Prevalence of naturally occurring viral infections, Mycoplasma pulmonis and Clostridium piliforme in laboratory rodents in Western Europe screened from 2000 to 2003.

In this report prevalence rates of rodent viruses in laboratory animals are presented based on routine serological screening of mouse and rat colonies from European institutes. The prevalences found during the period 2000-2003 are compared with those reported for 1981-1984 and 1990-1993. It is shown that some infections were eliminated from laboratory animal colonies (e.g. K-virus and polyomavirus) by taking preventative measures whereas other infections such as mouse hepatitis virus and parvoviruses remained at a high rate. Further decreases in prevalence rates in the last 10 years were found for infections such as pneumonia virus of mice, reovirus type 3, Sendai virus, sialodacryoadenitis/rat coronavirus and Mycoplasma pulmonis. The introduction of new detection methods showed that mouse parvovirus and rat parvovirus, both members of the Parvoviridae family, remain a major threat to laboratory mice and rats. Guinea pig cytomegalovirus and para-influenza virus appeared to be the most prevalent agents among laboratory guinea pigs. The importance of a standardized, up-to-date screening programme is discussed.

IFN-gamma-independent IgG2a production in mice infected with viruses and parasites.

After infection with some viruses and intracellular parasites, antibody production is restricted to IgG2a. We first observed that, whereas live viruses such as lactate dehydrogenase-elevating virus (LDV) or mouse adenovirus induced mostly an IgG2a response, a large proportion of antibodies produced against killed viruses were IgG1. This IgG1 antiviral response was suppressed when live virions were added to inactivated viral particles. These results indicate that the IgG2a preponderance is related to the infectious process itself rather than to the type of antigen involved. Since IFN-gamma is known to stimulate IgG2a production by activated B lymphocytes and to be secreted after infection, we examined the role of this cytokine in the antibody isotypic distribution caused by LDV. Most IgG2a responses were relatively unaffected in mice deficient for the IFN-gamma receptor or treated with anti-IFN-gamma antibody. A similar IFN-gamma-independent IgG2a secretion was observed after infection with the parasites Toxoplasma gondii and Trypanosoma cruzi. However, the IFN-gamma-independent IgG2a production triggered by infection still required the presence of functional T(h) lymphocytes. Therefore, signal(s) other than IFN-gamma secretion may explain the T(h)-dependent isotypic bias in antibody secretion triggered by viruses and parasites.

Polyclonal B lymphocyte activation induced by mouse hepatitis virus A59 infection.

Mouse hepatitis viruses (MHV) diversely affect immune responses, depending on the viral strain and the mouse genetic background. Here, we studied the effect of MHV-A59 infection on B cell responses of 129/Sv and CBA mice. Our results indicate that in these strains, MHV-A59 induces spleen cell activation that leads to enlargement of the spleen without structural alteration. Infection triggers production by B lymphocytes of large amounts of immunoglobulin G2a, mostly without viral specificity. This polyclonal immunoglobulin production is dependent on the presence of functional T helper cells. This polyclonal B lymphocyte activation induced by MHV-A59 infection can have pathological implications, such as the enhancement of concomitant autoimmune reactions.

Mutual viral and bacterial infections after housing rats of various breeders within an experimental unit.

Fifteen athymic rat strains from 11 breeding colonies were housed within an experimental facility for an immunological study. Health status records supplied with 14 of the strains listed infections by Kilham's rat virus (KRV), Clostridium piliforme (Bacillus piliformis) and Pasteurella pneumotropica for 2, 2 and 1 colonies respectively. In sera taken previous to the study from euthymic rats of 10 strains, antibodies to KRV were detected in 3 strains, to Pneumonia virus of mice (PVM), Rat corona virus (RCV) and Sendai virus in one strain each and to P. pneumotropica in 2 strains. Only 2 of the KRV infections had been reported by the supplier. During the study rats of all 10 strains developed antibodies to 2-4 of viral antigens. Eight out of 10 rat strains seroconverted to 1-5 of the antigens C. piliforme (B. piliformis), Bordetella bronchiseptica, Haemophilus spp., P. pneumotropica and Streptobacillus moniliformis. Two rat strains housed in filtertop cages did not develop antibodies to bacterial antigens. The potential detrimental effects of intercurrent infections on the outcome of the comparative immunological study are discussed.

Demonstration of Mycoplasma contamination in cell cultures by a Mycoplasma group- specific polymerase chain reaction.

Cell cultures are widely used in both medical and biotechnical research centers, industries, and also as diagnostic tools in a clinical setting It has been reported that up to 50% of cell cultures are contaminated with mycoplasmas (1). Mycoplasma contamination may alter cellular growth characteristics, enzyme patterns, and cell membrane composition, and can induce chromosomal abnormalities and cytopathogenic changes (2-5). In experimental results being published, it is now becoming standard practice to show that mycoplasma-free cell cultures have been used. For industrial production of biological materials derived from mammalian cells and intended for diagnostic or therapeutic use in humans, regulatory guidelines are emerging that are intended to guarantee the quality and safety of these products. Obligatory testing for mycoplasmas is therefore increasing (6).

Enzymatic amplification of lactate dehydrogenase-elevating virus.

To improve the detection of lactate dehydrogenase-elevating virus (LDV), we developed a PCR assay. Primers were selected from ORF7, encoding nucleocapsid protein VP1. No specific amplification was observed with any other common murine virus or with RNAs from the closely related Lelystad virus and equine arteritis virus. In experimentally infected mice, LDV could be detected in plasma in both the acute and the persistent phases. LDV was also detected by the PCR in contaminated pools of Plasmodium berghei parasites which were maintained in mice, both by a direct analysis of the samples and by testing of plasma from mice inoculated with these pools. There was a complete agreement between the results of the PCR assay and the lactate dehydrogenase (LDH) enzyme assay of plasma from the inoculated mice. In contrast to the results of the LDH enzyme assay, no false-positive reactions were obtained in the PCR assay with negative control samples showing visible hemolysis. Storage of plasma samples at room temperature and at 4, -20, and -80 degrees C for up to 8 days did not influence the results of the PCR. These results show that the PCR is a valuable technique which may replace the LDH test as a diagnostic tool.

16S rRNA based polymerase chain reaction compared with culture and serological methods for diagnosis of Mycoplasma pneumoniae infection.

The use of a 16S rRNA based polymerase chain reaction (PCR) for the detection of Mycoplasma pneumoniae infection was investigated. Sputum samples from 34 patients with respiratory illness and evidence of pneumonia as judged by chest X-ray were analyzed by PCR and microbiological culture. Throat swabs from 14 healthy individuals were used as controls. For serology, an enzyme immunoassay for the detection of immunoglobulin M antibodies and a complement fixation assay were performed. Evidence of Mycoplasma pneumoniae infection was obtained in ten patients (29%), eight of whom were found positive by both PCR and serology. Two of the sputum samples from these eight patients were negative by culture. Of the remaining two patients positive for Mycoplasma pneumoniae, one was positive by PCR and culture but negative by serology, and one was found positive by serology but negative by PCR and culture. Thirteen of the 14 controls were negative by both PCR and serology. One control, however, was negative by serology but positive by PCR, which was probably due to asymptomatic carriage of Mycoplasma pneumoniae. The results of this study indicate the suitability of the PCR for the detection of Mycoplasma pneumoniae in clinical samples as well as its potential value as an additional tool for the diagnosis of infection.

No evidence of mycoplasmas in peripheral blood mononuclear cell fraction of HIV-infected patients.

In this study, the prevalence of mycoplasmas in peripheral blood mononuclear cells (PBMC) from HIV-infected individuals was investigated using a mycoplasma genus-specific PCR assay. No mycoplasmas were detected in the PBMC samples from any of the 25 HIV-infected individuals (CDC 2, n = 8; CDC 3, n = 2; CDC 4, n = 15) or ten HIV-seronegative controls. As an internal control, HIV specific sequences were detected in the samples from all HIV-seropositives. These negative results do not support a suggested role of mycoplasmas as co-factor in the progression of HIV infection towards AIDS.

Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR.

The suitability of a 16S rRNA-based mycoplasma group-specific PCR for the detection of mycoplasma contamination in cell cultures was investigated. A total of 104 cell cultures were tested by using microbiological culture, DNA fluorochrome staining, DNA-rRNA hybridization, and PCR techniques. A comparison of the results obtained with these techniques revealed agreement for 95 cell cultures. Discrepant results, which were interpreted as false negative or false positive on the basis of a comparison with the results obtained with other methods, were observed with nine cell cultures. The microbiological culture technique produced false-negative results for four cell cultures. The hybridization technique produced false-negative results for two cell cultures, and for one of these cell cultures the DNA staining technique also produced a false-negative result. The PCR may have produced false-positive results for one cell culture. Ambiguous results were obtained with the remaining two cell cultures. Furthermore, the presence of contaminating bacteria interfered with the interpretation of the DNA staining results for 16 cell cultures. For the same reason the hybridization signals of nine cell cultures could not be interpreted. Our results demonstrate the drawbacks of each of the detection methods and the suitability of the PCR for the detection of mycoplasmas in cell cultures.

FRAR course on laboratory approaches to aging. Microbiological effects and quality control in laboratory rodents.

Numerous viruses, mycoplasmas, bacteria and parasites have been associated with infectious diseases in laboratory animals. It is clear that pathogenic agents causing overt disease represent a serious hazard to research results in both short- as well as long-term studies. However, these organisms may contaminate colonies without causing any clinical or pathological symptom. This makes research less reliable because of the more subtle effects of the silent infections, especially in long-term studies as in aging research. The establishment of animal colonies that were free from these (micro-) organisms has increased substantially the value of animals used in biomedical research. Characterization of the health status and microbiological monitoring of the animals in experiments are particularly important. This paper reviews many of the major considerations in the efforts to maintain animals free of unwanted organisms, including quality and sources of animals, transportation and quarantine, maintenance during experimentation, microbiological characterization and monitoring of animals and environment.

Detection of Mycoplasma pulmonis in experimentally infected laboratory rats by 16S rRNA amplification.

Recently, an rRNA-based polymerase chain reaction (PCR) has been developed for the detection of murine mycoplasmas at both the genus and species level (F. J. M. van Kuppeveld, J. T. M. van der Logt, A. F. Angulo, M. J. van Zoest, W. G. V. Quint, H. G. Niesters, J. M. D. Galama, and W. J. G. Melchers, Appl. Environ. Microbiol. 58:2606-2615, 1992). In this study, the diagnostic value of this PCR assay for the detection of Mycoplasma pulmonis in infected rats was studied. For this purpose, 25 Wistar rats were infected intranasally with M. pulmonis strain M72-138 and investigated for the presence of this pathogen by both in vitro isolation and PCR. Five rats were monitored longitudinally by screening of throat swabs at several time points for up to 248 days postinfection. The remaining 20 rats were killed between 3 and 87 days postinfection, and organism recovery from both throat and urogenital tract specimens was attempted. M. pulmonis could be detected in the throat for up to 248 days postinfection but not in the urogenital tract, either by culture or by PCR. PCR proved to be the optimal method for testing throat samples. All samples in which M. pulmonis was detected by culture were also positive by PCR. By PCR, M. pulmonis was also detected in 3.7% of the samples which were culture negative and in 9.9% of the samples from which cultures were overgrown with bacteria. The results of this study demonstrate the suitability of PCR for the detection of mycoplasmal infection in rodents.

Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification.

Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus- and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Acholeplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, Lactobacillus, Bacillus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.

Antibody-capture enzyme-linked immunosorbent assays that use enzyme-labelled antigen for detection of virus-specific immunoglobulin M, A and G in patients with varicella or herpes zoster.

Antibody-capture enzyme-linked immunosorbent assays (AC-ELISA) which use enzyme-labelled antigen were developed for detection of varicella-zoster virus-(VZV) specific IgM, IgA and IgG antibody in patients with varicella or herpes zoster and in sera from healthy individuals. All 18 patients with varicella developed a VZV-IgM and a VZV-IgG response, 17 also a VZV-IgA response. In contrast, all 19 patients with herpes zoster were shown to be positive for VZV-IgA whereas only 13 of these reacted positively for VZV-IgM. A VZV-IgM response was detected in only two sera from 100 healthy individuals and an IgA response in only one. The presence of virus-specific IgA and IgG in the cerebrospinal fluid as determined by AC-ELISA was a useful indicator of VZV infection of the central nervous system. By AC-ELISA, VZV-IgG was detected predominantly in sera from patients with acute or recent VZV infection. Only 14 sera from 100 healthy individuals were positive for VZV-IgG by AC-ELISA, whereas all were positive by an indirect ELISA. These results indicate that AC-ELISA's may be useful assays for determination for acute or recurrent VZV infection, but are not suitable for determination of past infection with this virus.

Prolonged increase of corticosterone secretion by chronic social stress does not necessarily impair immune functions.

The influence of a chronic social stress upon immunity was investigated in Wistar rats, submitted for four weeks to two different behavioral situations, balanced in a factorial design: housing with three females and membership rotation. The combination of these two factor led to adrenal enlargement (43.3%), thymus involution (39.5%) and increased basal corticosterone levels, all indices of activation of the hypothalamic-hypophysis-adrenal axis. However, neither natural killer cell activity, splenocyte reactivity to mitogen nor the rate of spontaneous development of antibodies against Mycoplasma pulmonis, a common pathogen of the respiratory tract, were changed in the endocrine activated animals. Analysis of the data on kinetics of stress at 1, 7 and 28 days after the initial mixing of the animals gave the same results. These data question the immunosuppressant activity usually conferred to corticosteroids, at least when adrenal hyperactivity is induced by chronic environmental stressors.

Necessity of a more standardized virological characterization of rodents for aging studies.

Characterization of the microbiological status is an important facet of a quality assurance program for laboratory animals. This paper addresses basic issues with regard to standardization of the characterization of murine viral status. Methods for such characterization include clinical signs, virus isolation, and serological tests. Significant considerations are screening profiles; sample collection, processing, and shipment; and sampling schedules. International standardization of programs and methods to control and characterize the microbiological status of laboratory animals is being developed, and will be highly significant in future efforts to produce, control, and maintain laboratory animals free of viral infections.

IgG subclass distribution of primary and secondary immune responses concomitant with viral infection.

Infection with mouse hepatitis virus was found to selectively increase the proportion of IgG2a in antibodies elicited by a concomitant administration of unrelated T cell-dependent protein Ag. In contrast, T cell-independent responses were only marginally affected. This isotypic bias, which occurred when the virus was inoculated shortly before or after a primary immunization, persisted in subsequent secondary responses. However, infection concomitant to secondary antibody responses did not affect their isotypic distribution. These observations suggest that the virus can durably modify unrelated T cell responses that are initiated at the time of infection, which could have implications in the pathogenesis of autoimmune reactions.

Rheumatoid factor production and polyclonal non-antiviral antibody responses elicited by infection with common mouse viruses.

Rheumatoid factor (RF) production and antibody responses directed against antigens unrelated to infectious agents were measured in the serum of 129/Sv mice infected with various viruses. Some of these viruses induced a polyclonal B lymphocyte activation reflected by the presence of serum antibodies reacting in ELISA with dinitrophenylated albumin and with transferrin, but failed significantly to trigger the production of RF capable of agglutinating IgG2a-coated latex particles. In contrast, high RF serum levels were detected only in mice infected with an intestinal agent which was not able to induce other non-antiviral antibody responses. This observation indicates that the strong RF production previously reported in 129/Sv mice is a phenomenon specifically elicited by infection with this particular intestinal agent that cannot be explained by mere polyclonal B lymphocyte activation similar to that induced by common viruses.

In vivo polyclonal B-lymphocyte activation elicited by murine viruses.

Viruses such as lactate dehydrogenase-elevating virus and adenovirus induce in vivo a polyclonal activation of murine B lymphocytes, followed by a marked increase in the production of immunoglobulin G2a (IgG2a). The role of T lymphocytes in this phenomenon was studied by injection of an anti-CD4 monoclonal antibody able to inhibit the T-helper function. This treatment profoundly depressed the production of IgG2a, whereas it had no effect on the proliferation of B cells. Activated B cells obtained from such infected and treated mice remained able to produce various immunoglobulin isotypes after exposure to an appropriate stimulus. In particular, gamma interferon, which is known to be secreted after viral infection, induced the production of IgG2a. These observations support the hypothesis that the influence of viruses on the switch of immunoglobulins is mediated by T-helper lymphocytes.

Diagnosis of herpes simplex virus encephalitis by detection of virus-specific immunoglobulins A and G in serum and cerebrospinal fluid by using an antibody-capture enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was evaluated for detection of intrathecal synthesis of immunoglobulin G (IgG) and IgA antibodies to herpes simplex virus (HSV) in patients with HSV encephalitis (HSVE). Since the antibody-capture principle was used and the assay was carried out at the saturation level of the anti-IgG- or anti-IgA-coated solid phase, correction for blood-brain barrier leakage was not needed. A total of 34 pairs of serum and cerebrospinal fluid specimens obtained from 20 patients with HSVE were examined. Intrathecal synthesis of HSV IgG and IgA was detected from day 7 after the onset of illness in patients with HSVE. Specimens from all 19 patients from whom paired serum and cerebrospinal fluid specimens were obtained at more than 10 days after the onset of illness were positive. Intrathecal synthesis of HSV IgG and IgA was not detected in patients with HSVE before day 7 of illness or in any of the 16 control patients with other causes of (meningo)encephalitis. Use of the antibody-capture enzyme-linked immunosorbent assay for HSV IgG and IgA allows the rapid diagnosis of HSVE during the second week of illness.