Visna/maedi virus-induced apoptosis involves the intrinsic mitochondrial pathway.

Visna/maedi virus (VMV) infection in sheep choroid plexus cells was associated with the appearance of apoptosis and the implication of a caspase-dependent mechanism. Sheep choroid plexus cells were mock-infected or infected with VMV to examine the time course of activation of the intrinsic pathway of apoptosis. The role of mitochondria and related apoptotic events were evaluated. A drop in mitochondrial potential was observed following mitochondrial membrane permeabilization using JC-1, a fluorescent probe, which shifted its fluorescence emission from green to red. Apoptosis Inducing Factor translocated to the nucleus of infected-cells and this translocation was concomitant with the release of cytochrome c in the cytosol of infected-cells and mitochondrial membrane permeabilization which seemed to be regulated by the p53 pathway. Following phosphorylated p53 induced downregulation of bcl-2. In addition, DNA flow cytometric analyses revealed a sub-G peak characteristic of an apoptotic population that gradually appeared as virus infection progressed. No cell cycle arrest was detected in infected cells while p21 expression increased. It was concluded that VMV apoptosis is mediated in part by the activation of p53 and the intrinsic mitochondrial apoptotic pathway.

AZT inhibits Visna/maedi virus-induced apoptosis.

Visna/maedi virus (VMV) causes severe encephalitis and a progressive demyelinating disease in sheep. Previous in vitro studies have demonstrated that VMV-infection leads to apoptosis in sheep choroid plexus cells (SCPC) via induction of both intrinsic and extrinsic pathways with subsequent activation of caspases. 3' azido-2',3'-deoxythymidine (AZT) is a potent and selective Human immunodeficiency virus 1 (HIV-1) reverse transcriptase inhibitor, widely used in antiretroviral therapy; however its effects on retrovirus-induced apoptosis are unknown. Using diverse strategies to detect apoptosis, we analysed the broad range effect of AZT treatment on inhibition of VMV-induced apoptosis. First, we found that AZT treatment inhibited the appearance of characteristic apoptotic morphologic changes documented by DAPI staining and oligonucleosomal DNA laddering. Secondly, AZT treatment inhibited caspase cascade and resulted in (i) diminished caspase-3, -8 and -9 activities and (ii) no fluorescein isothiocynate-[VAD]-fluoromethylketone (FITC-VAD-FMK) in situ labelling in VMV-infected cells treated with AZT. Finally, immunocytochemistry indicated that VMV-infection of SCPC induced the subsequent release of apoptosis inducing factor (AIF), whereas AZT treatment inhibited AIF leakage. Consequently, the anti-apoptotic effects of AZT are not restricted, since AZT treatment blocks all the apoptotic pathways induced during VMV-infection.

Implication of caspases during maedi-visna virus-induced apoptosis.

Maedi-visna virus (MVV) causes encephalitis, pneumonia and arthritis in sheep. In vitro, MVV infection and replication lead to strong cytopathic effects characterized by syncytia formation and subsequent cellular lysis. It was demonstrated previously that MVV infection in vitro induces cell death of sheep choroid plexus cells (SCPC) by a mechanism that can be associated with apoptotic cell death. Here, the relative implication of several caspases during acute infection with MVV is investigated by employing diverse in vitro and in situ strategies. It was demonstrated using specific pairs of caspase substrates and inhibitors that, during in vitro infection of SCPC by MVV, the two major pathways of caspase activation (i.e. intrinsic and extrinsic pathways) were stimulated: significant caspase-9 and -8 activities, as well as caspase-3 activity, were detected. To study the role of caspases during MVV infection in vitro, specific, cell-permeable, caspase inhibitors were used. First, these results showed that both z-DEVD-FMK (a potent inhibitor of caspase-3-like activities) and z-VAD-FMK (a broad spectrum caspase inhibitor) inhibit caspase-9, -8 and -3 activities. Second, both irreversible caspase inhibitors, z-DEVD-FMK and z-VAD-FMK, delayed MVV-induced cellular lysis as well as virus growth. Third, during SCPC in vitro infection by MVV, cells were positively stained with FITC-VAD-FMK, a probe that specifically stains cells containing active caspases. In conclusion, these data suggest that MVV infection in vitro induces SCPC cell death by a mechanism that is strongly dependent on active caspases.

Visna virus-induced cytopathic effect in vitro is caused by apoptosis.

Visna-Maedi virus (VMV), an ungulate lentivirus, causes a natural infection in sheep. In vitro, VMV infection and replication lead to strong cytopathic effects with subsequent death of host cells. We investigated, in vitro, the relative contribution of apoptosis or programmed cell death (PCD) to cell killing during acute infection with VMV, by employing diverse strategies to detect its common end-stage alterations. We demonstrated that VMV-infection in sheep choroid plexus cells (SCPC), is associated with apoptosis, characterized by morphological changes such as condensation of chromatin and the appearence of apoptotic bodies. DNA fragmentation was documented by TUNEL assay. Although the mechanism by which VMV activates this cell suicide program is not known, we examined the activation of caspases, the family of death-inducing proteases that resulted in cleavage of several cellular substrates. To study the role of caspases in VMV-induced apoptosis, we focused on several protease targets: procaspase-3 and procaspase-1. During VMV-infection, SCPC display active caspase-3 and no caspase-1 activity. In conclusion, our results suggest that VMV infection, in vitro, induces cell death of SCPC by a mechanism that can be characterized by many of the properties most closely associated with apoptotic cell death.

Sedimentation field-flow fractionation device cleaning, decontamination and sterilization procedures for cellular analysis.

In Sedimentation FFF (SdFFF) practice, it is known that a large number of cell elutions create aging phenomena of the separator, thereby reducing recovery and modifying elution characteristics. Systematic cleaning procedures are developed to enhance channel lifetime, together with microbial decontamination processes. Cells can be therefore reproducibly eluted for a large number of analyses and collected under sterile conditions, if needed. This is one of the most valuable aspect if further culture or transplantation is required. Decontamination was performed using, as contaminant probe, Staphylococcus aureus, highly adherent pathogenic bacteria that eluted from SdFFF as aggregates.

Xenobiotic-metabolizing enzymes in carp (Cyprinus carpio) liver, spleen, and head kidney following experimental Listeria monocytogenes infection.

Infection of carp with Listeria monocytogenes 4b resulted in decreased liver, spleen, and head kidney enzyme activities, involved in the metabolism of xenobiotics. After infection, cytochrome P-450 levels and ethoxyresorufin O-deethylase (EROD) activity were decreased while conjugation enzymes remained unaffected. The maximum decrease for phase I enzymes occurred on d 3. This loss of monooxygenase levels and activity could not be directly correlated with an increase in the number of organisms, as consistently high bacterial counts were observed in all three organs during infection. The effect of L. monocytogenes infection was also measured in carp exposed to 3-methylcholanthrene (MCA). Cytochrome P-450 levels and EROD activity were significantly reduced, especially on d 3. A significant decreased activity of conjugation enzymes such as glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT) was also observed for all days studied. Listeria infection inhibited MCA-induced increases in xenobiotic-metabolizing enzyme activities. These results indicate that infection may have deleterious effects on basal cytochrome P-450 monooxygenase levels. Furthermore, MCA treatment aggravates the insult to xenobiotic biotransformation enzymes by L. monocytogenes infection, by impairing a number of detoxification enzymes. These findings could result in significant changes in the susceptibility of fish to pollutants.

Comparative virulence between different strains of Listeria in zebrafish (Brachydanio rerio) and mice.

Listeriosis is a disease found in most animal species but is relatively uncommon in fish. We studied the relationship between Listeria and zebrafish by injecting Brachydanio rerio intraperitoneally with different Listeria strains having pathological or food-stuff origins. We then compared these results with those obtained in Swiss mice. Experimental Listeriosis in Zebrafish differs greatly from that observed in mice. The 50% lethal dose (LD50) previously determined was much higher than that observed in mice. In fish, a good correlation exists between infection found in renal tissue, an important lymphoïd organ and that present in whole fish (p < 0.001). Infection kinetics showed that, in contrast with mice, L. monocytogenes was unable to multiply in fish. Differential blood counts showed the development of an immune response in fish. The difference in the expression of Listeria virulence between Zebrafish and mice was also seen in their reactions to different wild strains inoculate i.p. Strains belonging the innocua, ivanovii, seeligeri and welshimeri were weakly or not virulent in mice but virulent in fish. Nevertheless, as in mice, differences in virulence existed between strains of L. monocytogenes belonging to serovars 4b, 1/2a, 1/2b and 1/2c.

Copper and zinc exposure of zebrafish, Brachydanio rerio (Hamilton-Buchaman): effects in experimental Listeria infection.

To investigate the effects of heavy metals on susceptibility of fish to Listeriosis, normal zebrafish, Brachydanio rerio (Hamilton-Buchanan), were exposed to varying concentrations of zinc (0.05, 0.15, and 0.25 mg/liter) and copper (0.05, 0.10, and 0.15 mg/liter). During copper exposure, this heavy metal did not accumulate in zebrafish kidney. Unlike copper, a small amount of zinc accumulated in kidneys of fish exposed at 0.25 mg/liter. To estimate the effects of this heavy metal on listerial infection, the mortality of fish and the number of viable bacteria in fish kidney were determined at various times (1, 4, 7, and 10 days) after ip challenge with Listeria monocytogenes (strain 31386, serotype 4b). The results indicate that the number of colony-forming units in zinc-exposed fish decreased at 4, 7, and 10 days after challenge with 0.2 x LD50 of viable bacteria. In contrast, copper-exposed fish indicated both decreases and increases in the number of colony-forming units depending on the concentration of L. monocytogenes used.

[Virulence of Listeria monocytogenes in pregnant mice]. / Virulence de Listeria monocytogenes sur des souris gestantes.

Serious food-borne outbreaks of listeriosis have been reported in North America and in Europe, during the past decade. The predominant risk groups appear to be immunocompromised adults, elderly people, newborn babies and pregnant women. In order to examine the relationship between alimentation, listeriosis and pregnant females, we developed an experimental model using Swiss mice fed ad libitum during 4 days with pellets containing a high concentration of Listeria monocytogenes serovar 4b (10(9) u.f.c/g). Samples were taken from many series of pregnant mice which had been infected respectively by L. monocytogenes from 6th, 10th, 14th and 18th day of pregnancy onwards. This was compared to non infected control series. The transmission of infection from mother to progeny and contamination of surviving progeny were evaluated by Listeria numeration in liver, brain and intestines. Females infected between day 6 and day 10, and between day 10 and day 14 after fertilization, aborted or died of encephalitis. Mice contaminated between day 14 and day 18, were the least prone to experimental listeriosis. On the other hand, some mice contaminated between day 18 and day 22, i.e. at the end of their pregnancy, may develop encephalitis a few days after parturition of a healthy litter. Series contaminated between day 6 and day 10, and between day 10 and day 14 turned out to be highly sensitive to the transmission of infection from mother to young. In two other series (day 14--day 18; day 18--day 22), the young mice contained generally no Listeria. Our experimental model shows the relationship between listeriosis and alimentation. In pregnant mice, sensitivity to infection depends on their gestational status with large individual variability.

Animal lentivirus replication and reverse transcriptase inhibitors.

We summarize the pathogenesis of animal lentiviruses (visna-maedi virus, caprine arthritis and encephalitis virus, and equine infectious anemia virus), which have raised considerable interest since the discovery of human lentiviruses. The human lentiviruses possess structural, genetic, and clinical properties similar to those of animal lentiviruses. We describe the different mechanisms of and the principal work on reverse transcriptase inhibitors of animal lentiviruses, such as HPA-23, phosphonoformate, or 2',3'-dideoxynucleosides. Animal viruses of this family may serve as models for infection with human lentiviruses such as human immunodeficiency virus, the etiologic agent of AIDS.

[Detection of visna-maedi virus antibodies and antigens by immunoblotting]. / Détection d'anticorps et d'antigènes du virus de Visna-Maedi par immunotransfert.

The visna-maedi virus was immunologically diagnosed using an immunoblotting technique from an antigenic preparation of the visna-maedi virus K796 purified by sucrose density gradient centrifugation. After SDS-PAGE electrophoresis and transfer onto a nitrocellulose sheet, the immunoblotting procedure was adapted to the search for specific antibodies in ovine serum samples. The results obtained showed that, in the natural visna-maedi disease, antibodies are not systematically detectable against every viral protein of the virus core. We have demonstrated the existence of antibodies directed against the proteins coded by the gag and the pol genes.

[Trial of pentamidine in the treatment of trypanosomiasis of sheep inoculated with Trypanosoma brucei brucei]. / Essai de la pentamidine dans le traitement de la trypanosomiase du mouton inoculé par Trypanosoma brucei brucei.

An experimental study was carried out using 5 Trypanosoma brucei brucei subcutaneously infected sheep. Pentamidine treatment appeared in the CSF. Two animals were injected daily for 10 days and they died with drug intoxication. Two animals received drug every 3 days for 15 days: one of them died, the other was cured after 3 weeks of set-back. This preliminary study should be completed by experimentation made over a more important period of time.