Yaakoub Gharbi

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.

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.

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.

FIRST REPORT OF CYTOSPORA PUNICAE ASSOCIATED WITH WOOD CANKER AND BRANCH DIEBACK DISEASE OF POMEGRANATE IN TUNISIA

Pomegranate (Punica granatum L.) is an economically important crop in Tunisia with an annual production of 23.000 tons. During May and June of 2014, severe branch dieback was observed in pomegranate plants cv. Gabsi located in Gabes region with about 8% of disease incidence. Symptoms of the disease included leaf yellowing, wood lesion and canker formation. Isolation of the pathogen was performed from 10 samples of active cankers plated onto PDA medium amended with 100 mg l-1 of tetracycline hydrochloride. Fungal colonies were then sub-cultured onto PDA medium at 22°C. All isolates were identified as Cytospora sp. based on colony morphology, conidial characteristics and pycnidia formation (Palavouzis et al., 2015). The isolates developed white mycelium, which turned green to dark brown with hyaline, allantoid, aseptate conidia (average 4-5 μm x 1.75 μm) and production of dark coloured pycnidia 300 to 450 μm in diameter after 15 days (Peduto Hand et al., 2014). Identity of these isolates was confirmed by sequencing the internal transcribed spacer region. The ITS sequences were deposited in GenBank (Accession No. KT272402). These sequences revealed 99% genetic identity with those of Cytospora punicae species available in GenBank (KJ621689; KJ621688). Pathogenicity of Cytospora punicae was evaluated by inoculation of two isolates in 1-year-old shoots of pomegranate cv. Gabsi (Palavouzis et al., 2015). The inoculated shoots developed necrotic spots with vascular discoloration spreading downward and upward from the inoculation site. Cytospora punicae was recovered from 100% of the inoculated shoots. This is the first report of Cytospora punicae causing wood canker and branch dieback of pomegranate in Tunisia.

Molecular prevalence of Chlamydia and Chlamydia-like bacteria in Tunisian domestic ruminant farms and their influencing risk factors

Chlamydia and Chlamydia-like bacteria are well known to infect several organisms and may cause a wide range of diseases, particularly in ruminants. To gain insight into the prevalence and diversity of these intracellular bacteria, we applied a pan-Chlamydiales real-time PCR to 1,134 veterinary samples taken from 130 Tunisian ruminant herds. The true adjusted animal population-level prevalence was 12.9% in cattle, against 8.7% in sheep. In addition, the true adjusted herd-level prevalence of Chlamydiae was 80% in cattle and 25.5% in sheep. Chlamydiales from three family-level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant herds. Our results showed that Parachlamydia acanthamoebae could be responsible for bovine and ovine chlamydiosis in central-eastern Tunisia. Multivariable logistic regression analysis at the animal population level indicated that strata and digestive disorders variables were the important risk factors of bovine and ovine chlamydiosis. However, origin and age variables were found to be associated with bovine and ovine chlamydiosis, respectively. At the herd level, risk factors for Chlamydia positivity were as follows: abortion and herd size for cattle against breeding system, cleaning frequency, quarantine, use of disinfectant and floor type for sheep. Paying attention to these risk factors will help improvement of control programs against this harmful zoonotic disease.

Serological and molecular evidence of coxiellosis and risk factors in sheep flocks in central-eastern Tunisia

In this study, we conducted an investigation to determine the true prevalence of coxiellosis in sheep in central-eastern Tunisia. A total of 492 veterinary samples taken from 110 flocks were screened for coxiellosis using IS1111-based real-time PCR assay. Sheep sera were tested using an indirect enzyme-linked immunosorbent assay. Based on molecular and serological results, the true adjusted animal and herd-level prevalence of coxiellosis were 11.8% and 20.21%, respectively. Bacterial excretion was observed in 17 flocks, and 19 females showed evidence of Coxiella burnetii shedding (100%). In addition, a statistically significant association was found between vaginal and milk shedding for sheep. Multivariable logistic regression analysis at the animal-population level indicated that strata and vaccination variables were found to be associated with coxiellosis. Besides, it was shown that this infection increased when the intensive farm was exposed to carnivores and when the cleaning practices were not respected, while it decreased when a suitable quarantine was introduced for any introduction of a new animal. Good hygiene and sanitation practices on-farm should be handled as strategies to deal with this zoonotic pathogen in herds.

Phenotypic and Molecular Characterization of Verticillium dahliae , the Causal Agent of Verticillium Wilt of Olive in Tunisia

Aims: During the last two decades, verticillium wilt of olive has spread to young olive orchards were it causes severe yield losses and death of olive trees in southern and central regions of Tunisia. Therefore, identification of the causal agent as well as the study of its pathogenicity will be useful for design the appropriate management program. Place and Duration of Study: This work was performed in the Laboratory of Phytopathology at the Olive Tree Institute (Sfax, Tunisia) between July 2014 and June 2015. Original Research Article Gharbi et al.; JALSI, 4(3): 1-8, 2016; Article no.JALSI.23696 2 Methodology: This study was conducted using phenotypic and molecular methods to identify the causal agent of the decline and death of olive trees. The pathogen was recovered from infested tissues using a potato dextrose agar medium. Identity of the isolates was confirmed by ITS-RFLP and sequencing. Pathogenicity of the isolates was evaluated by infection bioassay on young olive plants. Results: All the fungal isolates were hyaline, flocculose and produced microsclerotia after 15 days of incubation at 25°C, which is in agreement with the identification key of Verticillium species. All the isolates were characterized by ITS-RFLP and sequencing of the internal transcribed spacer. All the isolates produced 470 bp using primers ITS1/ITS4. Digestion of the ITS product using EcoRI and HaeIII produced two fragment of 250 and 220 bp and three fragment of 300, 150, and 20 bp respectively. Pathogenicity was evaluated on two-year old olive plants using an artificial infection bioassay. After 15 days of inoculation, similar symptoms were produced as natural infection. Symptoms of wilt developed rapidly and caused the death of more than 50% of the inoculated plants. Conclusion: V. dahliae is present in almost all growing olive regions of Tunisia. However, local V. dahliae populations in Tunisia is predominated by a highly pathogenic clone which is able to overcome the resistance of the main olive cultivar.

Molecular Epidemiology of Verticillium Wilt of Olive in Southern and Central Tunisia: Evidence of Host Adaptation Hypothesis

Aims: During the last two decades, verticillium wilt of olive has spread to young olive orchards where highly susceptible crops such as potato, watermelon and tomato are cultivated near to olive orchards. It was therefore hypothesized that there is an adaptation phenomenon that drives the pathogenicity of V. dahliae in order to infect a broad range of hosts. Therefore, it will be useful to identify the factors that increase the severity of the pathogen which helps growers to implement the appropriate crop rotation program. Place and Duration of Study: This work was performed in the Laboratory of Phytopathology at the Original Research Article

First report of dieback of olive trees caused by Neofusicoccum australe in Tunisia.

In spring 2011, a severe disease resulting in tree dieback of olive tree cv. Chemlali was observed in an orchard in Hencha (south-east Tunisia). Symptomatic trees exhibited plenty of dead twigs and wilted leaves. On potato dextrose agar (PDA), a fungus isolated from symptomatic twigs and branches has an initially white mycelim that turned glaucous grey to greenish grey on the upper surface. The fungus was identified as Neofusicoccum australe, based on morphological characteristics and analysis of the ITS gene region (White et al., 1990). The sequence analysis of ITS region of the isolate revealed 100% homology with a reference sequence of N. australe (Strain E54 ML, GenBank accession No. KF702388.1). Pathogenicity tests were conducted on 10 two-year-old olive trees of cv. Chemlali. A mycelial plug was put in a shallow wound on the stem of each plant. Control plants were inoculated with sterile PDA plugs. All plants were kept in a greenhouse. Two months post inoculation, symptoms appeared with stems showing brown color. No symptoms developed on the control plants. Neofusicoccum was isolated from inoculated stems, thus fulfilling Kochs postulates. N. australe has been reported as responsible for cordon grapevine dieback in Italy (Linaldeddu et al., 2010). To the best of our knowledge, this is the first report of N. australe as a causal agent of dieback of olive trees in Tunisia.

FIRST REPORT OF NEONECTRIA RADICICOLA ASSOCIATED WITH ROOT ROT DISEASE OF OLIVE IN TUNIS

Olive (Olea europaea) is an economically important crop in Tunisia. During surveys of olive diseases conducted in 2013 in Tunisia, symptoms of leaf wilting and chlorosis, Brown-to- black discoloration of the wood in cross-sections of the stems and necrotic lesions in the roots were observed on young olive trees. Isolation of the pathogen was performed from 15 infected root and stem samples plated onto PDA medium amended with 50 μg ml-1 of streptomycin sulfate. Fungal colonies were then cultured on synthetic nutrient-poor agar medium. All isolates were identified as Cylindrocarpon sp. based on colony morphology and conidial characteristics (Booth, 1966). The isolates developed abundant floccose mycelium, which varied in color from brown-yellow to sepia. All isolates produced only macroconidia, which were hyaline, straight, and predominantly three septate measuring 15.75 to 29.50 μm × 3.25 to 4.75 μm. Identity of these isolates was confirmed by sequencing the internal transcribed spacer region, which was amplified using primer pair ITS1 and ITS4 (White et al., 1990). The ITS sequences were deposited in GenBank (KM503139). These sequences revealed 98% genetic identity with those ofNeonectria radicicola the anamorphic form of Cylindrocarpon species available in GenBank. Pathogenicity of N. radicicola in olive cv. Chemlali was evaluated using three isolates. Three months after inoculation, the inoculated plants developed wilting and root symptoms similar to those observed in the field. N. radicicola was recovered from all the symptomatic plants. This is the first report of N. radicicola causing root rot of olive in Tunisia, which may potentially affect the sustainability of olive nurseries.