Comparison of the sensitivity of in vivo antibody production tests with in vitro PCR-based methods to detect infectious contamination of biological materials.

Bacteria and viruses may be transmitted to laboratory rodents by contaminated biological materials such as transplantable tumours, cell lines, sera or other biological materials. Biological materials are currently being screened using the mouse or rat antibody production (MAP/RAP) test (serological testing). We decided to test and validate an alternative assay using polymerase chain reaction (PCR/realtime PCR) technology to detect viral contamination directly in biological material. The aim of this study therefore is the validation of our new PCR assays and the comparison of PCR and the MAP test. For 8/14 viruses, conventional PCR was more sensitive and more specific than the MAP test in detecting murine viruses. For 12/14 viruses, the realtime PCR was more sensitive than the MAP test. In 2/14 cases, all three detection methods had the same sensitivity. Furthermore, PCR screening clearly conforms to the principles of the 3Rs as a replacement technique because it eliminates the need for using animals to screen for murine viruses in biological material.

Hypotensive infarction of the spinal cord in a rhesus macaque (Macaca mulatta).

A 6.5-year-old male rhesus macaque (Macaca mulatta) was presented with acute paraplegia following a surgical procedure. During surgery, he experienced an acute hypotensive episode that required 3 hours of supportive therapy before hemodynamic stability was restored. Paraplegia remained unchanged until euthanasia 72 hours later. At necropsy, spinal cord segments T10-S3 had acute, massive panmedullary necrosis, which involved most of the central and middle spinal cord, sparing the peripheral white matter. Additional lesions included arterial border zone necrosis of the brain, centrilobular hepatic necrosis, and proximal renal tubular necrosis. The histologic findings of central spinal cord necrosis with a preserved rim of white matter is consistent with severely decreased flow in the anterior spinal artery. Such lesions can result from episodes of generalized hypoperfusion. Possible factors contributing to the hypotensive episode in this case included the addition of xylazine to ketamine and atropine in the preanesthetic medication, positioning during anesthesia, and decreased blood pressure during surgery.

Preparation and in vivo testing of porous alumina ceramics for cell carrier applications.

Microporous alumina was used to develop implantable cell carriers shaped as a hollow-sphere with a central opening to allow ingrowth of vascularised tissues. The carriers were produced by suspending the ceramic raw materials in water, homogenising and dropping the resulting slurry onto a heated plate (hot plate moulding, HPM). Morphological characteristics of the cell carriers were investigated by SEM and optical microscopy. Produced carriers had an average diameter of 4.9 mm. The material was highly porous (56 +/- 8%). For in vivo testing the cell carriers were implanted into abdominal wall of Zur: SIV rats for up to 50 weeks and investigated by light microscopy, SEM and TEM. The surface of the hollow carriers was in close contact with unirritated muscle tissue; no inflammation or capsule formation was observed. Loose connective tissue had grown into the hollow cell carrier, and after prolonged implantation >20 weeks adipocytes were observed. The absence of scar tissue formation around the implant and the vitality within the cavity of the hollow carriers indicate that porous alumina may be used for cell transplantation devices.

The generation of monoclonal antibodies in mice: influence of adjuvants on the immune response, fusion efficiency and distress.

The objective of this study was to find a reliable alternative to Freund's adjuvant in order to reduce the distress imposed on the animals without impairing the fusion efficiency for immune-positive clones. For this purpose several commercially available adjuvants and adjuvant formulations representing different classes of molecules were compared. Humoral responses and animals' distress evaluated by clinical assessment and histopathological examinations were investigated and compared to fusion efficiencies. In a first set of experiments seven adjuvants were tested essentially to determine their potential to induce distress. Poly(A).poly(U) and GERBU were selected for further investigations due to their low overall toxicity. They were combined with five different antigens and compared to the classic Freund's adjuvant system (CFA/IFA) and to control immunizations without adjuvant. The results showed that adjuvants of very low toxicity could induce a high fusion efficiency. According to a standardized immunization protocol, GERBU induced polyclonal titres similar to Freund's whereas animals treated with poly(A).poly(U) did not attain titres higher than mice immunized with antigen in saline. Poly(A).poly(U) however, exhibited the best fusion efficiency, Freund and GERBU were slightly less efficient. Therefore poly(A).poly(U) and GERBU may serve as valuable alternatives to Freund's adjuvant for generating monoclonal antibodies. Furthermore, these two adjuvants are very easy to use.

Pathogenesis of guinea pig adenovirus infection.

BACKGROUND AND PURPOSE: The existence of guinea pig adenovirus (GPAdV) has been suspected on the basis of histopathologic findings, but the virus has not yet been isolated. In susceptible animals, it may cause severe bronchopneumonia and death. Adenovirus-like inclusion bodies have been observed in the lungs of animals with clinical disease. Prevalence of the infection is unknown. Recently, a polymerase chain reaction (PCR) assay was described that was able to selectively detect GPAdV. METHODS: To investigate the pathogenesis of GPAdV, we inoculated eight guinea pigs with GPAdV; eight control animals were sham inoculated. The PCR assay was used to trace the infection. In a second experiment, transmission of GPAdV from an experimentally infected animal to five immune-naive cohorts was examined. RESULTS: None of the infected animals developed clinical disease. The GPAdV could be detected by PCR analysis of nasal-swab specimens on days 6 through 15 after infection. Infective virus could be recovered from the nasal mucosa during this period (as determined by inoculation of immune-naive animals). The virus was transmitted from an experimentally infected animal to two of five immune-naive cage mates. CONCLUSION: The GPAdV may cause transient subclinical upper respiratory tract infection that may descend to the lungs.

MHC-genotype of progeny influenced by parental infection.

In a previous series of in vitro fertilization experiments with mice we found non-random combination of major histocompatibility complex (MHC) haplotypes in the very early embryos. Our results suggested that two selection mechanisms were operating: (i) the eggs selected specific sperm; and (ii) the second meiotic division in the eggs was influenced by the type of sperm that entered the egg. Furthermore, the proportion of MHC-heterozygous embryos varied over time, suggesting that non-random fertilization was dependent on an external factor that changed over time. As a higher frequency of heterozygous individuals correlated with an uncontrolled epidemic by MHV (mouse hepatitis virus), we suggested that MHV-infection might have influenced the outcome of fertilization. Here, we present an experiment that tests this hypothesis. We infected randomly chosen mice with MHV and sham-infected control mice five days before pairing. We recovered the two-cell embryos from the oviduct, cultured them until the blastocyst stage, and determined the genotype of each resulting blastocyst by polymerase chain reaction. We found the pattern that we expected from our previous experiments: virus-infected mice produced more MHC-heterozygous embryos than sham-infected ones. This suggests that parents are able to promote specific combinations of MHC-haplotypes during fertilization according to the presence or absence of a viral infection.

Prevalence of enterotropic and polytropic mouse hepatitis virus in enzootically infected mouse colonies.

Mouse hepatitis virus (MHV) causes the most prevalent viral infection in contemporary laboratory mouse colonies. According to their primary replication site, different strains of MHV segregate into two overlapping biotypes, enterotropic and polytropic. These two groups vary greatly in disease pattern, pathogenicity, immune response, and duration of infection. Historically, the polytropic MHV strains represent the extensively studied prototype strains that have minimal enterotropism, whereas enterotropic MHV strains have been less characterized. Anecdotal reports suggest that most MHV strains encountered today belong to the enterotropic biotype. We have identified 15 isolates of MHV from 19 independent enzootically infected mouse colonies. Sequencing of a variable region of the nucleoprotein (N) gene of each isolate confirmed that all were independent genetic variants. The principal tissue tropism of the new isolates was determined by experimental inoculation of infant mice and examination of intestine, liver, spleen, and brain for lesions. Nine isolates infected only intestine; four isolates infected intestine and liver; one isolate infected intestine, liver, and brain; and one isolate infected liver. These results confirm that the enterotropic MHV biotype is predominant in contemporary laboratory mouse colonies. The MHV biotype features need to be taken into consideration when dealing with MHV infections.

Enterotropic mouse hepatitis virus.

Mouse hepatitis virus [MHV], the coronavirus of the mouse, is the most common viral pathogen in contemporary laboratory mouse colonies throughout the world. It is highly contagious with variable clinical manifestations. The majority of infections are subclinical, but can still significantly influence biological responses, thus interfering with research, mainly in the field of immunology. MHV has been intensively studied from a number of research perspectives and has become the prototype for studying the molecular biology of coronaviruses. MHV contains a single-stranded, positive-sense RNA genome ranging from 27 to 31 kb, which is divided into seven genes. Virions consist of four to five structural proteins. There are many MHV strains that vary in virulence, organotropism and cell tropism, and are constantly evolving by naturally occurring mutation and recombination. Based on pathogenesis studies MHV strains are usually grouped according to their primary tissue tropism into two biotypes: polytropic and enterotropic. Enterotropic strains of MHV replicate in the intestinal mucosa and only rarely spread to other tissues. No morphological structure of the virion has as yet been identified that is responsible for enterotropism. The course of an MHV infection is dependent on the virus strain and host factors. Generally, MHV causes an acute, self-limiting infection which is inapparent in adult mice. Neonates are highly susceptible to disease and show high mortality. In an enzootically infected colony, however, they are protected by maternally derived passive immunity. Detection of MHV infections depends on serological screening of colonies. MHV is controlled by culling and rederivation of the affected colony using hysterectomy or embryo transfer or by elimination through cessation of breeding.

[Mouse hepatitis virus]. / Mäusehepatitis-Virus.

Mouse hepatitis virus (MHV), the coronavirus of the mouse (mus musculus), is one of the most important viral pathogens in contemporary laboratory mouse colonies. It is a highly mutable virus consisting of numerous antigenically distinct serotypes with different pathology. These can be divided according to their tissue tropism into respiratory and enterotropic strains. The course of an MHV infection is dependent on virus strain and host factors. Generally MHV causes an acute, self limiting infection which is inapparent in adult mice. Neonates are highly susceptible to disease and show high mortality. In an enzootically infected colony however, they are protected by maternally derived passive immunity. MHV's importance in biomedical research on one hand stems from its potential as an interfering agent, mainly in the field of immunology. On the other hand MHV serves as a model for coronaviruses of other species including man in studies on virus replication and tissue tropism. Since MHV infections are usually subclinical, detection depends on serological screening of colonies using Enzyme-linked immunosorbent assay (ELISA) or immunofluorescence. MHV is controlled by culling and rederivation of the affected colony using hysterectomy or embryo transfer or by elimination by cessation of breeding.

-Entrance of PCR for studying murine coronaviruses-. / Der Einsatz der PCR zum Studium muriner Coronaviren.

This paper describes a number of applications of the Polymerase Chain Reaction (PCR) in the study of enterotropic murine coronaviruses [mouse hepatitis virus (MHV)]. A diagnostic PCR was developed which detected all of 11 different MHV strains. This fast and reliable method was also able to differentiate MHV from other non-murine coronaviruses. On the basis of this assay a quantitative PCR was designed using a mutant template containing a point mutation which competed for the PCR primers. The amplification and cloning of the structural protein genes of enterotropic MHV strains it plasmid vectors for subsequent sequencing is described. In addition an RT PCR was developed which was able to selectivity detect artificially generated recombinant coronavirus.

Enzyme-linked immunosorbent assay for detection of antibody to lymphocytic choriomeningitis virus in mouse sera, with recombinant nucleoprotein as antigen.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibody to lymphocytic choriomeningitis virus (LCMV) in mouse sera. This assay is based on recombinant LCMV nucleoprotein generated in a baculovirus system. Sera from experimentally and naturally infected as well as noninfected mice were tested, and the results were compared with those obtained from an established immunofluorescence assay (IFA) that uses infected cells as antigen. An excellent correlation was found; the ELISA specificity and sensitivity were calculated to be 100 and 95% respectively. Unlike the IFA, this ELISA does not require the handling of infective virus. It eliminates the need to work with a zoonotic agent in the laboratory while allowing effective screening of laboratory mouse populations for LCMV antibody.

Molecular characterization of the S proteins of two enterotropic murine coronavirus strains.

Enterotropic strains of murine coronaviruses (MHV-Y and MHV-RI) differ extensively in their pathogenesis from the prototypic respiratory strains of murine coronaviruses. In an effort to determine which viral proteins might be determinants of enterotropism, immunoblots of MHV-Y and MHV-RI virions using anti-S, -N and -M protein-specific antisera were performed. The uncleaved MHV-Y and MHV-RI S proteins migrated slightly faster than the MHV-A59 S protein. The MHV-Y S protein was inefficiently cleaved. The MHV-Y, MHV-RI and MHV-A59 N and M proteins showed only minor differences in their migration. The S genes of MHV-Y and MHV-RI were cloned, sequenced and found to encode 1361 and 1376 amino acid long proteins, respectively. The presence of several amino acids changes upstream from the predicted cleavage site of the MHV-Y S protein may contribute its inefficient cleavage. A high degree of homology was found between the MHV-RI and MHV-4 S proteins, whereas the homology between the MHV-Y S protein and the S proteins of other MHV strains was much lower. These results indicate that the enterotropism of MHV-RI and MHV-Y may be determined by different amino acid changes in the S protein and/or by changes in other viral proteins.

Sequence analysis of the nucleoprotein genes of three enterotropic strains of murine coronavirus.

The nucleotide sequences of the nucleoprotein genes of three enterotropic strains of the murine coronavirus mouse hepatitis virus (MHV-Y, MHV-RI and DVIM) were determined and compared with previously reported sequences of three polytropic (respiratory) strains (MHV-A59, MHV-JHM and MHV-S). Greater than 92% homology was found among the six strains by pair-wise comparison at the nucleotide level. The genes encoded proteins of 451 to 455 residues and the deduced amino acid sequences were more than 91% homologous. A unique deletion of twelve nucleotides was found at the carboxy terminus of MHV-Y and a three nucleotide deletion was found in MHV-RI, which corresponded to the one previously reported in MHV-A59 and MHV-S. Two internal open reading frames were found within the coding region of the nucleoprotein, the smaller one was specific for the enterotropic strains. It could potentially encode a truncated version of the hypothetical protein described for MHV-A59 and MHV-S. Sequence relationship of the N gene showed no correlation with tissue tropism and no sequence or even single amino acid change unique to either tropism group was found. This indicates that the nucleoprotein of MHV probably has no part in the determination of the primary tissue tropism of an MHV strain. The role of the potential internal protein warrants further investigation.

[Procedures for health monitoring in a breeding facility for specific pathogen free mice and rats]. / Vorgehen bei der Hygiene-Uberwachung in einer Zuchtanlage für spezifiziert pathogen freie Mäuse und Ratten.

Causes of variation in animal experiments include differences in the genotype of the animals as well as a number of environmental factors. Through standardisation of the physical, chemical and biological components of the environment the quality of the results of the experiments can be improved, which in turn leads to a reduction of the number of animals used. One of the means to achieve this goal is the use of specified pathogen free (SPF) animals. To assure the microbiological quality of these animals the population and its environment needs to be screened thoroughly on a routine basis. This publication describes the necessary quality assurance procedures. These include bacteriological, parasitological, virological and histological examinations of the animals themselves, as well as environmental screens such as microbiological examinations of feed, control of water quality or the testing of the efficacy of disinfectants.

Transmission of murine viruses and mycoplasma in laboratory mouse colonies with respect to housing conditions.

Pathogen-free sentinel mice were placed in 7 animal rooms with different housing conditions and were serologically screened for antibodies to mouse hepatitis virus (MHV), pneumonia virus of mice (PVM), Sendai virus, reovirus 3, Theiler's mouse encephalomyelitis virus (TMEV), ectromelia virus and Mycoplasma pulmonis by enzyme-linked immunosorbent assays, at intervals after introduction. The most commonly detected antibody was against MHV, which was found in mice from 4 rooms, followed by PVM antibody in mice from 3 rooms. Seroconversion to Sendai virus and TMEV was detected in mice from one room each. No seroconversion to any of the antigens was found in 2 rooms. The common criteria of these 2 rooms were that they housed pathogen-free animals from a single source and that the access to the rooms was, purposely or not, restricted to people who had no contact to other mice. The study demonstrated the importance of husbandry and hygienic regimen on the prevalence of infectious agents in laboratory mice.

Nucleotide sequence comparison of the membrane protein genes of three enterotropic strains of mouse hepatitis virus.

The nucleotide sequences of the membrane (M) protein genes and their deduced amino acid sequences of three enterotropic strains of the coronavirus mouse hepatitis virus (MHV) -Y, -RI and -DVIM were determined and compared with the previously reported sequences of two respiratory MHV strains -A59 and -JHM. The five MHV strains shared extensive nucleotide (95.2-99.0%) as well as amino acid homology (95.6-98.7%). A variable region, including a 15 nucleotide deletion unique to MHV-RI, could be identified at the 5'-terminus of the gene. This region of the M protein may be immunogenic and may contribute to the antigenic diversity of the MHV strains. Sequence relationships between the strains showed no correspondence with the primary cell tropism. This may suggest that evolution of enterotropism was not a single occurrence among different MHV strains. No sequence unique to either tropism group could be identified, indicating that the M protein of MHV probably has no part in the determination of MHV tissue tropism.