In vitro probiotic and safety attributes of Bacillus spp. isolated from beebread, honey samples and digestive tract of honeybees Apis mellifera.

Bacillus species isolated from honeybee Apis mellifera gut, honey and bee bread samples were characterized for their in vitro probiotic and safety attributes. Alpha and γ haemolytic cultures were tested for their antibiotic resistance, antibacterial spectrum, acid and bile tolerance, adhesion ability (auto-aggregation, co-aggregation and hydrophobicity) and phenol tolerance. Safety criteria included evaluation of virulence genes and cytotoxicity percentages. Bacillus isolates inhibited both Gram-positive and Gram-negative pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and Streptococcus mutans, while none could inhibit Listeria monocytogenes. Among the isolates, Bacillus subtilis ZH05, ZB03 and ZG025 showed resistance to most of the tested antibiotics and were considered unsafe. B. subtilis (4) and B. licheniformis (1) tolerated acidic pH and bile conditions, never the less were more tolerant in simulated intestinal conditions vis-a-vis gastric conditions. In 0·5% phenol concentrations, B. licheniformis ZH02 showed highest growth, while, B. subtilis ZG029 demonstrated highest auto-aggregation (65 ± 4·6) and hydrophobicity (23 ± 3·6) percentages (P < 0·05). The isolates lacked virulence genes (hblA, hblC, hblD, nhe, cytK and ces), and their cytotoxic percentage on Caco-2 cell lines was ˂15%. Overall, honeybees appear to be a good source of Bacillus species exhibiting typical in vitro probiotic properties, which could be of commercial interest.

Use of fractal analysis in dental images for osteoporosis detection: a systematic review and meta-analysis.

Fractal dimension (FD) calculated on oral radiographs has been proposed as a useful tool to screen for osteoporosis. This systematic review and meta-analysis firstly aimed at assessing the reliability of FD measures in distinguishing osteoporotic patients (OP) from healthy controls (HC), and secondly, to identify a standardized procedure of FD calculation in dental radiographs for the possible use as a surrogate measure of osteoporosis. A comprehensive search was conducted up to September 2020 using PubMed, Web of Science, and SCOPUS databases. The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement was followed. Meta-analysis was performed on FD values calculated for HC and OP. Overall, 293 articles were identified. After a three steps screening, 19 studies were included in the qualitative appraisal and 12 were considered for meta-analysis. The methodological quality of the retrieved studies was generally low. Most of the studies included used White and Rudolph and box counting to process the images and to calculate FD, respectively. Overall, 51% of the studies found a meaningful difference between HC and OP groups. Meta-analyses showed that to date, FD measures on dental radiographs are not able to distinguish the OP from HC group significantly. From the current evidence, the use of FD for the identification of OP is not reliable, and no clear conclusion can be drawn due to the heterogeneity of studies. The present review revealed the need for further studies and provided the fundamentals to design them in order to find a standardized procedure for FD calculation (regions for FD assessment; images processing technique; methods for FD measurement). More effort should be made to identify osteoporosis using dental images which are cheap and routinely taken during periodic dental examinations.

Evaluation of Marginal and Internal Adaptation of Endocrowns Fabricated from Three Different CAD/CAM Materials.

OBJECTIVES: This in vitro study aimed to compare the internal and marginal discrepancies of endocrowns fabricated from different ceramics before and after adjustment. METHODS AND MATERIALS: Thirty-six endocrowns were fabricated from lithium disilicate (IPS e-max CAD), zirconia-reinforced lithium silicate (Suprinity), and polymer-infiltrated ceramic network (Enamic) using a CEREC intraoral scanner and milling unit. A reference point matching scan protocol was used to measure the endocrowns fit. The mixed ANOVA was used to analyze the data, and α=0.5 was considered as the significance level. RESULTS: There was no significant interaction between the fabrication materials and adjustment (P⟩0.05). Type of the materials did not change discrepancies at cavosurface, line angle, cavity wall, and pulpal floor (P⟩0.05). On the contrary, adjustment significantly decreased the discrepancies at all four sites (P⟨0.001). There were significant differences between every two sites before adjustment (P⟨0.001). After adjustment, except for cavosurface and cavity wall (P=0.058), the differences between other sites remained significant (P⟨0.001). CONCLUSIONS: IPS e-max CAD, Suprinity, and Enamic endocrowns were not significantly different regarding internal and marginal discrepancies. However, it is suggested that these materials be adjusted to fall in a clinically acceptable range to minimize the risk of caries and periodontal disease.

Oral health-related quality of life in patients with temporomandibular disorders: A case-control study considering psychological aspects.

OBJECTIVES: This case-control study aimed to compare patients with temporomandibular disorders (TMD) and healthy controls in terms of oral health-related quality of life (OHRQoL) considering Graded Chronic Pain Scale (GCPS) scores, pain duration, psychological impairment and demographic characteristics. METHODS: A total of 75 patients with TMD and 75 healthy controls were recruited. The short version of Oral Health Impact Profile (OHIP-14) was administered for evaluating the OHRQoL. Psychosocial impairments were assessed using the General Health Questionnaire-28 (GHQ-28). The Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) axis I and II were also used for patient diagnosis and collecting GCPS scores, pain duration, age and gender. Independent-sample t tests, Pearson's chi-square tests and multiple logistic and linear regression models were applied for statistical analysis. RESULTS: The mean age of the patients was 34.3±12.4 years. A female-to-male ratio of 6:1 was seen in the TMD group. The prevalence and severity of the OHIP were significantly different between the TMD and control groups (66.7% vs 12.0% and 18.0 vs 9.2, respectively). According to multiple logistic regression for OHIP prevalence and multiple linear regression for OHIP severity in the TMD group, GCPS scores and pain duration, followed by psychological impairment, were the most important predictors of the OHRQoL. CONCLUSION: TMD negatively affected the OHRQoL, particularly in patients with psychological impairments. Meanwhile, age and gender did not seem to have a serious effect. Hence, promoting the quality of life of patients with TMD requires emphasis on chronic pain management and maintaining good mental health.

Anti-Bacterial Activity of Four Distinct Propolis Extracts against P. larvae and M. plutonius; Etiological Agent of American and European Foulbrood Disease of Honeybees.

Four different propolis samples obtained from different regions of Iran were evaluated for their antibacterial effects against the bacterial agents responsible for two important honeybee diseases. Paenibacillus larvae (P. larvae) and Melissococcus plutonius (M. plutonius), as the etiological agents of American foulbrood (AFB) and European foulbrood (EFB) diseases, were subjected to propolis ethanolic extracts in the agar well diffusion assay. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) of the antibacterial effects of the samples against the two indicator organisms were determined by the microdilution technique using different concentrations of the propolis extracts. Finally, the synergistic antibacterial actions of the mixed propolis samples were determined, and their MIC and MBC values were recorded. A two-way analysis of variance was used to evaluate correlations among the diameters of the inhibition zones, the bacterial agents, and the propolis extracts. Based on our results, three of the propolis samples showed significant antibacterial effects against P. larvae and M. plutonius during the agar well diffusion assay. Furthermore, the antibacterial capacity of the propolis samples, when mixed in equal proportions, was significantly enhanced, as indicated by the obtained MIC and MBC values. Approximately, 0.02 mg/mL of mixed propolis samples was required for inhibiting the growth of both pathogens. A direct correlation was observed between propolis concentrations and their antibacterial activity. The results of the study are conclusive of the significant antibacterial actions of Iranian propolis samples against the etiological agents of the mentioned honeybee diseases, suggesting their probable use as a safe biological agent to control AFB and EFB diseases.

Diagnosis of Pebrine Disease in Silkworm Using Molecular Methods.

Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the disease and eliminating it completely from livestock production has been associated with numerous problems. This project aimed to identify the molecular cause of pebrine disease in silkworms using a sensitive, specific, and accurate method. To this purpose, a 136 bp fragment was selected based on the Nosema bombycis partial SSU rDNA sequence, and a pair of primers was designed. Afterward, using the conventional polymerase chain reaction (PCR) method, the target fragment was amplified and sequenced. After that, to determine the detection sensitivity, using the Real-Time PCR method, 5-fold serial dilutions of N. bombycis DNA were prepared, and the last dilution that produced a fluorescent signal was considered the minimum detection limit. All tests were performed in duplicates. Based on the results of the sensitivity test, the standard curve including Ct values ​​and DNA concentration was used for analysis. Moreover, 80 unknown samples examined by light microscope were evaluated using conventional PCR and Real-Time PCR. Both PCR results showed no amplification for the negative control samples. The findings demonstrated that the lowest detection limit for N. bombycis was less than 6 pg of DNA, while, this amount was 8 ng for conventional PCR. Out of 80 samples examined, 55, 60, and 62 samples were positive for light microscope, conventional PCR, and Real-Time PCR methods, respectively. The findings suggested that the Real-Time PCR method had a higher ability to detect the causative agent of pebrine disease than the conventional PCR method, and both methods were superior to light microscopy. Therefore, due to the fewer steps and higher accuracy of Real-Time PCR, it can be introduced as a suitable method for diagnosing pebrine disease.

Detection and Characterization of Nosema bombycis Using TEM and SEM Techniques.

Pebrine disease is the most important and dangerous disease of silkworm caused by Nosema bombycis as an obligate intracellular parasitic fungus. It has caused tremendous economic losses in the silk industry in recent years. Given the fact that light microscopy method (with low accuracy) is the only method for diagnosing pebrine disease in the country, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) methods were adopted in this study for accurate morphological identification of the spores causing pebrine disease. Infected larvae and mother moth samples were collected from several farms (Parand, Parnian, Shaft, and Iran Silk Research Center in Gilan province, Iran). The spores were then purified using the sucrose gradient method. From each region, 20 and 10 samples were prepared for SEM and TEM analysis, respectively. In addition, an experiment was performed to evaluate the symptoms of pebrine disease by treating fourth instars with the spores purified for the present study, along with a control group. The results of SEM analysis showed that the mean±SD length and width of spores were 1.99±0.25 to 2.81±0.32 µm, respectively. Based on the obtained results, the size of spores was smaller than the Nosema bombycis (N. bombycis) as the classic species that cause pebrine disease. In addition, transmission electron microscopy (TEM) pictures showed that the grooves of the adult spores were deeper than those of other Nosema species, Vairomorpha, and Pleistophora, and resembled N. bombycis in other studies. Examination of pathogenicity of the studied spores indicated that the disease symptoms in controlled conditions were similar to those in the sampled farms. The most important symptom in fourth and fifth instrars were the small size and no growth in the treatment group compared with the control group. Findings of SEM and TEM analysis showed better morphological and structural details of parasite compared with light microscopy, and demonstrated that the studied species were a native strain of N. bombycis specific to Iran, whose size and other characteristics were unique and introduced for the first time in this study.

Role of Honey Bee Gut Microbiota in the Control of American Foulbrood and European Foulbrood Diseases.

American foulbrood (AFB) and European foulbrood (EFB) are the two most important honey bee brood diseases which impose heavy economic losses to the apiculture industry worldwide by reducing bee population and honey production. Treatment with antibiotics has led to the emergence of antibiotic-resistant strains, calling for alternative safe treatment procedures that could control these diseases. Honey bee gut microbiota is known to affect the overall health of honey bees by enhancing their resistance to a number of diseases via modulation of the immune response and production of different antimicrobial metabolites. The majority of these gut resident bacteria are identified as probiotic bacteria and secure the health of these tiny insects. In the present review, we highlighted the significance of the honey bee gut microbial community and their probiotic potency for the prevention of AFB and EFB diseases in honey bees.

Molecular Identification of Six Honeybee Viruses in Iranian Apiaries.

The identification of honeybee viruses is of serious importance, particularly considering the lack of information on the natural incidence of viral infections in honeybee populations worldwide. Moreover, the global spread of Varroa destructor in honeybee colonies has a significant effect on the viral infection. In the present study, 160 samples of adult bee from apparently healthy colonies but with a background of parasitic diseases, tremor, and paralysis, were collected during 2011-2012. The samples belonged to 23 different provinces of Iran. They were sent to Razi Vaccine and Serum Research Institute, Karaj, Iran, for further analysis, and examined for the presence of viruses using reverse transcription polymerase chain reaction assay. According to the results, out of 160 samples, 9 (5.8 %), 40 (25.6 %), 12 (7.8 %), 34 (21.8 %), 7 (4.5 %), and 29 (18.5%) cases were positive for acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV). The samples collected from 18 provinces (78 %) were positive for at least one virus. Among all samples, 83 (53.2 %) specimens were infected with at least one virus. The highest prevalent virus was BQCV, followed by DWV, SBV, CBPV, ABPV, and KBV, respectively.

Prevalence of American foulbrood in asymptomatic apiaries of Kurdistan, Iran.

AIM: Paenibacillus larvae subsp. larvae is the etiological agent of American foulbrood (AFB), the most virulent bacterial disease of honey bee brood worldwide. In many countries, AFB is a notifiable disease since it is highly contagious, in most cases incurable, and able to kill affected colonies. The aim of this study was to determine the prevalence of P. larvae subsp. larvae in Kurdistan province apiaries by polymerase chain reaction (PCR) technique. MATERIALS AND METHODS: A total of 100 samples were randomly purchased from apiaries in Kurdistan, Iran. Apiaries were randomly sampled in accordance with the instructions of the veterinary organization from different provinces and were tested using PCR method and an exclusive primer of 16S rRNA for the presence of P. larvae subsp. larvae. RESULTS: The results of this study indicated a low level of contamination with P. larvae subsp. larvae in the Kurdistan province. The number of positive samples obtained by PCR was 2%. CONCLUSION: Therefore, monitoring programs for this honeybee disease in Kurdistan should be developed and implemented to ensure that it is detected early and managed.

The contribution of the arginine dihydrolase pathway to energy metabolism by Trichomonas vaginalis.

The enzymes of the arginine dihydrolase pathway were measured in Trichomonas vaginalis hydrogenosome-deficient lines MR-5 and MR-100, and the parent strain TV 10-02. The activities and substrate affinities of arginine deiminase, carbamate kinase and ornithine decarboxylase were similar for the hydrogenosome-deficient lines and the parent TV 10-02. The activity of catabolic ornithine carbamyltransferase, however, was found to be 5-7-fold elevated in the hydrogenosome-deficient lines; the apparent K(m) for citrulline was similar for all of the lines. Putrescine biosynthesis by the hydrogenosome-deficient cell lines was found to be significantly higher than the parent. Incubation of strain MR-100 with U-[14C]-arginine resulted in a 5-fold greater amount of 14CO2 liberated compared to the parent strain TV 10-02. Inclusion of the ornithine decarboxylase inhibitor difluoromethylornithine in these incubations reduced the CO2 production of strain TV 10-02 by 42%, but only inhibited the MR-100 strain by 14.5%, indicative that the majority of the CO2 liberated from arginine by this strain is derived from the elevated activity of ornithine carbamyltransferase. Despite the increased flow through the arginine dihydrolase pathway, the energy gain to the parasite is approximately 10% of that from glucose, thus, under the growth conditions used in this study carbohydrate metabolism provides the bulk of the ATP for the parasite.

Inhibition of Trichomonas vaginalis ornithine decarboxylase by amino acid analogs.

Ornithine decarboxylase (ODC) from Trichomonas vaginalis was inhibited irreversibly by several substrate analogs. Of these, DL-alpha-monofluoromethyldehydroornithine (MFMDO) and DL-alpha-monofluoromethylornithine (MFMO) were the most potent. The enzyme was unaffected by putrescine analogs suggesting that differences exist between the regulation of the trichomonad enzyme and that in other eukaryotes. In culture the ornithine analogs strongly inhibited putrescine synthesis and increased the generation time after 24 hr of exposure. In a semi-defined growth medium MFMDO methyl and ethyl esters increased the generation time from 4.5 hr to 9.0 and 8.2 hr, respectively. In standard undefined growth medium the trichomonad ODC was fully induced only after 15 hr (late log) and had an extended half-life of greater than 8 hr.

Trichomonas vaginalis: characterization of ornithine decarboxylase.

Ornithine decarboxylase (ODC), the lead enzyme in polyamine biosynthesis, was partially purified from Trichomonas vaginalis and its kinetic properties were studied. The enzyme appears to be of special significance in this anaerobic parasite, since the arginine dihydrolase pathway generates ATP as well as putrescine from arginine. ODC from T. vaginalis had a broad substrate specificity, decarboxylating ornithine (100%), lysine (1.0%) and arginine (0.1%). The enzyme had a pH optimum of 6.5, a temperature optimum of 37 degrees C and was pyridoxal 5'-phosphate-dependent. Attempts to separate ornithine- from lysine-decarboxylating activity by thermal-stability and pH-optima curves were not successful. Although Km values for ornithine and lysine were 109 and 91 microM respectively, and the Vmax values for these substrates were 1282 and 13 nmol/min per mg of protein respectively, the most important intracellular substrate is ornithine, since intracellular ornithine levels are 3.5 times those of lysine and extracellular putrescine levels are 7.5 times those of cadaverine. Ornithine was also an effective inhibitor of lysine-decarboxylating activity (Ki 150 microM), whereas lysine was relatively ineffective as inhibitor of ornithine-decarboxylating activity (Ki 14.5 mM). Crude ODC activity was localized (86%) in the 43,000 g supernatant and 3303-fold purification was obtained by (NH4)2SO4 salting and DEAE-Sephacel, agarose-gel and hydroxyapatite chromatography steps. The enzyme bound difluoro[3H]methylornithine ([3H]DFMO) with a ratio of drug bound to activity of 2500 fmol/unit, where 1 unit corresponds to 1 nmol of CO2 released from ornithine/min. The enzyme had a native M(r) of 210000 (gel filtration), with a subunit M(r) of 55,000 (by SDS/PAGE), suggesting that the trichomonad enzyme is a tetramer. From the subunit M(r) and binding ratio of DFMO, there is about 137 ng of ODC per mg of T. vaginalis protein (0.013%). The significant amount of ODC protein present supports the view that putrescine synthesis in T. vaginalis plays an important role in the metabolism of the parasite.

Subcellular localization of the enzymes of the arginine dihydrolase pathway in Trichomonas vaginalis and Tritrichomonas foetus.

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56-80%) in the non sedimentable fraction of both species. A large proportion (35-40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non-ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.