Mohamed Barkallah

Survey of Infectious Etiologies of Bovine Abortion during Mid- to Late Gestation in Dairy Herds

Bovine abortion of unknown infectious etiology still remains a major economic problem. Thus, we investigated whether Brucella spp., Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Coxiella burnetii are associated with abortion and/or stillbirth in Tunisian dairy cattle. Using a pan-Chlamydiales PCR, we also investigated the role of Chlamydiaceae, Waddlia chondrophila, Parachlamydia acanthamoebae and other members of the Chlamydiales order in this setting. Veterinary samples taken from mid to late-term abortions from twenty dairy herds were tested. From a total of 150 abortion cases collected, infectious agents were detected by PCR in 73 (48.66%) cases, 13 (8.66%) of which represented co-infections with two infectious agents. Detected pathogens include Brucella spp (31.3%), Chlamydiaceae (4.66%), Waddlia chondrophila (8%), Parachlamydia acanthamoebae (5.33%), Listeria monocytogenes (4.66%) and Salmonella spp. (3.33%). In contrast, Campylobacter spp. and Coxiella burnetii DNA were not detected among the investigated veterinary samples. This demonstrates that different bacterial agents may cause bovine abortion in Tunisia. This is the first report suggesting the role of Parachlamydia acanthamoebae in bovine abortion in Africa. Further studies with a larger number of samples are necessary to confirm whether this emerging pathogen is directly linked to abortion in cattle.

Genetic diversity of food-isolated Salmonella strains through Pulsed Field Gel Electrophoresis (PFGE) and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR).

All over the world, the incidence of Salmonella spp contamination on different food sources like broilers, clams and cow milk has increased rapidly in recent years. The multifaceted properties of Salomnella serovars allow the microorganism to grow and multiply in various food matrices, even under adverse conditions. Therefore, methods are needed to detect and trace this pathogen along the entire food supply network. In the present work, PFGE and ERIC-PCR were used to subtype 45 Salmonella isolates belonging to different serovars and derived from different food origins. Among these isolates, S. Enteritidis and S. Kentucky were found to be the most predominant serovars. The Discrimination Index obtained by ERIC-PCR (0.85) was slightly below the acceptable confidence value. The best discriminatory ability was observed when PFGE typing method was used alone (DI = 0.94) or combined with ERIC-PCR (DI = 0.93). A wide variety of profiles was observed between the different serovars using PFGE or/and ERIC-PCR. This diversity is particularly important when the sample origins are varied and even within the same sampling origin.

First detection of Waddlia chondrophila in Africa using SYBR Green real-time PCR on veterinary samples.

Waddlia chondrophila is a strict intracellular microorganism belonging to the order Chlamydiales that has been isolated twice from aborted bovine fetuses, once in USA and once in Germany. This bacterium is now considered as an abortigenic agent in cattle. However, no information is available regarding the presence of this bacterium in Africa. Given the low sensitivity of cell culture to recover such an obligate intracellular bacterium, molecular-based diagnostic approaches are warranted. This report describes the development of a quantitative SYBR Green real-time PCR assay targeting the recA gene of W. chondrophila. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing pathogens that can cause abortion in cattle. The PCR exhibited a good intra-run and inter-run reproducibility. This real-time PCR was then applied to 150 vaginal swabs taken from Tunisian cows that have aborted. Twelve samples revealed to be Waddlia positive, suggesting a possible role of this bacterium in this setting. This new real-time PCR assay represents a diagnostic tool that may be used to further study the prevalence of Waddlia infection.

Endocrine disrupting potential and reproductive dysfunction in male mice exposed to deltamethrin

Pesticide exposure may affect semen quality and male fertility in humans. The aim of the present work was to elucidate the adverse effects of deltamethrin (Delta), a synthetic pyrethroid, on exposed male mice and their offspring. Adult male Albino/Swiss mice received deltamethrin (5 mg/kg) daily for 35 days and mated with untreated females to produce offspring. Classical measurements of ejaculate and sperm quality and testicular histopathological changes were assessed. Deltamethrin treatment affects sperm quality and quantity in the ejaculated semen of mice that had also markedly impaired libido as measured by indices of mating and fertility and number of pregnant females housed with male mice exposed to this pesticide. Exposure mice to deltamethrin significantly decreased their testosterone and inhibin B levels and affected reproductive performance. Testes of exposed mice showed marked histopathological alterations as compared to the control group. The mice exposed to 5 mg/kg body weight/day of deltamethrin showed severe alterations of the seminiferous tubules, sloughing of the germ cells, the vacuolization of germ cell cytoplasm, and the disruption of spermatogenic cells compared to the control group. Altered pregnancy outcomes were directly attributed to damage of sperm of male mice exposed to deltamethrin compared to the control group. We concluded that exposure to deltamethrin affected the reproductive system of male mice explored by altered total sperm density, motility, and morphology in mice spermatozoa.

Protective effect of vanillin against carbon tetrachloride (CCl4)-induced oxidative brain injury in rats

This study investigated the protective effects of vanillin against acute brain damage induced by carbon tetrachloride (CCl4) in rats. The study was performed on 32 male rats divided into four groups: a control group, vanillin group ([Va] 150 mg/kg/day, intraperitoneally [i.p.]) and CCl4 toxication groups received a single injection of CCl4 (1 ml/kg, i.p.; CCl4 and Va + CCl4 groups). The degree of protection in brain tissue was evaluated by the levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase, glutathione transferase, glutathione peroxidase and nitric oxide (NO). Vanillin showed a significant brain-protective effect by decreasing the level of lipid peroxidation and NO2 and elevated the activities of antioxidative enzymes and level of GSH. Consequently vanillin blocked oxidative brain damage induced by CCl4 in rats.

Locked nucleic acid probe-based real-time PCR for the diagnosis of Listeria monocytogenes in ruminants

Because of its high fatality rate, listeriosis ranks among the most important infectious diseases worldwide. Although ruminants are known as natural reservoirs for Listeria monocytogenes and a possible source of human listeriosis, studies of the prevalence and risk factors associated with ruminant listeriosis are limited to some developed countries. Therefore, this report describes the development of a real-time PCR targeting the hly gene for the absolute quantification of L. monocytogenes based on circular and linear DNA standards. Results show that the PCR that uses circular plasmid as a template gave a 2.6-7.89 greater threshold cycle number than did equimolar linear standards. No cross-amplification was observed when bacteria commonly found in bovine and ovine diseases were tested. The PCR achieved good intra and inter-run reproducibility and a detection limit of 6.1 copies of linear plasmid per reaction. This PCR was then applied to 1134 samples taken from 378 Tunisian ruminants. Based on the test sensitivity (90%) and specificity (100%), the true individual animal prevalence of listeriosis was 5.7% in cattle and 10.2% in sheep. In addition, the true herd-level prevalence was 50.1% in cattle and 26.7% in sheep. A multivariable logistic regression analysis at the animal-population level indicated that for cattle, the variables strata and mastitis were important risk factors, whereas for sheep, the variables strata, age and abortion were found to be associated with listeriosis. At the herd level, risk factors for Listeria test-positivity they were: abortion, herd composition and silage storage for cattle, whereas for sheep were: management system, cleaning frequency, silage storage and floor type. Animal hygiene, food quality and sanitary practices on the farm should be applied as strategies to control this pathogen in ruminant herds.

Real time PCR gene profiling and detection of Salmonella using a novel target: The siiA gene.

The objective of this study was to develop and evaluate a SYBR Green real time PCR method for the specific detection of Salmonella spp using a novel target, the siiA gene. Primer specificity testing was done on a panel of 76 Salmonella strains and 32 non-Salmonella strains. The primers directed against the siiA gene amplified all Salmonella strains tested, while non-Salmonella strains were not amplified. The melting temperatures of the 107 bp amplicons were consistently specific as they gave melting peaks around 75.5°C. The precision of the assay, based on intra and inter-run variations, was shown to be widely acceptable. In the second part of this study, 45 Salmonella strains were screened for the presence of 6 virulence-associated genes (sopB, cat2, safC, sefB and SC1248) located in several Salmonella Pathogenicity Islands (SPIs) and the spvC gene from the Salmonella virulence plasmid. The prevalence of these genes ranged from 51% to 100%. Variable virulence gene profiles were obtained even within the same serotype.

Erythrocyte oxidative damage in rat treated with CCl4: protective role of vanillin.

Owing to the presence of hemoglobin and polyunsaturated fatty acids, erythrocytes are a convenient model to understand membrane oxidative damage induced by various xenobiotic pro-oxidants. This study investigated the antioxidant activity of vanillin, a naturally occurring food-flavoring agent, against carbon tetrachloride (CCl4)-induced erythrocyte damages in Wistar albino rats. A single injection of CCl4 (1 ml/kg, intraperitoneally [i.p.]) caused a significant induction of oxidative damage as evidenced by increased thiobarbituric acid reactive substances, protein carbonyl levels and osmotic fragility accompanied with a significant decrease in Na+/K+-ATPase and Ca2+-ATPase activities. Furthermore, catalase and superoxide dismutase activities were significantly elevated, while glutathione levels, glutathione-S-transferase and glutathione peroxidase activities were markedly reduced in the erythrocytes of CCl4-treated rats. Pretreatment of rats with vanillin (150 mg/kg/day, i.p.) for 3 consecutive days before CCl4 injection protected erythrocytes against the increase of lipid peroxidation and degradation of membrane proteins compared to CCl4-treated rats and exhibited marked prevention against CCl4-induced oxidative stress, alterations of membrane-bound enzymes as well as erythrocyte osmotic fragility. Our results suggest that vanillin plays a protective and curative role against the harmful effects of CCl4 on erythrocytes, thus ensuring membrane cell integrity.

Modelling Tetraselmis sp. growth-kinetics and optimizing bioactive-compound production through environmental conditions

The aim of this study is to predict Tetraselmis cells growth-kinetic and to induce the synthesis of bioactive compounds (chlorophylls, carotenoids and starch) with high potential for biotechnological applications. Using the statistical criteria, the Baranyi-Roberts model has been selected to estimate the microalgae growth-kinetic values. The simultaneous effects of salinity, light intensity and pH of culture medium were investigated to maximize the production of total chlorophylls, carotenoids and starch. The optimal culture conditions for the production of these compounds were found using Box-Behnken Design. Results have shown that total chlorophyll and carotenoids were attained 21.6mg·g-1DW and 0.042mg·g-1DW, respectively. In addition, the highest starch content of 0.624g·g-1DW has been obtained at neutral pH with high irradiance (182μmolphotonsm-2 s-1) and low salinity (20). A highly correlation (R2 = 0.884) has been found between the gravimetric and flow cytometric measurements of chlorophyll content.

Cyanobacteria as source of marine bioactive compounds: Molecular specific detection based on Δ9 desaturase gene

The blue-green microalga, Arthrospira sp., isolated from the sea of Kssour Essef in Mahdia (Tunisia), was purified and then identified both morphologically and genetically based on 16S rRNA gene sequence. Following physicochemical analysis, the prokaryotic microalga tested represented a competitive source of pigments and showed a considerable rate in protein (64%) which was confirmed by FTIR measurement. The lipid content (4%) was quantified by the gravimetric method and the intracellular lipid bodies were detected with the Nile red staining. Using gas chromatography coupled with flame ionization detector, the fatty acid profile revealed the presence of 27.4% and 32.88% of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs), respectively. Given the richness of the isolated microalga in unsaturated fatty acids, we have developed a SYBR Green real time PCR method for the specific identification of Arthrospira sp. Δ9 desaturase gene. This current method will be of great value for carrying out high-throughput studies like cloning, heterologous expression and structure-function relationship analysis.