Assessment of Microbial Reduction by Cage Washing and Thermal Disinfection using Quantitative Biologic Indicators for Spores, Viruses and Vegetative Bacteria

Cage washing is a key process of the biosecurity program in rodent facilities. For the current study, we developed systems (i. e., magnet attachments, quantitative biologic indicators (Q-BI), and measurement of thermal disinfection at equipment level) to assess the microbial decontamination achieved by a rodent equipment washer with and without thermal disinfection. 99% of the magnets remained in position to hold Q-BI and temperature probes inside cages, water bottles or at equipment level across a cabinet washer chamber with loads dedicated to either housing or drinking devices. Various types of Q-BI for Bacillus atrophaeus, Enterococcus hirae and minute virus of mice were tested. To simulate potential interference from biologic material and animal waste during cage processing, Q-BI contained test soil: bovine serum albumin with or without feces. As a quantitative indicator of microbial decontamination, the reduction factor was calculated by comparing microbial load of processed Q-BI with unprocessed controls. We detected variation between Q-BI types and assessed the washer's ability to reduce microbial load on equipment. Reduction factor results were consistent with the Q-BI type and showed that the washing and thermal disinfection cycle could reduce loads of vegetative bacteria, virus and spore by 5 log10 CFU/TCID50 and beyond. Thermal disinfection was monitored with temperature probes linked to data loggers recording live. We measured the period of exposure to temperatures above 82.2 °C, to calculate A0, the theoretical indicator for microbial lethality by thermal disinfection, and to assess whether the cabinet washer could pass the minimum quality standard of A0 = 600. Temperature curves showed an A0 > 1000 consistently across all processed equipment during thermal disinfection. These data suggest that, when sterilization is not required, a cabinet washer with thermal disinfection could replace an autoclave and reduce environmental and financial waste.

Parvovirus infection in children and pregnant women

Parvovirus (PV) is a widespread infection, despite the fact that this pathogen was discovered only recently. The therapeutic effect of PV, in particular its oncolytic activity, is being actively studied now. Notably, PVs causing infections in animals, such as rat PV H-1, caninae PV, and rodent protoparvovirus (minute virus of mice) suppress oncogenesis in these animals. There is an ex vivo evidence of rat glioblastoma and gliosarcoma sensitivity to PV. The affinity of PV B19 to P-antigen located primarily on the membranes of erythroid cells is crucial for the disease pathogenesis. The teratogenic effect of PV B19 is associated with its ability to infect placental cells (P-antigen is present on the cells of chorionic villi and surface of the trophoblast). PV infection can be acquired or congenital, typical or atypical. The outcome of intrauterine infection with PV B19 largely depends on the gestation age when the infection occurred. Women infected during the second trimester are at higher risk of vertical transmission and severe intrauterine pathology with a poor outcome than those infected during the third trimester. Constant contact with young children significantly increases the risk of PV B19 infection among pregnant women with no immunity to this virus. Serum is the most convenient biomaterial for detecting both PV DNA and virus-specific antibodies. One test for anti-PV IgG using enzyme-linked immunosorbent assay is sufficient to determine the immune status of a patient. Polymerase chain reaction with amniotic fluid is used to diagnose intrauterine infection with PV B19. Blood components and products should be checked for PV B19. High frequency of PV B19 detection in the blood of donors necessitates the development of special measures aimed at prevention of virus transmission. Key words: pregnant women, children, parvovirus B19, parvovirus infection

Non-Invasive Fecal Based PCR Assays for Detection of Mouse Parvo Virus and Minute Virus of Mice in Laboratory Mice

Background: Murine parvoviruses are a few of the most common pathogens of laboratory mice posing potential threats to mice colonies and experiments conducted in mice. We initiated this study to develop the fecal based PCR assays as an alternative to serology and to check its feasibility to detect the infections in mice caused by Mouse Parvo Virus (MPV) and Minute Virus of mice (MVM).Methods: Primers targeting the VP2 gene of MPV and MVM were selected and their sensitivity was analysed in tenfold serially diluted gene template in the presence of negative mouse fecal DNA. Selected thirty-seven mice at the age of 6 to 18 weeks randomly and collected blood samples for serology and fecal samples for PCR assays. Result: PCR assays of MPV and MVM detected as low as 0.4 fg of the target plasmid DNA. PCR assays in fecal samples of mice detected the presence of natural infections of MPV and MVM and their respective prevalence was 24% and 30%. Diagnostic sensitivity of MPV and MVM were 74% and 76% respectively. Our findings indicate that the fecal-PCR assay may be a useful, non-invasive and sensitive diagnostic tool in the ante-mortem detection of MPV and MVM in the health monitoring program.

Binding of CCCTC-Binding Factor (CTCF) to the Minute Virus of Mice Genome Is Important for Proper Processing of Viral P4-Generated Pre-mRNAs

Specific chromatin immunoprecipitation of salt-fractionated infected cell extracts has demonstrated that the CCCTC-binding factor (CTCF), a highly conserved, 11-zinc-finger DNA-binding protein with known roles in cellular and viral genome organization and gene expression, specifically binds the genome of Minute Virus of Mice (MVM). Mutations that diminish binding of CTCF to MVM affect processing of the P4-generated pre-mRNAs. These RNAs are spliced less efficiently to generate the R1 mRNA, and definition of the NS2-specific exon upstream of the small intron is reduced, leading to relatively less R2 and the generation of a novel exon-skipped product. These results suggest a model in which CTCF is required for proper engagement of the spliceosome at the MVM small intron and for the first steps of processing of the P4-generated pre-mRNA.