Antemortem detection of mouse parvovirus and mice ... - SAGE Journals

Antemortem detection of mouse parvovirus and mice minute virus by polymerase chain reaction (PCR) of faecal samples B A Bauer and L K Riley Research Animal Diagnostic Laboratory, University of Missouri, Columbia, MO 65211, USA

Summary Parvoviruses remain one of the most common viral infections seen in laboratory mouse colonies. The purpose of this study was to develop an antemortem polymerase chain reaction (PCR) assay to detect mice infected with mouse parvovirus-1 (MPV) and mice minute virus (MMV) using faecal samples. The MMV PCR assay consistently detected as few as 100 plasmid copies of MMV in faecal samples, while the MPV PCR assay detected as few as 10 plasmid copies of MPV. Faecal pellets from infected mice held at room temperature from 1 to 7 days tested positive by MMV and MPV PCR, respectively. This demonstrates that parvovirus DNA is stable in faecal samples kept at room temperature. PCR assays were also used to follow the length of MMV and MPV shedding in faeces from SENCAR mice, which were endemically infected with multiple agents. MMV faecal shedding was detected in 60–70% of the mice 5–7 weeks old, and by 13 weeks of age, faecal samples from all mice were negative for MMV. MPV faecal shedding was detected in 90–100% of the mice 5–11 weeks old; however, by 19 weeks of age, faecal samples from all mice were negative for MPV. These findings confirm that faecal shedding occurs for a limited time and suggest that 5–9-week-old mice are the most appropriate age group in endemically infected mice for faecal testing by MMV and MPV PCR. Keywords Parvovirus; mouse parvovirus; mice minute virus; polymerase chain

reaction; faecal

Mus musculus, the laboratory mouse, is the natural host for mouse parvovirus-1 (MPV) and mice minute virus (MMV), also known as minute virus of mice (MVM) (Jacoby et al. 1996, Berns et al. 2000). Natural infections of MMV in mouse colonies are not associated with clinical disease, but experimental infections produce runting in neonatal mice, and in some instances, a lethal disease with renal papillary necrosis, myeloid depression, and cerebellar lesions Correspondence: B A Bauer, Research Animal Diagnostic Laboratory, University of Missouri, E108 Veterinary Medicine Building, 1600 East Rollins Street, Columbia, MO 65211, USA. Email: [email protected] Accepted 24 August 2005

(Kilham & Margolis 1970, Kimsey et al. 1986, Brownstein et al. 1991, Segovia et al. 1995, Ramirez et al. 1996, Segovia et al. 1999). In contrast, MPV has not been associated with disease in either natural or experimental infections (Smith et al. 1993, Shek et al. 1998). Even though natural infections are not associated with clinical disease in mice, complications to research projects can result from the use of parvovirus-infected mice or tissues. Both MMV and MPV have a predilection for lymphoid tissue and have been shown to alter T cell function in vitro as well as potentiating rejection of tumour allografts

r Laboratory Animals Ltd. Laboratory Animals (2006) 40, 144–152

Parvovirus faecal PCR assay

in vivo (Engers et al. 1981, McKisic et al. 1993, 1996, 1998). Parvoviruses remain the most common viral pathogens found in mouse colonies and in biological materials (Collins & Parker 1972, Nicklas et al. 1993, Jacoby & Lindsey 1997, Garnick 1998, Livingston & Riley 2003). While serological assays are commonly used as indirect screens to detect MPV and MMV infection in mice, polymerase chain reaction (PCR) assays can be used to directly detect DNA of parvoviruses in mouse tissues and cell lines (Besselsen et al. 1995, Besselsen 1998, Redig & Besselsen 2001, Bootz et al. 2003, Bauer et al. 2004). PCR assays can detect parvoviruses in early infections prior to serological conversion or in immunocompromised mice that are not capable of developing a serological response. Current murine parvovirus PCR assays are based on amplification of viral DNA contained in mouse tissues such as mesenteric lymph node or spleen and necessitate euthanasia of mice prior to testing (Besselsen et al. 1995, Besselsen 1998, Redig & Besselsen 2001). In this study, PCR assays were developed and validated for the detection of MPV and MMV in faecal samples. These PCR assays offer the advantages of easy sample collection and antemortem testing of mice. The developed assays were used to examine the length of faecal shedding for MPV and MMV in an endemically infected mouse colony.

Materials and methods Viral isolates and clones

Three MMV viral isolates (MMVp, MMVi and MMVc) were used to demonstrate specificity of the MMV primers developed in this study. MMVp was propagated in murine A9 fibroblasts (ATCC CCL-1.4), MMVi in 324 K simian virus 40-transformed human newborn kidney cells (David Pintel, University of Missouri, MO, USA) and MMVc in BHK cells (ATCC CCL-10). Other rodent parvovirus isolates used included MPV1b propagated in CTLL-2 cells (ATCC TIB-214), LUIII virus (LuIII) propagated in HeLa cells (ATCC CCL-2), Kilham rat virus

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(KRV) propagated in C6 cells (ATCC CCL107), H-1 virus (H-1) propagated in 324 K simian virus 40-transformed human newborn kidney cells (David Pintel, University of Missouri, MO, USA) and rat parvovirus (RPV) propagated in RNK-16 LGL cells (John Ortaldo, National Cancer Institute). Viral stocks of the above listed parvoviruses were made using previously published methods (Besselsen et al. 1995). Briefly, all cell cultures were infected with a multiplicity of infection of 0.1, and virus was harvested when cell cultures exhibited complete cytopathic effect. MMV and MPV VP2 plasmid clones (MMV-VP2-pFastBac HTb and MPV-VP2-pFastBac HTc) were used as the template DNA for the sensitivity determination of the MMV and MPV faecal PCR assay (Livingston et al. 2002). DNA isolation

Total faecal DNA was isolated using the Qiagen DNeasys kit (Qiagen, Valencia, CA, USA) with modifications for DNA isolation from faeces. Briefly, one faecal pellet was suspended in 1 mL of PBS to form a homogeneous faecal slurry, and the entire slurry was then subjected to centrifugation at 500 g for 5 min at room temperature. After centrifugation, 200 mL of supernatant was transferred to a clean 1.5 mL tube for DNA isolation using the animal tissue protocol with the following minor modifications: the wash step using buffer AW1 was repeated one time; centrifugation for the first AW2 wash was only performed for 1 min followed by a second AW2 wash in which centrifugation was performed for 5 min; the elution buffer was heated to 701C prior to elution and 200 mL was added to the column; the column with the elution buffer was incubated for 5 min at room temperature followed by centrifugation for 1 min to collect total faecal DNA in AE buffer from the column. DNA from mouse tissue or cultured cells was extracted following the directions for animal tissue or cultured cells, respectively using the Qiagen DNeasys kit. Plasmid DNA was isolated using the QIApreps miniprep kit (Qiagen, Valencia, CA, USA) following the kit directions for the Laboratory Animals (2006) 40

B A Bauer & L K Riley

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miniprep spin protocol. All DNA extractions were performed in a PCR/ultraviolet (UV) workstation (Coy Laboratory Products, Grass Lake, MI, USA). Cross contamination during extraction was assessed by extracting a known negative sample along with test samples for each extraction with subsequent PCR amplification of the negative control sample along with the test samples. DNA concentration in eluted samples was determined by absorbance at 260 nm using a Gene-Quantt spectrophotometer (Amersham Biosciences Corp, Piscataway, NJ, USA) and a NanoDrop ND-1000 (NanoDrop Technologies, Inc, Wilmington, DE, USA). No significant difference in concentration was found between the two different spectrophotometer units. PCR amplification

Primers used in this study are listed in Table 1. MMV-specific primers were designed using OMIGAs software (Oxford Molecular Group, Campbell, CA, USA) based on the alignment of the VP2 sequence of MMV to that of other known parvoviruses. The MPV-specific primer sequences used in this study were previously published (Besselsen et al. 1995). All primers were obtained from Integrated DNA Technologies (Coralville, IA, USA). PCR assays were performed in a final volume of 50 mL which contained 1 U of FastStart Taq DNA polymerase and 5 mL of the supplied buffer (Roche, Mannheim, Germany), 2 mM MgCl2, 1 mM of each primer, and 200 mM of each dNTP (Roche, Mannheim, Germany).

Table 1 PCR primer sequence Parvovirus

Primer name

MPV

3759f 4018r

MMV

2929f 3713r

Sequence 50 -GCA GCA ATG ATG TAA CTG AAG CT 50 -CCA TCT GCC TGA ATC ATA GCT AA 50 -AAA TTA CTG CAC TAG CAA CTA GAC 50 -CTT CAG GAA AGG TTG ACA GCA

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To determine the specificity of the MMV primers, 5 mL of purified viral DNA from various rodent parvoviruses (KRV, H-1, RPV1, rat minute virus (RMV), HaPV, MPV, MMV or LuIII) or cellular DNA (isolated from uninfected mouse spleen, kidney, ileum, mesenteric lymph node or faeces; or from cultured cells of rat, mouse, human or non-human primate origin) were added as template DNA to each PCR reaction. To assess the sensitivity of the assay, 10-fold serial dilutions of quantitated plasmid DNA containing either MPV or MMV VP2 sequence were made using the elution buffer from the QIApreps miniprep kit. To assess the sensitivity of the PCR assays in the presence of tissue or faecal material, 5 mL of faecal DNA or mesenteric lymph node DNA isolated from uninfected mice were added to each reaction along with 5 mL of quantitated purified plasmid DNA in serial 10-fold dilutions. For all other PCR assays, 10 mL of faecal origin DNA or 5 mL of tissue origin DNA was added to each PCR reaction mixture. PCR reactions were performed using a GeneAmps PCR System 9700 thermal cycler (Applied Biosystem, Foster City, CA, USA). An initial denaturation step at 951C for 4 min was performed, followed by either 45 cycles for the faecal PCR assays or 40 cycles for the tissue PCR assays in which each cycle consisted of the following three steps: (1) denaturation for 10 s at 941C; (2) annealing for 10 s at 551C for the MPV PCR assay or 601C for the MMV PCR assay and (3) extension at 721C for either 30 s for the MPV PCR assay or 45 s for the MMV PCR assay. A final extension at 721C was performed for seven minutes. Amplicons obtained from PCR reactions were subjected to electrophoresis on a 2% agarose gel, ethidium bromide staining, and visualization by UV light. The predicted product size for the MPV PCR assay was 260 base pairs and the predicted product size for the MMV PCR assay was 735 base pairs. Molecular weight markers were used to confirm amplicons size and all PCR assays contained a negative control and a positive control reaction in addition to the test samples. Select MPV PCR amplicons obtained from SENCAR mice were


sequenced by SeqWright (Houston, TX, USA) and sequence data deposited with GenBank was assigned accession numbers AY870797 and AY870798. Animals

All animal studies were performed in accordance with the University of Missouri’s Animal Care and Use Committee guidelines and the ILAR Guide for the Care and Use of Laboratory Animals. Outbred SENCAR mice obtained from an endemically infected colony containing multiple pathogens were used in this study. This colony was selected because animals were readily available and the colony contained both MMV and MPV. This colony was also positive for murine hepatitis virus (MHV), Sendai virus and Theiler’s murine encephalomyelitis virus (TMEV) as evidenced by repeated serological testing. The colony was also infested with Syphacia obvelata as evidenced by positive perianal tape testing and microscopic examination of caecal contents. Pasteurella pneumotropica and multiple Helicobacter species (including H. bilis, H. rodentium and H. typhlonicus) were also endemic in this colony. Mice for the faecal shedding experiment were group housed in microisolator cages, fed a standard pelleted rodent chow, and housed in an environmentally controlled room with a 12:12 h light/dark cycle. Animals were checked daily by the animal care staff, and no signs of clinical illness or disease were noted in any of the mice during the entire study. Faecal pellets were collected from individual mice starting at five weeks of age and ending at 35 weeks of age. Collected faecal pellets were stored at 801C until processed for DNA isolation. Mice were euthanized with an inhaled overdose of CO2 followed by blood collection by cardiocentesis. Mesenteric lymph node and spleen were harvested using sterile technique and stored at 801C. Stability

To assess the stability of parvovirus DNA in faeces, faecal pellets were collected from 10 naturally infected mice and then ground

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together to form a homogeneous mixture. This mixture was then separated into eight equal aliquots in 1.5 mL tubes that were held at room temperature for one to seven days. At the end of the holding time, the faecal material was stored at 801C until processed for DNA isolation. Serology

Whole blood collected by cardiocentesis was diluted 1:5 with phosphate buffered saline. Serum was collected from the clotted whole blood samples by centrifugation, and ELISA evaluation was performed using methods previously published (Livingston et al. 2002). Briefly, 10 mL of serum were added to each VP2 antigen or cell control coated well in a 96 well plate. Secondary antibody consisted of a goat antimouse immunoglobulin G (IgG) coupled to alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and p-nitrophenyl phosphate was used as the substrate. Absorbance was determined at the 420 nm wavelength using a Titertek S20 ELISA plate reader (Titertek Instruments, Huntsville, AL, USA). Positive and negative control samples were included with each assay. The baseline value was set at the mean plus 2 standard deviations of the absorbance value for at least 80 known negative samples from mice of various strains and ages. Samples were considered negative if the absorbance reading was less than the baseline. Samples with an absorbance of twice that of the baseline were considered positive. Equivocal results that were greater than the baseline value but were less than twice that of the baseline absorbance were re-evaluated by either haemagglutination-inhibition assay (Besselsen et al. 2000) or by indirect fluorescent assay (IFA). For IFA assays, 20 mL of serum was added to a microscope slide well coated with a 50/50 mixture of infected and uninfected cells. Fluorescein conjugated goat antimouse IgG (KPL, Gaithersburg, MD, USA) was used as the secondary antibody. Both a positive and a negative control well were included with each assay. A positive result was determined by comparing the intensity and distribution of Laboratory Animals (2006) 40


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fluorescence with that of the positive and negative control wells. Equivocal ELISA results were considered positive only if the secondary test results were also positive.

Results PCR assay development

Three different DNA protocols were used to extract DNA from faeces based on previous work on the isolation of faecal DNA for the detection of Helicobacter (Beckwith et al. 1997). All three methods used Qiagen’s DNeasys kit and varied by different amounts of faecal slurry used (100 or 200 mL) for total DNA isolation or the amount of buffer used (100 or 200 mL) during the final elution step. Optimal results were obtained when DNA was isolated from 200 mL of faecal slurry and eluted in a final volume of 200 mL (data not shown). The primers used in the MPV assay are shown in Table 1 and have been previously published (Besselsen et al. 1995). In initial experiments, a previously published primer set for MMV was used to amplify faecal DNA samples from known positive MMV mice (Besselsen 1998). However, very few faecal samples yielded amplicons, and when present, amplicons consisted of multiple weak bands that were difficult to visualize. Examination of the reverse primer location revealed that this primer was in close proximity to the left hand hairpin of the MMV viral genome. Because secondary structure of this nature can inhibit or reduce PCR amplification, a new set of primers was designed within the VP2 region of MMV for use in the MMV PCR assay (see Table 1). Specificity of the new MMV primer set was tested by amplifying DNA extracted from various parvovirus species and parvovirus negative mouse tissues and cell lines from various species (data not shown). The MMV primer set amplified all three known isolates of MMV, which included MMVc, MMVi and MMVp, but did not amplify other parvovirus species (MPV, HaPV, LuIII, KRV, RPV-1 and RMV) or the negative tissues and cell lines of rat, mouse, human and hamster origin. Amplicon size was consistent with the Laboratory Animals (2006) 40

predicted product size as assessed by molecular weight markers. Sensitivity was determined for both the MMV and MPV PCR assay by using 10-fold serial dilutions of plasmids containing either the sequence of the MMV VP2 gene or the MPV VP2 gene. An equal volume of parvovirus free mouse mesenteric lymph node DNA or mouse faecal DNA was added to each reaction containing plasmid DNA to simulate diagnostic samples and to allow for a direct comparison of sensitivity between the use of DNA extracted from mesenteric lymph node and DNA extracted from faeces. The MMV PCR assay consistently amplified as little as 10 plasmid template copies in the presence of mesenteric lymph node DNA and 100 template copies of DNA in the presence of faecal DNA (data not shown). The MPV assay amplified as little as 10 template copies in the presence of both mesenteric lymph node DNA and faecal DNA (data not shown). Stability of virus in faeces

To determine the stability of parvovirus DNA in faecal material, MPV and MMV positive faecal samples were held at room temperature for 1–7 days. Figure 1 shows that DNA from both MPV and MMV were amplified from faeces held at room temperature at all time points examined. This finding shows that parvovirus DNA is stable in faeces for at least 7 days when held at room temperature. Detection of parvovirus from various biological samples

Mesenteric lymph node and faeces were collected from 19 female and 16 male SENCAR mice 6–8 weeks of age obtained from a colony with a history of endemic MPV and MMV infection. Table 2 contains the results of the PCR assays and serology performed in this study. All mesenteric samples (35/35, 100%) were positive for MPV by PCR. In contrast, only 26 out of 35 (74%) faecal samples were positive for MPV. For MMV, 22 out of 37 (63%) mesenteric lymph node samples were positive, but only


12 out of 35 (34%) faecal samples were PCR positive for MMV. Serology results were identical to the mesenteric lymph node PCR results for MPV and serology detected one additional animal in comparison with the MMV mesenteric lymph node PCR results. These findings indicate that while mesenteric lymph nodes yield a higher percentage of positive results, faecal samples can be tested by PCR when an antemortem test for the detection of parvovirus is required. Diagnostic sensitivity was calculated by using serology results as the true positive results. Diagnostic sensitivity of the MPV faecal PCR assay was 74% and the MMV faecal PCR assay was 52%. In order to calculate diagnostic specificity,

Figure 1 Agarose gel results of PCR products obtained from faeces held at room temperature. Panel (a) contains MMV PCR assay results and panel (b) contains MPV PCR assay results. Lanes 1-7 contain PCR results from faeces held at room temperature for one to seven days. Lanes: 1, 1 day; 2, 2 days; 3, 3 days; 4, 4 days; 5, 5 days; 6, 6 days; 7, 7 days. Negative control reactions are indicated by a minus symbol (negative extraction control, negative faecal DNA control, no DNA template control) and the positive control reaction is indicated by a plus symbol. The lane containing the molecular weight marker is indicated by MW

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parvovirus PCR was performed on 49 faecal samples obtained from multiple negative mouse colonies. All 49 samples were negative for both MPV and MMV resulting in a diagnostic specificity of 100% for both PCR assays.

Duration of faecal shedding

In this study, the parvovirus PCR assay performed on faecal samples was used to determine the duration of viral shedding in faeces collected from a colony of mice endemically infected with multiple murine pathogens. Faeces collected over seven months from 10 SENCAR female mice were tested by PCR for the presence of both MPV and MMV. The percent of mice with faeces positive for MPV and MMV as assessed by PCR assay is presented in Figure 2. MMV was detected in 70% of the mice tested at

Figure 2 Duration of MMV and MPV faecal shedding as determined by PCR assay. The percent of samples that tested positive is indicated on the Y axis and the age of the mice in weeks is indicated on the X axis. MPV positive samples are indicated by a square and MMV positive samples are indicated by a triangle

Table 2 PCR and serology results Parvovirus

Test material (assay)

No. positive/No. tested

% positive

MPV

Mesenteric lymph node (PCR) Faeces (PCR) Serum (ELISA)

35/35 26/35 35/35

100 74 100

MMV

Mesenteric lymph node (PCR) Faeces (PCR) Serum (ELISA)

22/35 12/35 23/35

63 34 66

MPV=mouse parvovirus, MMV=mice minute virus, PCR=polymerase chain reaction, ELISA=enzyme linked immunosorbent assay


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five weeks of age and steadily decreased until 13 weeks of age at which time all mice were negative. MPV was detected at a higher percentage and for a longer duration. From five weeks of age until 11 weeks of age, 90–100% of the mice tested were PCR positive for MPV. After 11 weeks of age, detection of MPV in the faeces declined, and all animals were negative by 19 weeks of age. None of the faecal samples collected after 19 weeks of age were positive for MPV. To confirm that the mice in the study had been infected with MPV and MMV, serum and mesenteric lymph nodes were collected from the 35-week-old mice. All of the animals had positive serological results for MPV and MMV as well as MHV and Sendai virus (nine out of 10 animals were positive for TMEV). MPV PCR positive results were obtained from 90% of the mesenteric lymph nodes, and MMV positive results were obtained from 80% of the samples. These PCR assay results demonstrate that while older animals (35 weeks of age) are not shedding detectable virus in the faeces, the virus is still present in mesenteric lymph node in the vast majority of the animals.

Discussion Serology remains the most cost effective method to screen mouse colonies for the presence of parvoviruses, but PCR-based assays offer an independent method to confirm serological results. Several parvovirus PCR assays have been previously published for mouse tissue or biological materials such as cell lines (Besselsen et al. 1995, Yagami et al. 1995, Chang et al. 1997, Besselsen 1998, Redig & Besselsen 2001). This study provides validated PCR assays that detect the presence of MMV and MPV in mouse faeces. The assays detect as little as 10 template copies of MPV and 100 template copies of MMV in the presence of DNA extracted from faeces. A faecal-based PCR assay offers several advantages to that of a tissue-based PCR assay. Major advantages are that faeces can be collected from live mice, and faecal collection is easy to perform. Another advantage is that the faecal sample does not require any special Laboratory Animals (2006) 40


handling during shipping. Also, the results of this study show that the viral DNA is stable in faeces for up to seven days at room temperature. The main limitation to the faecal assay is that the sample will only be positive while the animal is shedding virus. Unlike the persistent infection seen in mesenteric lymph nodes, faecal shedding is transient. Therefore, faecal PCR assay for the detection of parvovirus cannot be used to screen animals for past exposure but should be used for the detection of parvovirus during the active phase of the infection when viral replication and faecal shedding is occurring, such as during an initial viral outbreak. Faecal-based PCR assays may be the preferred confirmatory test for the individual valuable animal, such as genetically unique founder mice and animals on research studies since the assay is an antemortem test. Selection of the animals for faecal collection to detect the presence of MPV or MMV is the most challenging aspect in the utilization of this assay. The results of this study show that faecal shedding is not persistent and only occurs for a finite period of time. Many variables have been identified that can influence the course of viral shedding for both MMV and MPV. Genetic background and age are significant variables for both MPV and MMV (Brownstein et al. 1991, Smith et al. 1993, Besselsen et al. 2000). Pathogenesis and epidemiology of the different viral isolates is also another important variable. Many of the parvovirus studies that characterize the course of parvovirus infection have been performed with experimental inoculations of virus obtained as cell culture contaminants. A recent study suggests that these cell culture contaminants may not have the same infectivity or duration of shedding as field strains of the virus (Compton et al. 2004). Infectivity rates and the duration of shedding may be significantly shorter with these cell culture contaminants compared with field strain isolates. For instance, experimental inoculation of the cell culture contaminant MPV-1a in BALB/c mice demonstrated that intestines were only positive for up to three weeks post inoculation (Jacoby et al. 1995).


However, similar studies in BALB/c mice from a naturally infected colony containing a field strain of MPV demonstrated that intestines became positive at two months of age and continued to be positive in some animals up to six months of age (Shek et al. 1998). Other pathogens present in the infected mouse may also influence the course of parvoviral infection. In addition, maternal antibody is known to be protective against parvovirus infection in endemic colonies; the age at which viral infection occurs in an endemic colony is between four and six weeks (Parker et al. 1970). This large number of variables makes selection of which animals to sample in a potentially endemic colony seem like a difficult task, but in reality a few general guidelines can be followed to assist in this task. First, select animals that are greater than four weeks of age, when maternal antibody is likely to have waned and the animal is more likely to have recently been infected with parvovirus. Second, avoid selecting animals older than 12 weeks of age because viral shedding occurs for only a limited time and animals over the age of three months are unlikely to still be shedding virus in the faeces in an endemically infected colony. The results of this study suggest that 5–9 weeks of age is the most appropriate age group to evaluate endemically infected mice by faecal PCR (see Figure 2). In summary, faecal-based PCR assays offer an independent antemortem method of confirming serological test results for MMV and MPV. These assays can be used as adjunct diagnostic assays for the detection of MMV and MPV. Though parvovirus PCR assays performed with faecal material should only be used during early infections because faecal shedding is transient. PCR assays for detection of mouse parvoviruses in faecal samples are advantageous due to the ease of sample collection, the stability of the sample and the sensitivity of the assay. Parvovirus faecal PCR assays are particularly advantageous for testing genetically unique mice or mice on research studies in which euthanasia and tissue testing are not an option. Another use for these assays is in the early stage of a viral outbreak in which

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screening of the colony by faecal PCR assay may yield more information than testing of exposed sentinel animals. Acknowledgements This study was funded by the Mutant Mouse Regional Resource Center NIH Grant U42 RR14821.The authors gratefully acknowledge the assistance provided by the technical staff in the RADIL necropsy, molecular biology and serology laboratories.

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