RESOURCE Volume 32, No. 5 Lab Animal May 2003

Diagnostic Testing of Mouse and Rat Colonies for Infectious Agents

Robert S. Livingston, DVM, PhD and Infectious diseases of mice and rats are determine the type of test selected for each Lela K. Riley, PhD important to the scientific community infectious agent. Common primary testing because they can introduce unwanted vari- methods and available confirmatory testing ables that can alter experimental outcomes1. methodologies for the various infectious Rodent health monitoring programs The goal of laboratory animal facilities is to agents of mice and rats appear in Table 1. make an essential contribution to maintain disease-free animals so as to elimi- biomedical research by identifying nate these unwanted variables, and the goal Serology the presence of infectious agents of rodent health monitoring programs is to Serology continues to be the primary determine the presence or absence of patho- means of testing rodent colonies for expo- that might confound animal genic microbes (viruses, , endopara- sure to all viruses and a few bacteria, largely research. The authors discuss the sites, ectoparasites, protozoa, and fungi) because serological tests are sensitive and types of diagnostic tests available, within colonies of laboratory rodents. specific, are relatively inexpensive, and allow which agents deserve monitoring, Because most of the pathogens of laboratory screening for a multitude of infectious animals do not cause overt clinical disease, agents with one serum sample. Standard and the appropriate frequency for identification of these important pathogens serological assays used for rodent health such interventions. depends on a variety of specialized diagnos- monitoring detect antibodies against infec- tic tests. Characteristics central to the success tious agents. Of the many available serolog- of monitoring mouse and rat colonies for ical formats, enzyme-linked immunosor- infectious agents include knowledge of the bent assays (ELISA) are the most common- pathogenesis, epizootiology, and prevalence ly used primary tests because they are easily of the infectious agents; also necessary are automated for high-throughput testing and familiarity with available diagnostic tests the results obtained provide an objective and access to specific information about the measure of the immunoreactivity of the husbandry conditions of individual animal sample. Although indirect fluorescent anti- groups. This information will ultimately dic- body (IFA) assays are the most commonly tate which agents to survey, how frequently used secondary serological tests because of testing is needed, and what method of test- their sensitivity, they are more labor-inten- ing to employ. This article focuses on avail- sive than ELISAs, and interpretation of the able testing methodologies, and general results is subjective. issues to consider when choosing the agents Development of pathogen-specific to be monitored and the frequency of test- serum antibodies requires the generation of ing. In addition, the reader will obtain cur- an immune response that usually occurs rent information on the prevalence of infec- 5–10 days after exposure to a pathogen2. The authors are affiliated with the tious agents as an indicator of the potential Circulating antibody titers typically persist University of Missouri Research Animal risk of these infectious agents invading a for months after infection. In practice, test- Diagnostic Laboratory, Department of research mouse or rat colony. ing animals less than two weeks after expo- Veterinary Pathobiology, College of sure to a pathogen often will result in nega- Veterinary Medicine, University of Testing Methodology Options tive results because the animals have not Missouri, E115A Veterinary Medicine The tests available for evaluating mice had sufficient time to develop detectable Building, 1600 E. Rollins Rd., Columbia, and rats for infectious agents are diverse, and titers of pathogen-specific antibodies. Thus, MO 65211. Please send reprint requests variables such as cost, sample requirements, serology is a valuable tool that provides an to Livingston at the above address, or and the potential for the test to detect the indirect measure of past exposure to an email: [email protected]. presence of or exposure to the agent will infectious agent, but can fail to identify an

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TABLE 1. Commonly used testing methodologies for mouse and rat pathogens

Agent (species) Speciesa Primary testing Confirmatory methodology testing (sample tested) methodology Viruses Ectromelia M Serology (serum) PCR, Histology Hantaan (HTN) R Serology (serum) PCR K virus M Serology (serum) PCR Lymphocytic choriomeningitis virus (LCMV) M, R Serology (serum) PCR Lactate dehydrogenase-elevating virus (LDEV) M Bioassay (serum) PCR Mouse adenovirus 1 (MAD 1) M, R Serology (serum) PCR Mouse adenovirus 2 (MAD 2) M Serology (serum) PCR, Histology Mouse cytomegalovirus (MCMV) M Serology (serum) PCR, Histology Mouse hepatitis virus (MHV) M Serology (serum) PCR, Histology Mice minute virus (MMV) M Serology (serum) PCR Mouse parvovirus (MPV) M Serology (serum) PCR Mouse rotavirus (MRV) M Serology (serum) PCR, Histology Mouse thymic virus (MTV) M Serology (serum) PCR virus of mice (PVM) M, R Serology (serum) PCR Polyoma virus (polyoma) M Serology (serum) PCR Rat coronavirus (RCV) R Serology (serum) PCR, Histology Rat parvoviruses (rat parvos) R Serology (serum) PCR Reovirus type 3 (REO 3) M, R Serology (serum) PCR Sendai virus (Sendai) M, R Serology (serum) PCR Theiler’s murine encephalomyelitis virus (TMEV) M, R Serology (serum) PCR Bacteria Cilia-associated respiratory M, R Serology (serum)/PCR (trachea) Histology (CAR) bacillus Citrobacter rodentium M Culture (cecal contents) PCR, Histology Corynebacterium kutscheri M, R Culture (NP)b PCR spp. (any) M, R PCR (feces) Culture M, R PCR (feces) Culture Helicobacter bilis M, R PCR (feces) Culture Helicobacter typhlonius M, R PCR (feces) Culture Helicobacter rodentium M, R PCR (feces) Culture Helicobacter sp. unidentified M, R PCR (feces) Culture Mycoplasma pulmonis M, R Serology (serum)/PCR (NP) Culture Pasteurella pneumotropica M, R Culture (NP) PCR M Culture (cecal contents) PCR M Culture (cecal contents) PCR Salmonella spp. M, R Culture (cecal contents) PCR Clostridium piliforme M, R ELISA (serum) PCR, Histology Parasites Aspiculuris tetraptera M Direct exam (colon contents) Direct exam (fecal floatation) Myobia musculi M Direct exam (pelage) Myocoptes musculinus M Direct exam (pelage) Radfordia affinis M, R Direct exam (pelage) Radfordia ensifera M Direct exam (pelage) Rodentolepis nana M, R Direct exam (small intestine) Syphacia obvelata M Direct exam (cecal contents) Direct exam (perianal tape test) Syphacia muris R Direct exam (cecal contents) Direct exam (perianal tape test) Fungus Pneumocystis cariniic M, R PCR (lung) Histology Protozoan Encephalitozooan cuniculi M, R ELISA (serum) PCR, Histology aM, Mouse; R, rat. bNP, nasopharynx. cMonitored only in immunodeficient mice and rats. acutely infected animal and does not give PCR assays directly test for the presence of nucleic information regarding the current infection The need to detect viral, bacterial, fungal, acids from the infectious agent. PCR will status of an animal. Another limitation of and protozoal infections in laboratory mice detect DNA, and reverse transcriptase PCR serology is that it can only be used with con- and rats has led to the development of poly- (RT-PCR) will detect RNA. In this discus- fidence to test immunocompetent animals. merase chain reaction (PCR) assays3.These sion, the term PCR will refer to both PCR

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FIGURE 1. Microbiological reporting results of mouse samples tested at the University of Missouri Research Animal Diagnostic Laboratory from 1 November 2001 through 31 October 2002. The methodology for testing was the primary one listed for each agent in Table 1 (see Table 1 for definitions of abbreviations). (A) Viral pathogen testing results. For HTN, K virus, LDEV, MCMV, and MTV, between 1,400 and 6,000 sam- ples were tested. For all other viruses, between 17,000 and 61,000 samples were tested. (B) Results of bacterial pathogen and P. carinii and E. cuniculi testing. For M. pulmonis 45,609 samples were tested, and for all other bacteria between 5,400 and 13,500 samples were tested. For parasites 14,323 samples were tested, for P. carinii 719 samples were tested, and for E. cuniculi 10,194 samples were tested. (*) Tested only in immunodeficient mice. (**) This test is a bioassay that lacks specificity, in that other infectious agents, neoplasia, hemolyzed serum, liver trau- ma, eye bleeding, and even shipping stress can lead to positive serum lactate dehydrogenase (LDH) values.Thus, the prevalence of LDEV may be overestimated. and RT-PCR assays. Thus, PCR provides using PCR tests, that the tissue sample ynx and cecum is a routine method of evalu- information about the current infection sta- required for testing is specific to the pathogen ating mice and rats for most bacterial tus of the animal. For agents that produce of interest. For example, the mesenteric pathogens. Standard bacteriology relies on chronic infections such as helicobacters, cilia- lymph nodes are the target tissue for mouse cultivation of the organism on a variety of associated respiratory (CAR) bacillus, parvovirus (MPV) PCR, feces is the proper cell-free media; bacterial identification Pneumocystis carinii, and Mycoplasma pul- sample for helicobacter PCR, and the trachea depends on bacterial colony morphology, monis, PCR is an excellent primary test to or tracheal swab samples are the appropriate Gram stain characteristics, and biochemical monitor for infection because of the large samples to select for M. pulmonis PCR. profiles of the isolate. Standard bacteriology amount of time available to detect the Generally, PCR assays are extremely sensitive can lack sensitivity in the detection of fastid- pathogen. In contrast, for agents that pro- and specific, but they are labor-intensive, rel- ious bacteria or bacteria present in low num- duce only acute infections in immunocom- atively expensive, and susceptible to cross- bers. Also, bacteriology can lack specificity in petent mice, such as ectromelia virus and contamination due to high test sensitivity. that not all bacteria of the same species have mouse hepatitis virus (MHV), PCR is not as the same biochemical profile and closely valuable in defining the infection status of a Direct Examination related bacterial species can have the same colony because of the limited time period Direct microscopic examination is the morphological characteristics and biochemi- during which PCR can detect these agents in primary means of detecting parasites of lab- cal profiles. For these reasons, scientists are a given animal. However, if the goal of testing oratory animals. Examination of intestinal developing and increasingly using alternative is to determine if an individual animal is contents, fecal floatations, or perianal tape tests such as PCR that can provide greater test actively infected, PCR is an excellent tool for tests are common procedures for the detec- sensitivity and specificity than culture, to many agents, provided that samples are avail- tion of endoparasites, such as pinworms. detect murine bacterial pathogens. Diag- able from appropriate animals and tissues for Examination of the pelage allows possible nostic laboratories do not routinely use cul- testing. It is important to consider this differ- detection of ectoparasites, such as fur . tures to detect viral pathogens of mice and ence, because PCR and serology evaluation Whereas these tests provide a definitive diag- rats largely because the process is laborious of a given animal may yield conflicting nosis and are relatively inexpensive to con- and expensive and, for routine testing pur- results. For example, in rats infected with rat duct, they offer only moderate sensitivity. poses, does not offer advantages over other, coronavirus (RCV), virus is present in the Thus, false-negative results may occur when less expensive, and higher throughput assays Harderian, parotid, and exorbital glands 7 evaluating animals harboring low numbers such as serology and PCR. days after infection, but absent by 14 days4, of organisms. which is about the time that detectable RCV- Histopathology specific serum antibody titers are beginning Culture Histopathological examination of select- to appear. It is important to assure, when Microbiological culture of the nasophar- ed target tissues of mice and rats is an excel-

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chronic disease in these ani- al issue often overlooked when developing an mals that is detectable by agent exclusion list is the proposed plan for histopathological examina- dealing with the agent if it is detected. One tion. In addition, can argue that if no action is to be taken once histopathology has been a pathogen is detected in a colony of animals, central to the identification then there may be no justification for testing of novel murine pathogens. for it. However, defining the infection status For example, histopathologi- of a group of animals from which the agent cal identification of hepatitis need not be excluded is justifiable if these in A/JCr mice was the first animals are a potential source of contamina- clue leading to the identifica- tion to other animal groups from which the tion of Helicobacter hepaticus agent must be excluded. as a pathogen of mice5,6. More recently, histopatho- Frequency of Monitoring logical examination of the Once an agent exclusion list exists, the lungs of rats revealed a novel next question regards the frequency of test- interstitial pneumonia7,8,for ing. Although the potential for a pathogen to which a viral pathogen, rat affect ongoing research negatively or to pose respiratory virus (RRV), is a threat to the health of personnel influences the suspected cause9. the frequency of testing, the potential risk of the infection being introduced into a colony Agents to Monitor of animals is also a consideration. A way to The decision as to which evaluate this risk is to examine the current agents to monitor, often and recent historical prevalence of these termed creating an agent infectious agents not only in individual ani- FIGURE 2. Microbiological reporting results of rat samples tested at the University of Missouri Research Animal exclusion list, will differ mals, groups, and rooms within the research Diagnostic Laboratory from 1 November 2001 through 31 among rodent populations facility, but also in the local surrounding October 2002. The methodology for testing was the primary and is largely affected by the research campus and among research mouse one listed for each agent in Table 1 (see Table 1 for definitions of abbreviations). (A) Viral pathogen testing results. For all potential negative influence and rat populations in general. This informa- viruses, between 3,800 and 8,200 samples were tested. (B) of a particular infectious tion, along with animal husbandry condi- Results of bacterial pathogen and P. carinii and E. cuniculi test- agent on ongoing research tions and the movement of animals and peo- ing. For M. pulmonis 8,132 samples were tested, and for all other bacteria between 950 and 3,600 samples were tested. For or, for zoonotic agents, the ple among groups of animals, can give an parasites 3,691 samples were tested, for P. carinii 1,264 sam- potential risk the agent may indication of the risk that a particular infec- ples were tested, and for E. cuniculi 878 samples were tested. pose to the health of animal tious agent will gain entry into a population (*) Tested only in immunodeficient rats. care personnel and of animals. It follows that the greater the researchers. Inclusion of an potential for an infectious agent to gain entry lent tool for the investigation of disease out- agent on an exclusion list depends on the into an animal population, the more fre- breaks, because it permits multiple organ sys- nature of the pathogen, the species of ani- quently should a facility monitor the popula- tems to be evaluated quickly and inexpensive- mals in the colony, the genotype of the ani- tion. ly for pathogens that produce pathognomon- mals in the colony (i.e.,immunodeficient vs. Information about local prevalence data ic lesions, allowing for the identification of immunocompetent), and the type of will vary between research institutions, the etiology of disease. However, for routine research being conducted. Another practical which must collect and analyze these data on screening, histopathological examination consideration is the potential ability to keep a case-by-case basis. In this discussion, we lacks sensitivity, because many rodent the pathogen out of the animal colony in will provide current information on the pathogens do not produce histological evi- light of the uncertain health status of animals prevalence of microbes in research mice and dence of disease or, when a pathogen induces entering the colony or surrounding colonies rats in general as one indicator of the risk lesions, there is often a short period of as well as the ability and availability of diag- that an infectious agent will invade a naive detectability. Nevertheless, for immunodefi- nostic tests to detect the pathogen. This is (clean) rodent population. Figures 1 and 2 cient rodents, in which serological monitor- currently an issue with the novel agent RRV, provide a summary of the microbiological ing has little value, histopathological moni- which causes interstitial pneumonia in labo- reporting data from the University of toring is a productive alternative protocol ratory rats and for which no sensitive com- Missouri Research Animal Diagnostic because several rodent pathogens produce mercial diagnostic assays exist9.An addition- Laboratory (MU RADIL) from 1 November

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prevalent agents and less frequently for the TABLE 2. General mouse pathogen testing recommendations rarest agents, while considering the impact Pathogens to monitor Pathogens to monitor Pathogens to monitor that each of these agents would have on the frequently less frequently infrequently ongoing research or the research staff if Viruses Viruses Viruses detected in the rodent population. Using Mouse hepatitis virus Ectromelia Lactate dehydrogenase- mouse pathogens as an example, it seems rea- elevating virus Mice minute virus Lymphocytic chorio- Mouse cytomegalovirus sonable to test frequently for MHV,MPV,and meningitis virus MRV, which are highly prevalent agents that Mouse parvovirus Mouse adenovirus 1 Mouse thymic virus 11–15 Mouse rotavirus Mouse adenovirus 2 can alter the physiology of infected mice . Sendai virus Pneumonia virus of mice LCMV and ectromelia are not very prevalent Theiler’s murine encephalo- Polyoma virus myelitis virus agents, but because LCMV poses a human Reovirus type 3 health risk16 and ectromelia is a highly conta- Bacteria Bacteria Bacteria gious virus that causes devastating disease in Helicobacter spp. Pasteurella pneumotropica Cilia-associated respiratory mice17,one could argue that these agents bacillus Mycoplasma pulmonis Pseudomonas aeruginosa Citrobacter rodentium should be monitored, but perhaps less fre- Salmonella spp. Clostridium piliforme quently. In contrast, while agents such as E. Corynebacterium kutscheri 18 19 Proteus mirabilis cuniculi and the CAR bacillus can cause Parasites Parasites severe disease, they are not highly contagious Pinworms Fur mites and are not prevalent among research mice Fungi Protozoa and therefore could be subject to infrequent Pneumocystis cariniia Encephalitozoon cuniculi testing. The definitions of “frequently” and aMonitored only in immunodeficient mice and rats. “infrequently” will also vary substantially among research facilities and will depend on 2001 to 31 October 2002, and include prima- Sendai virus and rat coronavirus (RCV) several of the variables mentioned earlier; ry test results for viral, bacterial, fungal, pro- being detected less frequently. It should be generally, though, frequently may mean tozoal, and parasitic infectious agents of lab- noted that seroreactivity to TMEV in rats has monthly or quarterly and infrequently may oratory mice or rats, respectively. It is note- not been linked to infection with the TMEV mean semiannually, annually, or only on a worthy that these data represent a biased viruses of mice and likely represents infection case-by-case basis. Respectively, Tables 2 and sample population in that animals tested and with other viruses in the Picornaviridae fam- 3 provide general recommendations regard- tests requested were selected by the clients of ily of unknown significance. As in mice, heli- ing which pathogens of mice and rats to the RADIL and do not represent a randomly cobacters were the most common bacteria monitor and the frequency to test for these selected population. However, the MU identified and pinworms were the most com- agents. Readers should recall that these are RADIL carries out testing for many academ- mon parasites detected. These data are con- only general guidelines that can aid in devel- ic and private research institutions in geo- sistent with the 1998 study by Jacoby and oping specific testing needs and frequencies at graphically varied regions across North Lindsey that used surveys to investigate the each research institution for the rodent sub- America, so these data likely provide a valid prevalence of infectious agents in North populations of interest. In particular, if relative measure of pathogen prevalence in American research institutions10.Interest- colonies of mice or rats are at an increased research mouse and rat colonies in the ingly, in our prevalence study, some agents in risk of exposure to a specific agent because of United States and Canada. research mice and rats were not detected. a previous or current outbreak, it would be Figures 1 and 2 show that most of the These agents were Hantaan virus, K virus, prudent to monitor more frequently for this murine pathogens that are subject to routine mouse thymic virus, Citrobacter rodentium, agent in vulnerable populations. monitoring were detected in research rodent Corynebacterium kutscheri, and colonies during the past year. In mice, mouse Encephalitozooan cuniculi for mice, and An Example of a Testing parvovirus (MPV), mouse hepatitis virus Hantaan virus, lymphocytic choriomeningi- Program (MHV), and mouse rotavirus (MRV) were tis virus (LCMV), mouse adenovirus 1, After considering the budget available for the most prevalent viruses, helicobacters Salmonella spp., Rodentolepis nana, and testing, one must prioritize the list of agents were the most prevalent bacteria, and pin- Encephalitozooan cuniculi for rats. for monitoring in terms of prevalence and worms were the most commonly detected If one uses these prevalence data to aid in potential animal or human health impact, parasites. In rats, the most frequently detect- developing a list of agents for which to test then determine the frequency and type of ed viruses were the parvoviruses, pneumonia and to help determine the frequency of test- testing compatible with the budget and avail- virus of mice (PVM), and Theiler’s murine ing, a cost-conscious and justifiable approach able diagnostic tests. For example, a logical encephalomyelitis virus (TMEV), with would be to test frequently for the most course of action on a quarterly basis might be

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to collect serum to monitor for antibodies to TABLE 3. General rat pathogen testing recommendations the most prevalent and serious viral and bac- terial pathogens, collect feces for Helicobacter Pathogens to monitor Pathogens to monitor Pathogens to monitor frequently less frequently infrequently spp. testing by PCR, and concomitantly check for evidence of pinworm infection by Viruses Viruses Viruses conducting perianal tape tests to detect Rat parvoviruses Lymphocytic choriomeningitis virus Hantaan Pneumonia virus of mice Reovirus type 3 Mouse adenovirus 1 Syphacia spp. and fecal floatations to identify Rat coronavirus Aspiculuris tetraptera in mice. On an annual Sendai virus Theiler’s murine encephalo- basis, institutions should consider a more myelitis virus comprehensive monitoring approach in Bacteria Bacteria Bacteria which whole animals are submitted to a Helicobacter spp. Pasteurella pneumotropica Cilia-associated respiratory diagnostic laboratory for a more exhaustive bacillus Mycoplasma pulmonis Salmonella spp. Clostridium piliforme serological testing battery, bacterial culture Streptococcus pneumoniae Corynebacterium kutscheri for enteric and respiratory pathogens, a more Parasites Parasites detailed parasitological examination that Pinworms Fur mites includes both ecto- and endoparasites, and Fungus a possibly histopathological screening of a bat- Pneumocystis carinii tery of tissues for evidence of pathogens or aMonitored only in immunodeficient mice and rats. disease processes. It should be stressed that this is a general recommendation, and indi- to sentinel mice rather quickly28.However,in proteins as antigens to detect anti-MPV anti- vidual testing needs and frequencies will dif- another study, sentinel mice failed to become bodies in infected mice with high sensitivity fer between rodent subpopulations. infected by H. hepaticus from some known and specificity31,32,providing highly accurate, positive groups of mice29.These researchers low-cost, high-throughput assays for the Specific Testing Methodologies also reported delayed and inconsistent trans- detection of MPV-infected mice. Subsequent for Highly Prevalent Mouse mission of Helicobacter bilis to sentinel IFA assays are in common use as secondary Pathogens mice28,29 and we have seen failed transmis- serological tests for samples positive for MPV Helicobacters. From the data cited earlier, sion of H. bilis and H. rodentium to sentinel by ELISA. These IFA tests are sensitive, but 32% of all mouse fecal samples tested were mice exposed to contaminated bedding from lack specificity and are more labor-intensive positive for a helicobacter, making helicobac- infected colonies. Recognizing the potential than ELISA. Hemagglutination-inhibition ters the most prevalent pathogen in mice for false-negative results with sentinels and (HAI) assays also serve as secondary serolog- used for biomedical research. This is not sur- recognizing that fecal PCR assays are ante- ical confirmation for MPV. Although MPV prising, given that the first reports of patho- mortem tests, we recommend screening of HAI tests are specific, they lack sensitivity, are genic intestinal helicobacters were in 1994 colony animals for helicobacter infections. To labor-intensive, and must be subjectively (refs. 5, 20), and testing and control measures help minimize the cost associated with heli- interpreted. PCR of mesenteric lymph nodes to exclude or eliminate these pathogens from cobacter PCR testing, one can pool ≤10 fecal can also detect MPV-infected mice33,because research mouse colonies have not been pellets from several mice of the same micro- MPV persists in lymphatic tissue of infected extensively used until recently. The patho- biological status and evaluate the composite mice34.Investigators usually reserve PCR for genic potential of the different Helicobacter sample for helicobacter infection (L.K. Riley, confirming serological test results because of spp. varies, but in general, they all can cause unpublished data). Serological assays for its expense and because serology appears to or have been associated with intestinal or murine helicobacters are also available29,30, have slightly greater sensitivity in detecting liver inflammation, depending on the geno- but thus far there are no serological assays infected mice33 (B. Bauer, unpublished data). type of the infected mouse6,21–25.Conducting that are known to detect all murine heli- For example, in an endemically infected PCR on DNA extracted from feces is the pri- cobacters. Additionally, helicobacter serolog- colony, all of ten 7.5-month-old Sencar mice mary method used to test mice for heli- ical assays lack species specificity29,30. tested positive for MPV by serology, but only cobacter infection. The usual approach is to Mouse parvovirus. MPV is among the nine of ten mice were positive for MPV by do a helicobacter PCR assay that will detect most prevalent mouse viral pathogens in PCR (B. Bauer, unpublished data). all bacteria of the Helicobacter genus26,27.If research mice and can adversely affect Laboratories also use MPV PCR to test for positive samples are detected, then one can research data obtained from infected environmental contamination, to aid in do species-specific PCR assays to define mice12,13.Serology is the preferred primary detecting MPV-contaminated equipment, which Helicobacter species are present. testing method for detecting MPV-infected and to assess effectiveness of decontamina- Livingston et al. showed that Helicobacter mice. Recently, several investigators have tion efforts after an MPV outbreak. hepaticus was transmitted from colony mice developed ELISA assays that use viral capsid Mouse hepatitis virus. MHV is highly

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prevalent among research mouse colonies obtained more rapidly, and they do not itive serologically, but to have since cleared and continues to have a severe impact on bio- require the use of animals. the infection and thus test negative for the medical research using mice. Serology contin- agent by PCR or histopathology. Conversely, ues to be the best method for screening Confirmatory Testing it is possible for an animal to be acutely immunocompetent mice for MHV infection. Diagnosticians continually strive to infected with a pathogen, to lack a detectable Several investigators have also described PCR develop new and more accurate tests. antibody response, and to test negative for assays for MHV35–37; they can be used to con- However, it is essential to emphasize that, in the agent by serology, but still test positive for firm infection. In immunocompetent mice, practice, no diagnostic test is 100% sensitive the presence of the agent by PCR or show PCR assays can detect MHV in the feces36 or or 100% specific, so there is always a chance acute lesions suggestive of infection. mesenteric lymph nodes35 for as long as four of obtaining a false-negative or false-positive Understanding the epizootiology of infec- weeks after infection. However, immunocom- test result. Therefore, confirmation of unex- tious agents within a given animal popula- promised and genetically engineered mice pected or unusual test results should always tion and having knowledge of the advantages have shown persistent infection and shedding precede their use in making major colony and limitations of each diagnostic methodol- of MHV37,38.Researchers have used serologi- management decisions. The best way to ogy will help sort out these potential differ- cal and fecal PCR monitoring for MHV as a investigate unexpected or unusual results is ences, and can help in deciding on appropri- means to facilitate the re-establishment of to gather enough additional information to ate methods and samples to use for confir- MHV-free mice from infected colonies37.In substantiate or refute the initial finding. matory testing. In addition, experts at diag- this approach, a laboratory re-establishes the Options for confirmatory testing include nostic laboratories can provide insight on colony by selecting as breeders mice that were repeating the same test or doing a suitable test performance, help with data interpreta- seropositive for MHV (i.e.,indicating history alternative test on the same sample or a new tion, and aid in developing a plan for health of infection with MHV) but consistently neg- sample derived from the same animal, or monitoring and follow-up testing. ative for viral shedding as determined by fecal conducting the same or different tests on PCR (i.e., indicating clearance of infection). samples collected from cohort animals. In Conclusions practice, all of these approaches are often Although there is no single diagnostic Testing of Biological necessary. For example, if a serum sample testing plan that can be applied to all mouse Materials tests positive for an agent by ELISA, one and rat colonies, one can develop individual Experimental protocols involving mice or should attempt to confirm the result by testing programs by assessing the risk posed rats make use of a variety of transplantable retesting the sample using an alternative by the various infectious agents (i.e.,risk to tumors, cell lines, antibodies and other bio- serological assay, such as an IFA test. Testing research, risk to personnel, and risk of expo- logical materials that become potential additional animals from the colony by serol- sure of the colony to the infectious agents) sources of infectious agents for research ogy for the same agent is also prudent. In and by having a thorough knowledge of mouse and rat colonies. A complete rodent addition, if a suitable alternative test such as available diagnostic tests and testing method- disease prevention program should include PCR, culture, or histopathology exists for the ologies. One can obtain this information by screening these materials for the presence of pathogen, it is wise to test animals from the reading reference material1,10,40–44 and scien- pathogens to prevent the possible introduc- same colony using these techniques. tific literature, as well as by consulting with tion of infectious agents into research mice However, suitable alternative testing methods laboratory animal experts and laboratory and rats. Traditionally, one examines such are not available for all pathogens. Readers animal diagnosticians. biological materials for the presence of infec- should recall that when multiple diagnostic tious agents with antibody production tests, approaches are used to test for the same Received 2/5/03; accepted 3/6/03. such as the mouse or rat antibody production agent, it is possible to obtain conflicting (MAP or RAP) test, in which a portion of the results. This is most common when using References 1. Nicklas, W. et al. Implications of infectious biological material is inoculated into naive PCR assays or histopathology as confirmato- agents on results of animal experiments. animals and then, four to six weeks later, the ry tests for serologically detected agents that Lab Anim. 33, S1:39–S1:87 (1999). 2. Abbas, A.K. & Lichtman, A.H. in Cellular animal is tested for antibodies against various cause acute infections. It is essential to and Molecular Immunology 5th edn. (ed. mouse or rat pathogens. Recently developed remember that serology provides an indirect Abbas, A.K.) 191 (WB Saunders, PCR assays permit direct testing of biological measure of exposure to an agent, whereas Philadelphia, PA, 2003). 3. Compton, S.R. & Riley, L.K. Detection of materials for the presence of these pathogens PCR directly detects the presence of the agent infectious agents in laboratory rodents: tra- and provide an alternative to antibody pro- and histopathology detects pathological ditional and molecular techniques. Comp. 39 Med. 51, 113–119 (2001). duction tests .Advantages of these PCR- changes induced by the microbe. Therefore, 4. Compton, S.R., Smith, A.L. & Gaertner, based tests over antibody production tests are it is quite possible for an animal to have been D.J. Comparison of the pathogenicity in that they provide equal or greater sensitivity exposed to an agent, to have developed a rats of rat coronaviruses of different neu- tralization groups. Lab. Anim. Sci. 49, in detecting the pathogens, results can be detectable antibody response, and to test pos- 514–518 (1999).

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