Comparison of ELISA with shell vial cell culture method for the detection of human rotavirus in fecal specimens

The aim of the study was to compare an enzyme immunoassay method with shell vial cell culture method for detection of rotavirus in fecal specimens. In addition, the correlation between laboratory results and clinical scores of patients with gastroenteritis was evaluated. A total of 219 fecal specimens from children (ages 3 weeks to 5 years) with acute gastroenteritis submitted to pediatric emergency room were evaluated by both ELISA and shell vial cell culture. A Vesikari score was used for assessing the severity of the illness. Among 219 stool samples tested, 107 (48.9%) were determined to be positive. Two specimens were positive by shell vial cell culture method while they were ELISA negative. According to these results the calculated sensitivity, specificity, PPV, and NPV of ELISA were 98.1%, 100%, 100%, and 98.2%, respectively. The mean severity score for the 107 episodes of rotavirus diarrhoea was 11.0 +/- 3.6 compared to 4.5 +/- 1.9 for the 112 episodes of non-rotavirus diarrhea in the same population. Our study indicates that ELISA, which is easier to perform, faster and cheaper than cell culture methods may be suitable for routine diagnosis of rotavirus infections. The severity of rotavirus positive gastroenteritis was significantly higher than that of rotavirus negative patients.     

Mycotoxins in laboratory rodent feed

Twenty-one batches of fixed-formula rodent diets from three feed manufacturers were tested for the presence of five mycotoxins: deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin, T-2 toxin and ochratoxin A (OTA). Five batches were also tested for the presence of zearalenone (ZEN) and six batches for aflatoxins. Detectable levels of DON (up to 298 microg/kg), NIV (up to 118 microg/kg), OTA (up to 3.1 microg/kg) or ZEN (up to 26.7 microg/kg) were found in samples from all manufacturers. Three batches contained two (DON or NIV and OTA or ZEN) and one batch contained three (DON, OTA and ZEN) different mycotoxins. Aflatoxins, T-2 and HT-2 were not detected in any of the batches. The concentrations of mycotoxins detected in the feed were low, but indicated that feed ingredients, probably the cereal ingredients, were contaminated by mycotoxins. Since mycotoxins are known to have toxic and/or immunosuppressive effects, non-contaminated ingredients should be used for production of laboratory animal feed. The results imply that an improved quality control of ingredients used for laboratory rodent feed should be implemented.     

Identification and sequencing of a novel rodent gammaherpesvirus that establishes acute and latent infection in laboratory mice

Gammaherpesviruses encode numerous immunomodulatory molecules that contribute to their ability to evade the host immune response and establish persistent, lifelong infections. As the human gammaherpesviruses are strictly species specific, small animal models of gammaherpesvirus infection, such as murine gammaherpesvirus 68 (γHV68) infection, are important for studying the roles of gammaherpesvirus immune evasion genes in in vivo infection and pathogenesis. We report here the genome sequence and characterization of a novel rodent gammaherpesvirus, designated rodent herpesvirus Peru (RHVP), that shares conserved genes and genome organization with γHV68 and the primate gammaherpesviruses but is phylogenetically distinct from γHV68. RHVP establishes acute and latent infection in laboratory mice. Additionally, RHVP contains multiple open reading frames (ORFs) not present in γHV68 that have sequence similarity to primate gammaherpesvirus immunomodulatory genes or cellular genes. These include ORFs with similarity to major histocompatibility complex class I (MHC-I), C-type lectins, and the mouse mammary tumor virus and herpesvirus saimiri superantigens. As these ORFs may function as immunomodulatory or virulence factors, RHVP presents new opportunities for the study of mechanisms of immune evasion by gammaherpesviruses.     

Global harmonization of laboratory rodent health surveillance standards

The authors discuss the need for international standards for the microbiologic and genetic quality of rodents.     

Prevalence of antibodies to Sendai virus and rotavirus in laboratory rabbits

One hundred and sixty rabbit sera from 10 breeding colonies and 13 laboratory colonies were tested for antibodies to Sendai virus and rotavirus by enzyme-linked immunosorbent assay (ELISA). Antibodies were detected to Sendai virus in 53% and to rotavirus in 81%, indicating the prevalence of these viral infections in laboratory rabbit colonies.     

Evaluation of a latex agglutination test for rapid detection of rotavirus in faecal specimens

Two methods for detecting rotaviruses (latex agglutination, electron microscopy) have been compared on 80 faecal samples. These samples were obtained from infants between the age of four and 30 months hospitalized for acute gastroenteritis in the Children's Hospital, Karl Marx University at Leipzig, in 1982. Complete agreement among the two techniques was found in 75 specimens. Sensitivity of latex agglutination could be estimated at 95%, the specificity also at 95%. Only one sample reacted nonspecifically. Performance of the latex agglutination proved quite simple. The results indicate that latex agglutination is suitable for rapid screening of rotavirus induced gastroenteritis in clinical practice thus enabling the rate of nosocomial rotavirus infections in children's hospitals to be reduced.     

Breeding of wild-caught rodent cricetidae Holochilus brasiliensis under laboratory conditions

The breeding of wild-caught rodent Holochilus brasiliensis (Desmarest, 1819) was studied under laboratory conditions. The mean gestation length was 28.4 days. Litter size ranged from one to five. Males matured at 2-3 months and females at 2-4.5 months of age. The oestrous cycle lasted 6-8 days. Eleven pairs observed over 6-13 months increased to a population of 276 individuals (153 males and 123 females).     

Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome l-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using β-galactosidase/ ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. the described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.     

Identification of streptococci in a medical laboratory

A total of 965 cultures of streptococci received at a reference unit for identification were examined with API-20 Strep kits and also by established methods. The API method, although it needed to be supplemented with additional tests, largely overcame the difficulty that pyogenic streptococci are usually identified by their serological reactions and that biochemical tests are used for the identification of the other streptococci. Representatives of at least 24 established or possible species were identified.     

Identification of streptococci in a medical laboratory

A total of 965 cultures of streptococci received at a reference unit for identification were examined with API-20 Strep kits and also by established methods. The API method, although it needed to be supplemented with additional tests, largely overcame the difficulty that pyogenic streptococci are usually identified by their serological reactions and that biochemical tests are used for the identification of the other streptococci. Representatives of at least 24 established or possible species were identified.     

Identification of neuropeptide FF-related peptides in rodent spinal cord.

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in mouse and rat spinal cord, by using reverse phase high pressure liquid chromatography with radioimmunoassay and electrospray mass spectrometry detection. In both species, two octapeptides, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPSF (Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were identified but a longer peptide NPA-NPFF (Asn-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was present at the highest concentration in rat spinal cord. In mouse, the homologous peptide, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was not detected. Both peptides NPFF and NPSF reverse morphine-induced analgesia in the tail flick test. Our data reveal species differences in the maturation of NPFF precursor.     

Identification of human hsp-70 expression in stably transfected rodent cells by isoelectrofocus immunoblots.

The major heat-shock protein, hsp-70, is synthesized by cells from a wide variety of organisms in response to heat shock or other stresses. It is assumed that hsp-70 may have an important thermal protective function. To test this hypothesis directly, we have transfected rat fibroblast cells with appropriate expression plasmids containing a cloned human hsp-70 gene. Stable transfectants expressing the human hsp-70 gene product were identified by Western blot analysis. During the course of selecting successful transfectants, we found that when standard methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunostaining were employed with the monoclonal antibody raised against the human hsp-70 antigen, we were unable to differentiate the human hsp-70 from the heat-inducible rat hsp-70. This was because the monoclonal antibody cross-reacts with the human and rat proteins, which have the same mobility in SDS-PAGE, and it is difficult to determine which protein is expressed. To improve the resolution of the Western blot technique, we performed additional immunoblot analysis of cellular proteins separated by slab gel isoelectrofocusing. Our study shows that the isoelectrofocusing technique, when combined with antibodies against hsp-70, gave a better resolution for the separation of exogenous human hsp-70 and the endogenous rat hsp-70 than the commonly used SDS-PAGE Western blot analysis. It provides a rapid and specific method to identify positive colonies that express the human hsp-70 gene in transfected rodent cells.     

Identification of clinical,epidemiological and laboratory risk factors for leprosy reactions during and after multidrug therapy

This cross-sectional retrospective study evaluated 440 leprosy patients; 57% (251/440) had leprosy reactions during and/or after multidrug therapy, 80.5% (202/251) of whom presented with multibacillary leprosy. At diagnosis, positive bacterial index (BI) [odds ratio (OR) = 6.39; 95% confidence interval (CI): 4.1-10.1)] or polymerase chain reaction (PCR) (OR = 9.15; 95% CI: 5.4-15.5) in skin smears, anti-phenolic glycolipid-1 (anti-PGL-1) ELISA (OR = 4.77; 95% CI: 2.9-7.9), leucocytosis (OR = 9.97; 95% CI: 3.9-25.7), thrombocytopenia (OR = 5.72; 95% CI: 2.3-14.0) and elevated lactate dehydrogenase (OR = 2.38; 95% CI: 1.4-4.0) were potential markers for the development of reactions during treatment. After treatment, positive BI (OR = 8.47; 95% CI: 4.7-15.3) and PCR (OR = 6.46; 95% CI: 3.4-12.3) in skin smears, anti-PGL-1 ELISA (OR = 2.25; 95% CI: 1.3-3.9), anaemia (OR = 2.36; 95% CI: 1.2-4.5), leucocytosis (OR = 4.14; 95% CI: 1.5-11.6) and thrombocytopenia (OR = 3.70; 95% CI: 1.3-2.2) were risk factors for the occurrence of reactions during the study period. The identification of groups with an increased risk for developing reactions will allow for the timely development of a treatment plan to prevent nerve damage and, therefore, the appearance of the disabling sequelae associated with the stigma of leprosy.     

The status and outlook of the laboratory diagnosis of rotavirus infection]

Rotavirus infection is not only the most distributed disease of children but a widely spread cause of their death. Nowadays considerable methodical potential which permits effectively detecting the whole virions, group A, antigens of human rotaviruses, virus-specific RNA in the clinical material is accumulated. There is a tendency to an increase of sensitivity and expressivity of the methods based on the use of immunological principles for detection and calculation of the antigen-antibody complex. Molecular-biological methods of the study are developed, which is important for the theory and practice of this infection control.     

Strain differences in avoidance reactions of septal laboratory rats

The effect of a lesion of the dorsal septum on active and passive type of avoidance reactions of adult male Wistar (W) and Long-Evans (LE) rats was studied. The rate of acquisition and extinction of the reaction was studied by 3 different testing methods. The animals were operated on when juvenile (30 days) or adult (90 days). The experiments were always started 50 days after the operation, when the "rage syndrome" was no longer present. In the three different tests we found a single common variable--the strain factor. It was this that determined whether the operation was effective, as well as the direction of deviations and the operation age which led to manifest changes. In W males the lesion did not impair either the acquisition or the extinction of an active avoidance reaction (AAR); the passive avoidance reaction (PAR) was acquired and extinguished more slowly after a lesion in adulthood. The spontaneous passive avoidance reaction (i.e. preference of a small, dark space) was likewise negatively affected by the operation. In intact LE animals the AAR was extinguished more slowly than in intact W males; after a lesion produced in juvenile or adult age extinction was speeded up, so that there were no differences compared either with intact or with septal W animals. Intact LE rats also acquired a PAR more slowly than W rats; a septal lesion led to faster acquisition, irrespective of the age at which the operation was performed, so that the rate for septal LE rats were the same as for intact W animals. The extinction of this reaction took longer after an operation at juvenile age and the rate for these septal LE rats were the same as for those of septal W individuals operated on at 90 days. The spontaneous PAR was qualitatively poorer in intact LE animals than in intact W rats, since only 40% of them preferred the small, dark space. A lesion was followed by improvement, which was especially marked after operation on the 30th day, when all the animals preferred this space, and in a very short time, so that they were equal to intact W males. Correlation of the acquisition and the extinction rate showed that there was imbalance of excitation and inhibition processes in the AAR of the W controls, that they were balanced in the PAR and that the lesion reversed these relationships. Both processes were balanced in the LE control and a septal lesion did not alter the situation.     

Identification and verification of novel rodent postsynaptic density proteins

The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission.