Abdeljelil Ghram

Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. A review.

Abstract In recent years there has been enormous interest in researching oxidative stress. Reactive oxygen species (ROS) are derived from the metabolism of oxygen as by-products of cell respiration, and are continuously produced in all aerobic organisms. Oxidative stress occurs as a consequence of an imbalance between ROS production and the available antioxidant defence against them. Nowadays, a variety of diseases and degenerative processes such as cancer, Alzheimer’s and autoimmune diseases are mediated by oxidative stress. Heat stress was suggested to be an environmental factor responsible for stimulating ROS production because of similarities in responses observed following heat stress compared with that occurring following exposure to oxidative stress. This manuscript describes the main mitochondrial sources of ROS and the antioxidant defences involved to prevent oxidative damage in all the mitochondrial compartments. It also deals with discussions concerning the cytotoxic effect of heat stress, mitochondrial heat-induced alterations, as well as heat shock protein (HSP) expression as a defence mechanism.

First serological investigation of peste-des-petits-ruminants and Rift Valley fever in Tunisia.

This study, carried out between September 2006 and January 2007, is the first cross-sectional serological investigation of peste-des-petits-ruminants (PPR) and Rift Valley fever (RVF) in Tunisia. The objective was to assess the potential need to develop a dual, recombinant PPR-RVF vaccine and how such a vaccine might be utilised in Tunisia. An overall PPR seroprevalence of 7.45% was determined, a finding supported by the high specificity (99.4%) and sensitivity (94.5%) of the ELISA used. On assessment of the diversity and density of mosquitoes in the sampling area, four species of RVF-vectors of the genus Aedes and Culex were identified. However, no serological evidence of RVF was found despite the use of a highly sensitive ELISA (99-100%). Larger scale investigations are underway to confirm these findings and the continuation of the emergency vaccination program against these two diseases remains valid.

Characterization of New Variants of Avian Infectious Bronchitis Virus in Tunisia

Abstract Three infectious bronchitis virus (IBV) strains, isolated from suspected Tunisian broiler flocks, were characterized as variant viruses using genotyping and serotyping techniques. They were compared with commonly used vaccine strains, including 793/B, D274, and Massachusetts types. Reverse transcription-PCR-restriction fragment length polymorphism, nucleotide sequencing, and GenBank BLAST database analyses of the hypervariable region of the S1 subunit of the virus spike gene showed that the three isolates, designated TN20/00, TN200/01, and TN335/01, share from 64% to 82% homologies between each other but are very different from the H120 strain, the only infectious bronchitis vaccine used in Tunisia. In addition, they showed from 57% to 78% similarities with the European genotypes, including D274 and 793/B. Phylogenetic data allowed classification of the three Tunisian isolates as new genotypes placed inside the same genetic group as the CR88121 and D274 genotypes but very distant from the Massachusetts genotype. Cross-virus neutralization tests confirmed the genotyping results and showed that both TN200/01 and TN335/01 isolates are serologically related, whereas the TN20/00 is closer to TN335/01 than to TN200/01. Moreover, all three Tunisian isolates are closely related to the European variant serotypes, including the CR88121 and the D274 strains, but none is serologically related to the H120 vaccine strain. These data demonstrated, for the first time in Tunisia, the cocirculation of IBV variant serotypes along with the Massachusetts type, causing severe clinical diseases and high economic losses to the poultry industry.

Risk Factors and Characteristics of Low Pathogenic Avian Influenza Virus Isolated from Commercial Poultry in Tunisia

Objective Estimate the seroprevalence of influenza A virus in various commercial poultry farms and evaluate specific risk factors as well as analyze their genetic nature using molecular assays. Materials and Methods This report summarizes the findings of a national survey realized from October 2010 to May 2011 on 800 flocks in 20 governorates. Serum samples were screened for the presence of specific influenza virus antibodies using cELISA test. Additionally, swab samples were tested by real time and conventional RT-PCR and compared with results obtained by others assays. Phylogenetic and genetic analyses of the glycoproteins were established for some strains. Results Out of the 800 chicken and turkey flocks tested by cELISA, 223 showed positive anti-NP antibodies (28.7%, 95% CI: 25.6–32.1). Significantly higher seroprevalence was found among the coastal areas compared to inland and during the autumn and winter. Broiler flocks showed significantly lower seroprevalence than layers and broiler breeders. The influenza virus infection prevalence increased after the laying phase among layer flocks. In addition, AIV seropositivity was significantly associated with low biosecurity measures. The Ag EIA and rRT-PCR tests revealed significantly higher numbers of AI positive samples as compared to cell cultures or egg inoculation. All new strains were subtyped as H9N2 by real time and conventional RT-PCR. Drift mutations, addition or deletion of glycosylation sites were likely to have occurred in the HA and NA glycoproteins of Tunisian strains resulting in multiple new amino acid substitutions. This fact may reflect different evolutionary pressures affecting these glycoproteins. The role of these newly detected substitutions should be tested. Conclusion Our findings highlight the potential risk of AIV to avian health. Strict enforcement of biosecurity measures and possible vaccination of all poultry flocks with continuous monitoring of poultry stations may ensure reduction of AIV prevalence and avoid emergence of more pathogenic strains.

S1 gene sequence analysis of new variant isolates of avian infectious bronchitis virus in Tunisia

Purpose Tissue samples were collected from suspected broiler flocks showing respiratory signs to identify infectious bronchitis virus (IBV), characterize emerging field strains, and study their relationships with the Massachusetts H120 strain, the only IB vaccine used in Tunisia. Samples and methods Several IBV isolates were identified from field samples collected from flocks located in different regions in the northeast of Tunisia. The IBV isolates were characterized and compared to commonly used vaccine strains (including 793B, D274, and H120 types), other reference IBV strains from Europe, and the recently characterized Tunisian field variants TN20/00, TN200/01, and TN335/01. Reverse transcription-polymerase chain reaction and nucleotide sequencing analyses of the hypervariable regions of the S1 gene were carried out. Results Four new IBV variants were isolated during the period 2007-10 and were designated TN295/07, TN296/07, TN556/07, and TN557/07. The amino acid sequence data showed 100% similarity between TN295/07 and TN296/07, suggesting that these two isolates are identical and belong to the same genotype. Similar results were demonstrated for TN556/07 and TN557/07. Sequence identity values indicated that TN296/07 and TN556/07 share 55% amino acid homologies between each other, but are very different from the reference IBV serotypes, in particular the H120 strain. It was also shown that they have 50%-77% similarities with the Tunisian virus isolated between 2000 and 2001. Phylogenetic clustering allowed classification of these Tunisian isolates as new genotypes that are closer to TN200/01, TN335/01 Tunisian field variants, and Italy02 variant than MassH120 vaccine strain. Conclusion S1 sequence analyses confirmed the cocirculation of H120 vaccine strain with novel IBV variants isolated from Tunisian field.

Non-cytotoxic Thymus capitata extracts prevent Bovine herpesvirus-1 infection in cell cultures.

BackgroundBovine herpes virus type 1 (BHV-1) still causes great economic loss to the livestock industry and trade because there aren’t any available drugs that proved to be fully effective against it. In this study, the cytotoxicity and the antiviral activities of the Thymus capitata extracts were evaluated for the development of new, non toxic and specific anti-herpesvirus drug. Aqueous extracts (AE), ethanolic extracts (EE) and essential oil (EO) of the aerial parts of Thymus capitata were analyzed to determine their chemical compositions by gas chromatography, and high performance liquid chromatography combined with mass spectrometry. Their cytotoxicity and antiviral activities against Bovine Herpesvirus type 1 (BHV-1) were evaluated by quantifying the reduction of the viral cytopathic effect using Madin-Darby Bovine Kidney cell line with colorimetric assay. T. capitata extracts were added at different stages of the viral infection to investigate and better quantify their potential inhibitory effects.ResultsPolyphenols and flavonoids were the major compounds found in T. capitata EO, EE and AE. The cytotoxic concentrations at 50% were 48.70, 189 and 289 μg ml−1 for EO, EE and AE, respectively. The inhibitor concentrations at 50% for the EO, EE and AE, were 3.36, 47.80 and 164 μg ml−1, respectively. The selectivity index anti-BHV-1 values were 14.49, 3.95 and 1.81 for EO, EE and AE, respectively. Thus, the EO extracts were the most efficient antiviral compounds. T. capitata extracts affect mainly the adsorption of BHV-1 virus to host cells.ConclusionT. capitata extracts inhibit the viral replication by interfering with the early stages of viral adsorption and replication. Thus, T. capitata is a potential candidate for anti-herpesvirus treatment.

Efficiency of refinery sludge biodegradation using municipal wastewater and activated sludge and effect of hydrocarbon concentration on culturable bacteria community

The efficiency of activated sludge as inoculum and municipal wastewater as diluent for the biodegradation of refinery sludge was investigated. At a laboratory scale, biodegradation experiments carried out in an aerobic batch reactor showed that toxic oily sludge was biodegradable after dilution. Compared with freshwater, the use of municipal wastewater for dilution clearly enhanced the biodegradation of this toxic pollutant. Using municipal wastewater, experiments showed that, starting from an initial total hydrocarbon concentration (THC) of 750 mg L−1, we could reach a final THC of 5 mg L−1 and a COD removal of 97% in only 19 days. In addition, we found that bacterial dominance was affected by hydrocarbon load. An absolute dominance of Gram-positive strains with high hydrocarbon load (7500 mg L−1) was found. However, with a hydrocarbon load of 750 mg L−1, some Gram-negative strains were also active. This bacterial dominance also changed in the course of treatment. We conclude that municipal wastewater and activated sludge, less often used nowadays in oily sludge biodegradation processes, contained bacterial strains that are efficient and able to work in a community. Municipal wastewater is also strongly recommended as a diluent as it offers an effective low-cost solution.

Accurate Detection of Avian Respiratory Viruses by Use of Multiplex PCR-Based Luminex Suspension Microarray Assay

ABSTRACT A novel oligonucleotide suspension microarray (Luminex microsphere system) was developed for the rapid detection of avian respiratory viruses of major clinical importance. This test was optimized and validated with 70 clinical samples. The developed tool was accurate for high-throughput detection and differentiation of the most important avian respiratory viruses: avian influenza virus (AIV), Newcastle disease virus (NDV), infection bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV) in single- and mixed-virus infections. A multiplex reverse transcriptase PCR (RT-PCR), followed by a monoplex or a multiplex Luminex assays, were realized using a Luminex 200 analyzer instrument. The sensitivity, specificity, and reproducibility of the multiplex DNA suspension microarray system were evaluated. The results showed no significant differences in the median fluorescence intensity (MFI) value in monoplex and multiplex Luminex assays. The sensitivity and specificity proved to be completely concordant with monoplex real-time RT-PCR. We demonstrated that the multiplex DNA suspension microarray system is an accurate, high-throughput, and relatively simple method for the rapid detection of the main respiratory viruses of poultry.

Simultaneous alteration of residues 279 and 284 of the VP2 major capsid protein of a very virulent Infectious Bursal Disease Virus (vvIBDV) strain did not lead to attenuation in chickens

BackgroundCell culture adaptation of very virulent infectious bursal disease virus (vvIBDV) was shown to be mainly associated with the VP2 capsid protein residues 253, 279, and 284. The single mutation A284T proved critical for cell culture tropism, but did not confer efficient virus replication, which at least required one additional mutation, Q253H or D279N. While the double mutation Q253H/A284T was unambiguously shown to confer both efficient replication in cell culture and attenuation in chickens, conflicting results have been reported regarding the replication efficiency of vvIBDV mutants bearing the D279N/A284T double mutation, and no data are hitherto available on their virulence in chickens.FindingsHere we used an in vivo reverse genetics system to assess the impact of the D279N/A284T double mutation on the replication and attenuation of a chimeric IBDV virus, whose polyprotein derived from a non-culturable vvIBDV clinical isolate. We found that the D279N/A284T double mutation did indeed confer efficient replication in chicken embryo fibroblast (CEF) cell culture, but the mutant virus remained highly pathogenic to chickens.ConclusionsThe double mutation D279N/A284T of the VP2 major capsid protein of vvIBDV is sufficient to confer cell culture tropism and replication efficiency, but does not necessarily lead to virus attenuation.

Molecular characterization of rabies virus isolated from dogs in Tunisia: Evidence of two phylogenetic variants

In an attempt to explain temporal and geographical rabies incidence fluctuations in Tunisia, a molecular epidemiological study of rabies virus (RV) was carried out. A panel of RV isolates from dogs, collected between 1992 and 2003, from different regions in Tunisia have been analysed by direct sequencing of PCR-amplified products coding for the nucleoprotein gene. New sequences have been compared to prototype sequences of Lyssavirus species and nine lineages of species 1. All Tunisian isolates belonged to species rabies virus and segregated into two rabies lineages geographically distinct: NCS lineage characterizing Northeast, Central and Northern areas of the country and NW lineage more restricted to the North-Western regions. Phylogenetic analyses showed that Tunisian RV clustered most closely to Africa 1a lineage: NCS lineage showed nucleic affiliation with isolates from Algeria and Morocco, whereas, NW lineage shared a strong relationship with Ethiopian and Sudanese strains.