible for personnel, which is sometimes considered a disadvantage by experimenters. Finally, monitoring programmes help to detect and control potential sources of contamination and may therefore be of crucial importance for the management of a facility housing animals of a good microbiological quality. o a% m g Keeping rodents free of pathogens is a much more complex problem in research facilities than in breeding units. It is necessary that all potential sources of infections are considered and evaluated. They have been discussed in more detail by Nicklas (1993) .

Unwanted microorganisms may be introduced into a barrier unit by various routes and materials. The most important sources of infections are infected animals of the same or closely related species (e.g. mice). In addition, biological materials (e.g. cell lines, sera, monoclonal antibodies, transplantable tumours, isolated organs, virus strains or parasites after animal-to-animal passages) may be contaminated (Collins and Parker, 1972; Nicklas et al., 1993a) . The contaminating agents may survive for years or decades when contaminated samples are stored frozen or freeze-dried. Therefore, such materials must be included in regular health monitoring programmes to avoid transmission of unwanted

Monitoring is usually done by the rat antibody production test (RAP test). This test is based on the immune response to rat viruses which is stimulated in pathogen-and antibody-free animals if the material injected is contaminated. A short protocol is given in Table 8 .5; for more details see Nicklas eta/. (1993a) . The polymerase chain reaction (PCR) can also be used to demonstrate the presence or absence of microorganisms in such materials but is more expensive and time consuming to perform.

All additional materials that have been in contacz with infected animals may be contaminated and may act as potential vectors. However, many of them (e.g. cages, feeders, bottles, etc.) can easily be decontaminated by hygienic procedures or appropriate disinfection.

Another important factor is human contact. Although the risk of transmitting rat pathogens by humans is very low if all personnel (caretakers, technicians, researchers) are properly educated and motivated, in practice pathogens are often transmitted as a consequence of a lack of discipline or thoughtlessness. 

The: main purpose of health monitoring is to detect or prevent infections which might influence physiological characteristics of animals or their health. Appropriate health monitoring helps to avoid imprecise results and allows all the experiments necessary to be carried out with a minimum number of animals. In contrast to troubleshooting, which means an ad hoc search and identification of unknown causes of abnormalities in an experiment, health monitoring describes a scheduled programme for monitoring the microbiological status of an animal population. The health monitoring programme aims at determining the microbiological status of a population before and during an experiment through regular and repeated examination and monitoring for previously defined, known infectious agents. Another aim of health monitoring is prevention of the introduction of unwanted organisms.

As the major risk factor, the animal remains the main target of the monitoring laboratory. We must emphasize that all diseased or dead animals should be examined in addition to regular and scheduled monitoring of clinically healthy animals. They are a valuable source of information about the hygienic status of the colony.

The Federation of the European Laboratory Animal Science Associations (FELASA) publish recommendations dealing with health monitoring of either breeding colonies or experimental units (Kraft et al., 1994; Rehbinder et al., 1996) . In experimental units in particular, the monitoring programme will differ between institutions or between different units of the same facility in its dependence on (a) research objectives, (b) physical conditions and the layout of the animal house, (c) husbandry methods, (d) sources of animals, (e) staff quantity and qualification, (f) diagnostic laboratory support, (g) finances.

An overview on monitoring of experimental rodent colonies has been given by Nicklas (1996) .

In most experimental units, animals of appropriate ages will not always be available for random sampling to monitor the microbiological status. Furthermore, diverse special experimental animals -transgenic, immunodeficient, pretreated-which are only available in small quantities, have been used increasingly during recent years. The use of sentinel animals is therefore advisable. Sentinels are animals from a breeding colony of known hygienic status (negative for all known pathogens) which aid in the evaluation of the microbiological status of the colony. They must be housed in the population to be monitored for a sufficiently long time (minimum of 4-6 weeks) in order to develop detectable antibody titres or parasitic stages. Sentinels should be kept in such a way that they receive maximum exposure to potential infections (on bottom shelves of different racks within an animal room, open cages, use of 'dirty bedding') (National Research Council, 1991) .

A sufficient number of animals has to be monitored to obtain relevant information on a given population. Clearly, infections with an attack rate of 50% or more (Sendai virus, Rat coronavirus/sialodacryoadenitis virus, RCV/SDAV) require far fewer animals to detect their presence than infections with low attack rates.

It has been recommended by the ILAR Committee on Long-term Holding of Laboratory Rodents (1976) that at least eight randomly sampled animals should be monitored, which is (theoretically) sufficient to detect an infection with a 95% probability if at least 30% of a population is infected. The formula which can be used to calculate the number of animals for an estimated prevalence rate is given in Table 8 .6.

In breeding units these animals should be at least 10 weeks old, which ensures that they have reached immunological maturity and had sufficient time to develop detectable antibody titres or parasitic stages (e.g. worm eggs). For experimental units, the time animals have been housed in the unit to be monitored may be more important than their age. As already mentioned for the sentinel animals, they should have been housed in the respective population for a minimum of 4-6 weeks before serological monitoring is conducted.

According to the FELASA recommendations two additional weanlings should be monitored because they may be better suited for the detection of specific parasites or bacterial pathogens than older animals.

Monitoring must be performed on a regular basis to detect unwanted microorganisms in good time. The recommended frequency is every 12 weeks. Most commercial breeders test more frequently (e.g. every 6 weeks). In most multipurpose experimental units animals are regularly bought and introduced 

into a facility. It may, in such cases, be reasonable to tesl-with a higher frequency (e.g. 3-5 animals every 4--6 weeks instead of 10 every three months) as this will result in the earlier detection of an infection (Kunstyr, 1992) .

For each facility or even for every single unit within a facility, the agents that are acceptable must be defined. Besides FELASA (Kraft et al., 1994) , various other organizations (Kunstyr, 1988; National Research Council, 1991; Waggie et al., 1994) have published similar lists of microorganisms which should be monitored for in routine programmes. The list will usually be restricted to organisms that pose a threat to animals (or humans) or organisms which are known to affect experiments and that can be eliminated. However, infections in immunodeficient animals frequently result in increased mortality due to reduced or lack of resistance to weakly pathogenic or even saprophytic microorganisms. It may therefore be necessary to include organisms with low pathogenicity in a monitoring protocol for immunodeficient animals.

On the other hand, some pathogens of laboratory rats have disappeared during domestication or gnotobiotic derivation (e.g. Francisella tularensis, Leptospira sp., Rickettsia sp., SpMllum minus) and are less likely to infect laboratory animals housed behind barriers. Some parasites (e.g. most cestodes) need an intermediate host not found in barrier units. Monitoring for these agents may therefore be less urgent or even unnecessary and may be performed less frequently. FELASA recommends testing once a year for such agents, i.e. agents of lower priority (Kraft et al., 1994) . Some of the most important bacteria, fungi and parasites for which rats should be monitored are given in Table 8 .

IJI @ A number of new organisms have emerged during recent years and are not included in existing lists. A number of Pasteurellaceae that have not yet been definitely classified seem to infect rats, in addition to the only known species, Pasteurella pneumotropica (Nicklas et al., 1993b) . Several Helicobacter species have been isolated recently from rats, such as /-/. muridarum (Lee et al., 1992) ,/-/. hepaticus (Fox et al., 1994; Riley et al., 1996) ,/-/. bilis (Fox et al., 1995; Riley et al., 1996) ,/-/. trogontum (Mendes et al., 1996) . A rat parvovirus has also been detected (Ueno et al., 1995 (Ueno et al., , 1997 Jacoby et al., 1996) in addition to those parvoviruses already known (IO'lham rat virus, . Other organisms, such as ClosMdium piliforme, have been renamed recently (Duncan et al., 1993) , which leads to some confusion in those scientists who are not sufficiently familiar with health monitoring of laboratory rats.

In general, the examination methods are: (a) necropsy-following after sacrifice, (b) serology, (c) bacteriology and (d) parasitology. Most of these methods are described in special publications (Feldman and Seely, 1988; Kunstyr, 1992; Owen, 1992; Kraft et al., 1994) and in various textbooks. Reliable results are only obtained if appropriate and sufficiently sensitive methods are used for health monitoring. It is therefore evident that the methods must be adapted to the actual 'state of art', i.e. to introduce new proven methods as they become available.

Microscopic methods such as stereomicroscopy are commonly used for monitoring for ectoparasites. Adhesive tape, flotation or direct microscopy of wet mounts taken from the intestinal tract are used for detection of endoparasites. Monitoring for bacteria is usually done by culture methods. However, serology or PCR may in some cases be superior or the only reliable approaches (e.g. for Streptobacillus moniliformis, Clostridium piliforme or Mycoplasma pulmonis) (van Kuppeveld et al., 1993; Goto and Itoh, 1994) .

Monitoring for viral infections is primarily done by serological methods. PCR, as an example of a new method, might be applicable in the case of acute infections (clinical disease) or for agents causing persistent infections (e.g. parvoviruses under specific conditions; Gaertner et al., 1995; Besselsen et al., 1995) . However, the lack of macroscopical changes during necropsy or lack of histopathological changes are still commonly used as the sole basis for declaring a population negative for a specific organism. This must be considered insufficient and unacceptable.

Serological methods must be selected properly as they may differ in their sensitivity and specificity (Smith, 1986; Lussier, 1991) . Unexpected serological results should always be confirmed by an independent method or, preferably, by virus isolation or antigen detection in order to avoid false-positive results. Some acceptable serological methods for the most common viral and some bacterial pathogens are given in Table 8 .8.

A health status report is usually requested and necessary when animals are shipped from breeders or between scientific institutions. It must contain sufficient data to provide reliable information on the quality of a population. Usually, each animal facility or breeder has its own style of report sheets which are sometimes difficult to read and to interpret. The FELASA (Kraft et al., 1994; Rehbinder et al., 1996) recommends using a uniform health report for breeding and for experimental colonies. Some additional information might be reasonable (e.g. housing conditions, treatment) and should be included. Table 8 .9 gives a checklist of the basic information that should be included in a health status report. Table 8 .9 Information which should be included in a health report when animals are shipped to external colonies

Viral and Mycoplasma Infections of Laboratory Rodents: Effects on Biomedical Research

Necropsy Guide: Rodents and the Rabbit

Handbook of Laboratory Animal Science

Long-term holding of laboratory rodents

List of Pathogens for Xpeci~cation in SI~ v Laboratory Animals

Diagnostic Microbiology for Laboratory Animals

Infectious Diseases of Mice and Rats

Mycoplasmal Infections of I~aboratory Rodents

Manual of Microbiologic Monitoring of s tory Animals