Phosphate solubilizing bacteria from soils with varying environmental conditions: Occurrence and function.

Phosphate solubilizing bacteria (PSB) is an advantageous way to supply phosphate (P) to plants. The Mediterranean climate of Morocco, especially the low-lying areas, is semi-arid with nutrient-depleted soils in which small-scale, low-income farmers dominate without access to expensive inorganic fertilizers. However, there is not a wide range of PSBs suitable for various agroecological situations. Furthermore, our understanding of the soil and climatic variables that influence their development is limited. This study aims to examine the impacts of specific environmental factors, such as climate and soil, on the abundance, potential, and diversity of PSBs in four agricultural regions of Morocco. To assess the possible impact of these factors on the P solubilization capacity of PSBs and plant growth-promoting (PGP) traits, we analyzed the soil and climate of each sample studied. Similarly, we tested the P solubilization efficiency of the isolates. The bacteria were isolated in a National Botanical Research Institute's phosphate (NBRIP) agar medium. A total of 51 PSBs were studied in this work. The P-solubilization average of Rock P (RP) and Tricalcium P (TCP) of all strains that were isolated from each of the four regions ranged from 18.69 mg.L-1 to 40.43 mg.L-1 and from 71.71 mg.L-1 to 94.54 mg.L-1, respectively. The PGP traits of the isolated strains are positively correlated with the PSBs abundance and the sample characteristics (soil and climate). The morphological and biochemical characteristics of the strain allowed us to identify around nine different bacterial genera, including Bacillus, Pseudomonas, and Rhizobium. The findings showed that bacterial communities, density, and potency are closely correlated to various edapho-climatic conditions such as temperature, precipitation, soil nutrient status, and soil texture. These findings could be used to improve an effective plant-PSBs system and increase agricultural output by taking into account their specific ecological traits and plant growth mechanisms.

Poxvirus sensitivity of a novel diploid sheep embryonic heart cell line.

Lumpy skin disease virus (LSDV), camelpox virus (CPV), and orf virus (ORFV) are members of the family Poxviridae. These viruses are usually isolated or produced in embryonated eggs or primary cells because continuous cell lines are less sensitive to infection. Disadvantages of the use of eggs or primary cells include limited availability, potential endogenous contaminants, and a limited ability to perform multiple passages. In this study, we developed a diploid cell culture from sheep embryonic hearts (EHs) and demonstrated its high proliferative and long-term storage capacities. In addition, we demonstrated its sensitivity to representatives of three genera of the family Poxviridae: Capripoxvirus (LSDV), Orthopoxvirus (CPV), and Parapoxvirus (ORFV). The cell culture had a doubling time of 24 h and reached 40 passages with satisfactory yield. This is comparable to that observed in primary lamb testis (LT) cells at passage 5 (P5). After infection, each poxvirus titer was 7.0-7.6 log TCID50/mL for up to five passages and approximately 6.8, 6.4, and 5.6 for the three viruses at P6-P25, P30, and P40, respectively. The sensitivity of sheep EH cells to poxvirus infection did not decrease after long-term storage in liquid nitrogen and was higher than that of primary LT cells, which are used for capripoxvirus and parapoxvirus detection and growth, and Vero cells, which are used for orthopoxvirus detection and growth. Thus, EH diploid cells are useful for poxvirus isolation and production without embryonated eggs or primary cells.

Growth stimulation of two legumes (Vicia faba and Pisum sativum) using phosphate-solubilizing bacteria inoculation.

The application of chemical fertilizers for plant growth and protection is one of the reasons for the environment and ecosystem destruction, thus, sustainable agriculture is gaining popularity in research and among farming communities. Although most soils are high in total phosphorus (P), a large portion is unavailable to plants and regarded as a growth-limiting factor. P-solubilizing bacteria (PSB) exploitation is a newly developed bio-solution for enhancing rhizosphere P availability; however, the effect of these bacteria on soil quality and the different phases of plant growth remains unknown. This study aims to evaluate the impact of five strains of PSB, isolated from legume rhizosphere, on the growth of two plants (Vicia faba and Pisum sativum) and certain soil properties. The efficient strains of PSB used are characterized by the P-solubilization, the ACC deaminase activity, the fixation of N, and the IAA, HCN, and siderophores production. The activity of these bacteria is tested in vitro and in vivo under controlled conditions on the growth of the two plants supplemented with the rock P (RP). According to our findings, all PSBs strains outperformed the control in terms of enhancing the growth of the tested legumes with a percentage ranging from 77.78 to 88.88%, respectively. The results showed that all treatments significantly improved plant parameters like nitrogen- (N) and P-content in the plants (67.50, 23.11%), respectively. Also, an increase in the fresh and dry weights of above- (41.17, 38.57%) and below-ground biomasses (56.6, 42.28%), respectively. Compared to the control, this leads to an increase of 72% in root length, 40.91% in plant dry weight, and 40.07% in fresh weight. Rhizospheric soil in PSBs treatments displayed high levels of N, P, and organic matter. All treatments were found to have significantly higher levels of alkaline phosphatase, basal soil respiration, and ß-glucosidase activity than the control. It is concluded that multi-traits PSB can be an alternative for utilizing chemical fertilizers to enhance soil quality and plant growth. Despite the potency of PSBs, its use as a source for the development of sustainable agriculture implies focusing on crop species and adaptation, stress tolerance and climate resilience.

Evaluation of the Sysmex UF-4000i urine analyzer as a screening test to rule out urinary tract infection and reduce urine cultures. / Évaluation de l'analyseur Sysmex UF-4000i comme test de dépistage pour exclusion des infections urinaires et diminution des mises en culture

INTRODUCTION: Urinary tract infection (UTI) diagnosis by urine culture is time- and labor- consuming. In the Ibn Rochd microbiology laboratory, up to 70% of urine culture samples yield no growth or insignificant growth. OBJECTIVE: To evaluate the new generation of Sysmex UF-4000i fluorescence flow cytometry analyzer with a blue semiconducting laser as a method to rule out negative urine samples for UTI, in comparison of urine culture. MATERIAL AND METHODS: Flow cytometry and microbiological analysis were performed on 502 urine samples included in the study. We used ROC analysis to determine cutoff points at which optimal sensitivity and specificity are achieved for clinical use. RESULTS: Our results showed that bacteria count at a cut-off of 100/µL, and/or the leucocytes count ≥ 45/µL are the optimal indicator for positive culture results. At these cut off, bacteria sensitivity (SE), specificity (SP), Positive predictive value (PPV) and negative predictive value (NPV) were 97,3%, 95%, 87,8% and 98,8% respectively. For leucocytes, SE, SP, PPV and NPV were 99,1%, 95,8%, 88,6% and 99,7% respectively. DISCUSSION AND CONCLUSION: The bacterial and leucocytes counts generated by UF-4000i analysis may be useful in our context as a rapid screening to exclude UTI by reducing about 70% of urines cultures and then workload. Nevertheless, further validation is needed for different patient groups especially with urological disease or immunocompromised patients.

Isolation and characterization of phosphate solubilizing bacteria naturally colonizing legumes rhizosphere in Morocco.

Low-cost and environmentally friendly agricultural practices have received increasing attention in recent years. Developing microbial inoculants containing phosphate (P) solubilizing bacteria (PSB) represents an emerging biological solution to improve rhizosphere P availability. The present study aims to explore PSB strains isolated from soils located at different bioclimatic stages in Morocco and present in various legumes rhizosphere to improve agronomic microbial fertilizer's effectiveness. It was also aimed to test the isolated strains for their ability to solubilize P in NBRIP medium with Tricalcium P (Ca3 (PO4)2) (TCP), rock phosphate (RP), and their combination as a source of phosphorus, by (22) experiment design. Bacterial strains with a high P solubility index (PSI) were selected, characterized, and compared to commercial control. The vanadate-molybdate method was used to estimate P solubilization activity. Stress tolerance to salinity, acidity, drought, and temperature was tested. From all isolated strains (64), 12 were screened as promising biotechnological interest because of their P solubilization and their good resistance to different drastic conditions. Besides, the strain WJEF15 showed the most P solubility efficiency in NBRIP solid medium with a PSI of 4.1; while the WJEF61 strain was located as the most efficient strain in NBRIP-TCP liquid medium by releasing 147.62 mg.l-1 of soluble P. In contrast, in the NBRIP-RP medium, the strain WJEF15 presented maximum solubilization with 25.16 mg.l-1. The experiment design showed that a combination of RP and TCP with max level progressively increases P solubilization by 20.58%, while the WJEF63 strain has the most efficient concentration of 102.69 mg.l-1. Indeed, among the selected strains, four strains were able to limit tested fungi growth. Thus, results reveal a potential effect of selecting PSBs to support cropping cultures as plant growth-promoting rhizobacteria (PGPR).

In Vitro Study of the Phytochemical Composition and Antioxidant, Immunostimulant, and Hemolytic Activities of Nigella sativa (Ranunculaceae) and Lepidium sativum Seeds.

The Moroccan flora abounds and is an important reserve of medicinal plants. Nigella sativa and Lepidium sativum are plants that are widely used in traditional medicine for their multiple therapeutic properties. The current study aims to highlight the biological activities that can justify and valorize the use of these plants. Flavonoids, total phenols, condensed tannins, and sugars were determined. The biological activities tested were antioxidant by determining the IC50 (defined as the concentration of an antioxidant required to decrease the initial concentration by 50%; inversely related to the antioxidant capacity), hemagglutination, and hemolytic activities. Phytochemical quantification of the seed extracts indicated that the total phenol content was largely similar for both plants and in the order of 10 mg GAE (Gallic acid equivalent)/g. On the other hand, L. sativum seeds registered a higher content of flavonoids (3.09 ± 0.04 mg QE (quercetin equivalent)/g) as compared to Nigella saliva (0.258 ± 0.058). Concerning condensed tannins, N. saliva seeds present a higher amount with a value of 7.2 ± 0.025 mg/g as compared to L. sativum (1.4 ± 0.22 mg/g). Concerning the total sugar content, L. sativum shows a higher content (67.86 ± 0.87 mg/g) as compared to N. sativa (58.17 ± 0.42 mg/g); it is also richer in mucilage with a content of 240 mg as compared to 8.2 mg for N. saliva. Examination of the antioxidant activity using a DPPH (2.2-diphenyl 1-pycrilhydrazyl) test revealed that the EButOH (n-butanol extract) and EAE (ethyl acetate extract) extracts were the most active, with IC50 values of 48.7 and 50.65 µg/mL for the N. sativa extracts and 15.7 and 52.64 µg/mL for the L. sativum extracts, respectively. The results of the hemagglutination activity of the different extracts of the two plants prepared in the PBS (phosphate-buffered saline) medium showed significant agglutination for the L. sativum extract (1/50) compared to the N. sativa extract (1/20). An evaluation of the hemolytic effect of the crude extract of the studied seeds on erythrocytes isolated from rat blood incubated in PBS buffer compared to the total hemolysis induced by distilled water showed a hemolysis rate of 54% for Nigella sativa and 34% for L. sativum. In conclusion, the two plants studied in the current work exhibited high antioxidant potential, which could explain their beneficial properties.

Evaluation of ELISA and VNT for sheeppox virus antibody detection and development of an immunoenzymatic quantitative method.

Vaccination against sheep pox (SPV) is the most efficient tool to control spread of the disease and virus neutralization test (VNT) is the gold standard for vaccination monitoring. In the presented study, we evaluated the use of ELISA and VNT for quantification of SPV humoral response post vaccination. Results confirmed that VNT is more sensitive since ELISA did not detect 22% of positive tested sera, and VNT weak positive sera were either negative or doubtful by ELISA. The most sensitive cells to perform VNT were ESH-L instead of Lamb primary cells. We also investigated immunoperoxidase IPMA and immunofluorescence IFA assays for detection of SPV specific antibodies and IPMA showed higher antibody titers comparatively to IFA. VNT using ESH-L cells with immune-enzymatic revelation provide specific quantitative SPV antibody titers, easier to read in shorter incubation time.

Comparative sensitivity study of primary cells, vero, OA3.Ts and ESH-L cell lines to lumpy skin disease, sheeppox, and goatpox viruses detection and growth.

Lumpy skin disease virus (LSDV), sheeppox virus (SPPV) and goatpox (GTPV) virus have been usually grown on primary cells for diagnosis, production and titration purposes. The use of primary cells present several inconvenient, heavy preparation, heterogeneous cell population, non-reproducible viral titration and presence of potential endogenous contaminants. Therefore investigating sensitivity of candidate continuous cell lines is needed. In this study, we compared the above Capripox viruses (CaPVs) sensitivity of primary cells of four origin (heart, skin, testis and kidney), with three cell lines (Vero, OA3.Ts and ESH-L). We tested sensitivity for virus isolation, replication cycle and titration, revealed by cytopathic effect (CPE), immunoenzymatic staining and immunofluorescence. Our results show that ESH-L cells and primary fetal heart cells present the highest sensitivity for CaPVs growth and detection. Vero cells can replicate those viruses but without showing any CPE while the titer obtained on OA3.Ts is lower than primary and ESH-L cells. ESH-L cells are an effective alternative to primary cells use for growing Capripoxviruses and their diagnosis.

Evaluation of the Sysmex UF-4000i urine analyzer as a screening test to rule out urinary tract infection and reduce urine cultures.

INTRODUCTION: Urinary tract infection (UTI) diagnosis by urine culture is time- and labor- consuming. In the Ibn Rochd microbiology laboratory, up to 70% of urine culture samples yield no growth or insignificant growth. OBJECTIVE: To evaluate the new generation of Sysmex UF-4000i fluorescence flow cytometry analyzer with a blue semiconducting laser as a method to rule out negative urine samples for UTI, in comparison of urine culture. MATERIAL AND METHODS: Flow cytometry and microbiological analysis were performed on 502 urine samples included in the study. We used ROC analysis to determine cutoff points at which optimal sensitivity and specificity are achieved for clinical use. RESULTS: Our results showed that bacteria count at a cut-off of 100/µL, and/or the leucocytes count ≥45/µL are the optimal indicator for positive culture results. At these cut off, bacteria sensitivity (SE), specificity (SP), Positive predictive value (PPV) and negative predictive value (NPV) were 97,3%, 95%, 87,8% and 98,8% respectively. For leucocytes, SE, SP, PPV and NPV were 99,1%, 95,8%, 88,6% and 99,7% respectively. DISCUSSION AND CONCLUSION: The bacterial and leucocytes counts generated by UF-4000i analysis may be useful in our context as a rapid screening to exclude UTI by reducing about 70% of urines cultures and then workload. Nevertheless, further validation is needed for different patient groups especially with urological disease or immunocompromised patients.

Production of small ruminant morbillivirus, rift valley fever virus and lumpy skin disease virus in CelCradle™ -500A bioreactors.

BACKGROUND: Animal vaccination is an important way to stop the spread of diseases causing immense damage to livestock and economic losses and the potential transmission to humans. Therefore effective method for vaccine production using simple and inexpensive bioprocessing solutions is very essential. Conventional culture systems currently in use, tend to be uneconomic in terms of labor and time involved. Besides, they offer a limited surface area for growth of cells. In this study, the CelCradle™-500A was evaluated as an alternative to replace conventional culture systems in use such as Cell factories for the production of viral vaccines against small ruminant morbillivirus (PPR), rift valley fever virus (RVF) and lumpy skin disease virus (LSD). RESULTS: Two types of cells Vero and primary Lamb Testis cells were used to produce these viruses. The study was done in 2 phases as a) optimization of cell growth and b) virus cultivation. Vero cells could be grown to significantly higher cell densities of 3.04 × 109 using the CelCradle™-500A with a shorter doubling time as compared to 9.45 × 108 cells in Cell factories. This represents a 19 fold increase in cell numbers as compared to seeding vs only 3.7 fold in Cell factories. LT cells achieved modestly higher cell densities of 6.7 × 108 as compared to 6.3 × 108 in Cell factories. The fold change in densities for these cells was 3 fold in the CelCradle™-500A vs 2.5 fold in Cell factories. The titers in the conventional system and the bioreactor were not significantly different. However, the Cell-specific virus yield for rift valley fever virus and lumpy skin disease virus are higher (25 virions/cell for rift valley fever virus, and 21.9 virions/cell for lumpy skin disease virus versus 19.9 virions/cell for rift valley fever virus and 10 virions/cell for lumpy skin disease virus). CONCLUSIONS: This work represents a novel study for primary lamb testis cell culture in CellCradle™-500A bioreactors. In addition, on account of the high cell densities obtained and the linear scalability the titers could be further optimized using other culture process such us perfusion.

EGFR Amplification and IDH Mutations in Glioblastoma Patients of the Northeast of Morocco.

Glioblastomas are the most frequent and aggressive primary brain tumors which are expressing various evolutions, aggressiveness, and prognosis. Thus, the 2007 World Health Organization classification based solely on the histological criteria is no longer sufficient. It should be complemented by molecular analysis for a true histomolecular classification. The new 2016 WHO classification of tumors of the central nervous system uses molecular parameters in addition to histology to reclassify these tumors and reduce the interobserver variability. The aim of this study is to determine the prevalence of IDH mutations and EGFR amplifications in the population of the northeast region of Morocco and then to compare the results with other studies. Methods. IDH1 codon 132 and IDH2 codon 172 were directly sequenced and the amplification of exon 20 of EGFR gene was investigated by qPCR in 65 glioblastoma tumors diagnosed at the University Hospital of Fez between 2010 and 2014. Results. The R132H IDH1 mutation was observed in 8 of 65 tumor samples (12.31%). No mutation of IDH2 was detected. EGFR amplification was identified in 17 cases (26.15%). Conclusion. A systematic search of both histological and molecular markers should be requisite for a good diagnosis and a better management of glioblastomas.

Prevalence of IDH1/2 Mutations in Different Subtypes of Glioma in the North-East Population of Morocco.

BACKGROUND: Genetic alterations in gliomas have increasing importance for classification purposes. Thus, we are especially interested in studying IDH mutations which may feature potential roles in diagnosis, prognosis and response to treatment. Our aim was to investigate IDH mutations in diffuse glioma patients diagnosed in university hospital centre of Fez in Morocco. MATERIALS AND METHODS: IDH1 codon 132 and IDH2 codon 172 were direct-sequenced in 117 diffuse glioma samples diagnosed and treated in University Hospital Hassan II between 2010 and 2014. RESULTS: The R132H IDH1 mutation was identified in 43/117 tumor samples and R172K IDH2 mutation was detected in only one anaplastic oligodendroglioma. IDH mutations were observed in 63.2% of astrocytomas, 73.3% of diffuse oligodendrogliomas and 12.90% of glioblastomas. CONCLUSIONS: Our results confirmed other studies published earlier for other populations with some small discrepancies.

Hematology reference intervals in Moroccan population.

BACKGROUND: Laboratory reference intervals are important for both clinical orientations and therapeutic decisions. In Morocco, no reference ranges are available for local population. The ranges commonly used in clinical laboratories and by physicians are those of Caucasian population. We have decided that it is relevant to undertake an epidemiological investigation on local adult healthy population, with the aim of establishing hematology reference intervals in Moroccan population. METHODS: Blood samples were taken from healthy adult volunteers of the regional transfusion center and measured on a Sysmex XE-2100 analyzer. We have grouped our data samples with regard to gender and retained donors aged between 18 and 45 years old according to ICSH guidelines 1982. Leucocyte, erythrocyte, and platelet parameters were analyzed. For any sample flagged by the automate or thrombopenia with platelets < 100000/microL, a systematic smear was done and checked. RESULTS: A significant difference between male and female was found with regard to the values for leucocyte, erythrocyte, and platelet parameters as well as for hemoglobin and hematocrit. These data were compared to normal values reported for Arabic, Caucasian, and African population. CONCLUSIONS: As part of this study, we have given a descriptive approach of normal blood cell count and its peculiarities in North African Arabian and Berber population not explored until now. We have established similarities and differences between our population and other African, Arab, and Caucasian populations.