Effects of Achillea tenuifolia Lam. hydro-alcoholic extract on anxiety-like behavior and reproductive parameters in rat model of chronic restraint stress.

INTRODUCTION: Achillea tenuifolia Lam (AT) has several biological activities and medicinal properties. In this study, we elucidated the impact of the AT on anxiety-related behaviors, reproductive parameters, antioxidant capacity in male rats subjected to chronic restraint stress (CRS). METHODS: 35 Wistar rats were divided into five groups: control, CRS-control (received normal saline) and three CRS-treated groups received AT extract (100, 150, and 200 mg/kg body weight) for 21 consequences days. To induce CRS rats, the rats were immobilized for 21 days and received the extract orally. On the last day of treatment, anxiety-related behaviors were assessed through the sucrose preference test (SPT) as well as elevated plus maze (EPM) tests. Corticosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), testosterone levels were evaluated to determine reproductive capacity. Sperm parameters including the total count, motility, and viability were also analyzed. Weight of body, testis and seminal vesicles was measured as well. RESULTS: The findings revealed that 100, 150, and 200 mg/kg of AT extract had anxiolytic effects in CRS rats, as confirmed by the EPM test and SPT. In addition, AT extract could improve fertile capacity and sperm quality to varying degrees. The level of corticosterone had decreased, whereas the level of LH, FSH and testosterone had increased in CRS-treated rats. Moreover, the reduced level of MDA coincided with an increased rate of antioxidant capacity. Our findings suggest that AT extract could alleviate stress-induced dysfunctions. CONCLUSION: Overall, these observations would infer that AT extract could improve fertility capacity and behavioral impairment in the stress conditions. GRAPHICAL ABSTRACT: Assumption pathway describing the probability underlying mechanism of CRS-induced anxiety and reproductive toxicity and protective effect of AT.

Prevalence and Early Detection of Hypodermosis in Goats using a Competitive ELISA System in Lorestan, Iran.

This study aimed to determine the prevalence and early detection of hypodermosis in goats by the investigation of Przhevalskiana larvae and sera collected from the infested animals. This study was conducted in Lorestan province, located in the South-West of Iran, from April 2017 up to April 2018. A total of 3350 goats slaughtered in Lorestan abattoirs were investigated by clinical-parasitological examinations in different periods. The larvae were collected from the back and flank regions of the slaughtered goats. The number of infested animals, gender and age, number of maggots present on the body of each animal, location, and larval stage of warble flies were recorded in this study. To detect an infestation in the early period, a total of 150 blood samples were randomly collected from the field animals in Lorestan, Iran. The morphological findings showed that out of 3350 goats examined, 706 (21.07%) goats were infested. Furthermore, three species of Przhevalskiana, including P. Silenus (n=726, 50.07%), P.crossii (n=440, 30.43%), and P. aegagri (n=284, 19.59%) were recognized as the causative agents of goat hypodermosis in this province. No significant difference was observed between genders and/or among the age groups (P>0.05). The anti-Przhevalskiana antibodies in the serum samples were detected using ELISA from August up to mid-September (summer). Clinical diagnosis of infestation was usually performed from late October until mid-March (winter) by visual observations and direct palpation of warbles in the back and flank regions of the animals. It could be concluded that the use of ELISA can help to detect hypodermosis among goats in the early stages.

Renoprotective effects of prazosin on ischemia-reperfusion injury in rats.

BACKGROUND: Renal ischemia-reperfusion (IR) injury is one of the main leading causes of acute kidney injury associated with inflammation, oxidative stress and cell apoptosis. We studied the effects of prazosin, as a specific blocker of α1-AR, on renal IR injury. METHODS: Rats were divided into normal control; untreated IR and prazosin-treated IR (1 mg/kg body weight). Prazosin was administered by intraperitoneal injection 30 min prior to IR induction. The level of urea/creatinine and oxidative factors were detected by colorimetric methods. Apoptosis-associated factors, inflammatory, and signaling proteins were analyzed in renal tissue. The abnormalities of renal histopathology were detected by immunohistochemistry. RESULTS: Administration of prazosin to IR rats ameliorated serum urea and creatinine and IR-induced histopathological damages. Lipid peroxidation was significantly improved after treatment by prazosin in IR injury rats, however, antioxidant status was not affected. Rats subjected to IR injury activated Bax protein and NF-κB mediated inflammatory response. Moreover, treatment with prazosin inhibited renal NF-κB activation, resulting in a significant decline in pro-inflammatory cytokine of IL-6. CONCLUSION: These findings suggest that prazosin could be a good candidate to attenuate renal IR injury due to its ability to modulate renal function, apoptosis and inflammation.

Serodiagnosis of Przhevalskiana spp. Infestation in Goats Using a Competitive ELISA.

Goat warble-fly infestation is one of the parasitic diseases caused by the larvae of przhevalskiana spp. It is known to be a major challenge for health and welfare in infested goats and causes high economic losses in livestock worldwide. The detection of goat grub was previously conducted by direct palpation of second and third stage larvae in the back and flank site of the live animals or visual evaluation of the carcasses in slaughterhouses. However, due to the small size of the first instar larvae of przhevalskiana (less than 1 mm during emerging from the egg), some of the infected cases were ignored and recorded as negative samples. Immunodiagnostic procedures as easy and cost-effective diagnostic methods provide early detection of myiasis in living animals (even when the larvae are still migrating or are undetectable in the animal body).This study was conducted to evaluate the competitive enzyme-linked immunosorbent assay (ELISA) system in order to detect the antibodies of przhevalskiana larvae in the goat sera. In order to prepare the larval antigen, 200 first instar larvae of przhevalskiana were collected from the subdermal region of the back and flanks of the infested goats in Khoramabad slaughterhouse, Khoramabad, Iran, from September 2017 to March 2018. Totally, 37 and 46 sera samples were taken from the infected and uninfected goats. The sensitivity and specificity at cut-off 3SD were obtained at 89.18% and 84.78%. Moreover, the measures of inter-and intra-assay coefficients of variability to express the precision or replicability of ELISA kit results were 5.33% and 2.81%, respectively.

UNRAVEL: big data analytics research data platform to improve care of patients with cardiomyopathies using routine electronic health records and standardised biobanking.

INTRODUCTION: Despite major advances in our understanding of genetic cardiomyopathies, they remain the leading cause of premature sudden cardiac death and end-stage heart failure in persons under the age of 60 years. Integrated research databases based on a large number of patients may provide a scaffold for future research. Using routine electronic health records and standardised biobanking, big data analysis on a larger number of patients and investigations are possible. In this article, we describe the UNRAVEL research data platform embedded in routine practice to facilitate research in genetic cardiomyopathies. DESIGN: Eligible participants with proven or suspected cardiac disease and their relatives are asked for permission to use their data and to draw blood for biobanking. Routinely collected clinical data are included in a research database by weekly extraction. A text-mining tool has been developed to enrich UNRAVEL with unstructured data in clinical notes. PRELIMINARY RESULTS: Thus far, 828 individuals with a median age of 57 years have been included, 58% of whom are male. All data are captured in a temporal sequence amounting to a total of 18,565 electrocardiograms, 3619 echocardiograms, data from over 20,000 radiological examinations and 650,000 individual laboratory measurements. CONCLUSION: Integration of routine electronic health care in a research data platform allows efficient data collection, including all investigations in chronological sequence. Trials embedded in the electronic health record are now possible, providing cost-effective ways to answer clinical questions. We explicitly welcome national and international collaboration and have provided our protocols and other materials on www.unravelrdp.nl .

Variation in bacterial endosymbionts associated with the date palm hopper, Ommatissus lybicus populations.

The date palm hopper, Ommatissus lybicus, is a key pest of the date palm, which is expected to be comprised of many allopatric populations. The current study was carried out to determine bacterial endosymbiont diversity in the different populations of this pest. Ten date palm hopper populations were collected from the main date palm growing regions in Iran and an additional four samples from Pakistan, Oman, Egypt and Tunisia for detection of primary and secondary endosymbionts using polymerase chain reaction (PCR) assay with their specific primers. The PCR products were directly sequenced and edited using SeqMan software. The consensus sequences were subjected to a BLAST similarity search. The results revealed the presence of 'Candidatus Sulcia muelleri' (primary endosymbiont) and Wolbachia, Arsenophonus and Enterobacter (secondary endosymbionts) in all populations. This assay failed to detect 'Candidatus Nasuia deltocephalinicola' and Serratia in these populations. 'Ca. S. muelleri' exhibited a 100% infection frequency in populations and Wolbachia, Arsenophonus and Enterobacter demonstrated 100, 93.04 and 97.39% infection frequencies, respectively. The infection rate of Arsenophonus and Enterobacter ranged from 75 to 100% and 62.5 to 100%, respectively, in different populations of the insect. The results demonstrated multiple infections by 'Ca. Sulcia muelleri', Wolbachia, Arsenophonus and Enterobacter in the populations and may suggest significant roles for these endosymbionts on date palm hopper population fitness. This study provides an insight to endosymbiont variation in the date palm hopper populations; however, further investigation is needed to examine how these endosymbionts may affect host fitness.

Association of angiogenic cytokines (VEGF-A and VEGF-C) and clinical characteristic in women with unexplained recurrent miscarriage.

INTRODUCTION: Recurrent miscarriage (RM) defined as 2 or more spontaneous miscarriage before 20 weeks of gestation, affects at least 1 % of couples trying to conceive. In over 50 % of cases, the cause of the loss of pregnancy remains unexplained. Reduced expression of Angiogenic factors such as: vascular endothelial growth factor (VEGF-A) and VEGF-C has been linked with spontaneous miscarriage, likely due to defective foetal and placental angiogenesis. AIMS AND OBJECTIVES: To investigate the relationships between serum level of VEGF-A and VEGF-C with clinical characteristic in women with URM and compare to pregnant and healthy women. MATERIALS AND METHODS: A case-control study, which was conducted between 90 non-pregnant women with history of RM, age-matched with 70 non-pregnant women without history of recurrent abortion with at least one child (controls) and 70 pregnant women without history of recurrent abortion with at least one child (controls). Those with unexplained RM were eligible. Demographic and Anthropometric data were retrieved by pre-test questionnaire and serum level of VEGF-A and VEGF-C measured by ELISA kit. RESULTS: This study showeds that maternal levels of VEGF-A and VEGF-C were distinctly lower in RSA (189.87±88.1 vs 238.8±99.6) compared to healthy (239.1±99.7 vs 275.5±133.08) and pregnant (301.5±76.4 vs 402.5±128.6) women as control groups. Univariate analysis demonstrated that clinical characteristic factors were significantly associated with concentration of VEGF-A and VEGF-C in cases and controls. CONCLUSIONS: Our findings suggest that these molecules could be used as potential predictive markers of miscarriage in these women presenting with URM (Tab. 4, Fig. 5, Ref. 40).

RNAi-induced silencing in floral tissues of Petunia hybrida by agroinfiltration: a rapid assay for chalcone isomerase gene function analysis.

Variegation in flower color is commonly observed in many plant species and also occurs on Petunia (Petunia hybrida) as an ornamental plant. Variegated plants are of highly valuable in the floricultural market. Agroinfiltration is an Agrobacterium-mediated transient assay for the analysis of gene function and genetic modification in leaves, flowers and fruit tissues of various plants. Transient RNAi-induced silencing by agroinfiltration has been developed in leaves and fruits of several plant species. Here we report the establishment of a transient hairpin RNAi-induced silencing system for color modification assay in floral tissues of Petunia with different colors. chiRNAi construct was cloned into the pBI121 vector under the control of 35S promoter. Transient RNA silencing of chi in the floral tissues of Petunia was induced by delivering 530 bp chi hairpin RNAs (hpRNAs) into the petals of flowers using agroinfiltration. Impaired anthocyanin accumulation and reduction of endogenous mRNAs of the corresponding targets were observed in the infiltrated areas of the petals of four colors of Petunia. Silencing of the endogenous chi mRNAs was highly effective in reduction of chi gene and anthocyanin accumulation. This transient silencing system is a prototype for modification of the anthocyanin biosynthetic pathway in Petunia through chi gene suppression.

Simultaneous determination of hydroquinone and catechol at gold nanoparticles mesoporous silica modified carbon paste electrode.

A new electrochemical sensor based on gold nanoparticles mesoporous silica modified carbon paste electrode (AuNPs-MPS) was developed for simultaneous determination of hydroquinone and catechol. Morphology and structure of the AuNPs-MPS were characterized by transmission electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. The electrochemical behavior of hydroquinone and catechol were investigated using square wave voltammetry and the results indicate that the electrochemical responses are improved significantly at the modified electrode. The observed oxidative peaks separation of about 120mV made possible the simultaneous determination of hydroquinone and catechol in their binary-mixture. Under the optimized condition, a linear dynamic range of 10.0µM-1.0mM range for hydroquinone with the detection limit of 1.2µM and from 30.0µM-1.0mM for catechol with the detection limit of 1.1µM were obtained. The applicability of the method was demonstrated by the recovery studies of hydroquinone and catechol in spiked tap water samples.

[Constitution of a New Specialised Pediatric Home Care Team - First Year Experience]. / Aufbau eines Palliativteams für Kinder und Jugendliche (SAPV-KJ).

BACKGROUND: Since the amendment of the Social Law V in Germany in 2007 the financial basis for a Specialised Home Palliative Care for Children (SHPC) for children was established. In Hesse 3 different SHPC teams entered into collective negotiations with health insurance companies. In 2014, the team of the University Children's Hospital in Giessen started to treat the first patient with a lead time of two months. METHODS: Thus in this paper the development of a SHPC team is described. After the first year anonymized patients data were retrospectively analyzed. RESULTS: Within 12 months 35 patients, 24 females and 11 males, were treated. All of the 6 patients who died, died at home. Calculated 48 weeks survival was 78%. 45% of the patients suffered from malignancies, 34% of malformations and 34% had metabolic disorders. 51% needed crisis intervention and 51% infusion therapy. Only 26% of parents denied cardiopulmonary resuscitation (CPR). Only 10% of the patients or their families received professional psychological care. CONCLUSION: Formation of a SHPC is feasible within a short time period once a financial basis is established. So, empathic guidance of families to help decision making for emergency situations are considered to be important. Analysis of patient's data after one year could help to improve the quality of care. Our data provides information for developing a palliative care team und could motivate colleagues to start the job.

Lower serum level of anti-tetanus toxin antibodies in patients with type 2 diabetes mellitus.

AIM: To evaluate the serum levels of anti-tetanus toxin antibodies (anti-TTA) in patients with type 2 diabetes mellitus (DM) and in a control group. METHODS: Totally, 100 patients with type 2 DM and 100 age- and sex-matched healthy individuals were enrolled to study. The presence of type 2 DM confirmed according to the clinical and para-clinical criteria such as fasting plasma glucose above 126 mg/dl. A peripheral blood sample was collected from all subjects. The serum samples of participants tested for the levels of anti-TTA by ELISA method. The serum antitoxin concentration 0.1 IU/mL was considered as a protective level of antibody. RESULTS: The seroprotective rate in healthy group was significantly higher than diabetic group (99% vs. 92%; p<0.02). The mean titer of anti-TTA in healthy group (5.32 ± 0.26 IU/ml) was also significantly higher than diabetic patients (3.46 ± 0.26 IU/ml; p>0.001). In diabetic men the mean titer of anti-TTA was significantly higher in comparison to diabetic women (3.94 ± 0.34 IU/ml vs 2.59 ± 0.36 IU/ml; p<0.01). In diabetic patients the seroprotective rate and the mean titer of anti-TTA in subjects with age >40 years was also lower in comparison to those with age <40 years (89.23% vs 97.14%; p<0.15 and 4.57 ± 0.38 IU/ml vs 2.86 ± 0.32 IU/ml; P<0.002, respectively). The mean titer of anti-TTA was significantly higher in patients with diabetes duration <5 years in comparison to patients with disease duration >5 years (3.91 ± 0.35 IU/ml vs 2.85 ± 0.38 IU/ml; p<0.04). CONCLUSION: these results showed lower levels of anti-TTA in patients with type 2 DM, in diabetic women, in patients aged >40 years and in diabetic patients with disease duration >5 years.

Pareto design of state feedback tracking control of a biped robot via multiobjective PSO in comparison with sigma method and genetic algorithms: modified NSGAII and MATLAB's toolbox.

An optimal robust state feedback tracking controller is introduced to control a biped robot. In the literature, the parameters of the controller are usually determined by a tedious trial and error process. To eliminate this process and design the parameters of the proposed controller, the multiobjective evolutionary algorithms, that is, the proposed method, modified NSGAII, Sigma method, and MATLAB's Toolbox MOGA, are employed in this study. Among the used evolutionary optimization algorithms to design the controller for biped robots, the proposed method operates better in the aspect of designing the controller since it provides ample opportunities for designers to choose the most appropriate point based upon the design criteria. Three points are chosen from the nondominated solutions of the obtained Pareto front based on two conflicting objective functions, that is, the normalized summation of angle errors and normalized summation of control effort. Obtained results elucidate the efficiency of the proposed controller in order to control a biped robot.

Factors influencing range of motion after total knee arthroplasty.

BACKGROUND: The range of motion after a total knee arthroplasty is an important clinical outcome affecting the life of the patient. The aim of this study was to determine the most important factors influencing the postoperative knee flexion in Tehran, Iran. METHODS: Between July 2007 and January 2009, on 95 cases of total knee joint replacement (89 patients), who were followed for 1 year postoperatively, the risk factors were assessed. Patient demographics (sex, age, body mass index, previous surgery, preoperative Knee Society System score, and preoperative range of motion) as well as radiographic measurement for preoperative tibiofemoral angle were statistically analyzed and the probable predictors entered into a linear regression model. RESULTS: Univariate analysis showed that age, preoperative flexion angle, preoperative flexion arc and preoperative tibiofemoral angle had significant correlation with the postoperative flexion angle. The linear regression model on the other hand revealed that preoperative flexion angle and preoperative tibiofemoral angle were the true predictors of the postoperative flexion angle with coefficients of 0.64 and -0.21, respectively. CONCLUSION: Better range of motion before the surgery with a lower tibiofemoral varus/valgus angle were more likely to result in a better range of motion after the surgery, suggesting that an appropriate timing for the surgery when the knee joint is still in a better function can lead to a better outcome.

Unerupted primary molar teeth positioned inferior to the permanent premolar: a case report.

Primary tooth impaction is a rare finding during the development of primary dentition. Several factors contribute to the impaction of a deciduous tooth. This report describes the diagnosis and treatment of a 10-year-old boy who presented an impacted second primary mandibular molar. This tooth, located inferior to the second premolar together with an odontoma, was positioned superior to the premolar teeth. Treatment consisted of surgical removal of the impacted deciduous tooth and odontoma and placement of a passive lower lingual holding arch. Periodic examination was indicated for follow-up. Early intervention was recommended to manage orofacial disfigurement and to avoid consequent problems.

Evaluation of PAN-based manganese dioxide composite for the sorptive removal of cesium-137 from aqueous solutions.

Hydrous manganese dioxide-polyacrylonitrile (MnO(2)-PAN) was chemically synthesized and evaluated, as an organic-inorganic composite material, for the removal of radio-contaminant cesium-137 from aqueous solutions. The physico-chemical characterization was carried out by Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), CHN elemental analysis, scanning electron microscopy (SEM), nitrogen adsorption-desorption studies and thermogravimetry-differential scanning calorimetry (TGA-DSC). Batch experiments were carried out as a function of contact time, interference of the coexisting ions and initial pH of adsorptive solution applying a radiotracer technique. The effect of temperature on the distribution coefficient of cesium has been utilized in order to evaluate the changes in the standard thermodynamic parameters. The results indicated that Cs(+) ions could be efficiently removed using MnO(2)-PAN composite in the pH range of 4-9 from aqueous solutions and the uptake of cesium is affected to varying degrees by the presence of some diverse co-ions. The equilibrium isotherms have been determined and the sorption data were successfully modeled using Freundlich model.

The clinical manifestation, evaluation and surgical management of sphenoid sinus mucoceles: a case series and literature review.

OBJECTIVE: This study was designed to describe clinical manifestation, treatment and outcomes in patients with sphenoid sinus mucocele presenting to Amiralam Hospital, Tehran, Iran. METHODS: A review of 43280 medical records covering a period of 10 years included just 12 patients with sphenoid sinus mucocele presenting to our teaching centre. RESULTS: The mean age of the patients was 35 years (ranging from 19 to 52 years). Headache was the most frequent symptom in 9 of the 12 patients. Visual disorders were noted in five patients. Two patients underwent external ethmoidectomy accompanied by intranasal sphenoidotomy while transseptal sphenoidotomy was performed for five patients and five patients underwent endoscopic endonasal sphenoidotomy. Wide spectrum antibiotics were given to all the patients for 3 to 5 days in order to prevent any iatrogenic infection. All patients did well after surgery without postoperative complications. CONCLUSIONS: Sphenoid sinus mucocele should be considered in the differential diagnosis of cystic lesions presenting as visual disturbance. Retro-orbital headache with or without visual symptoms is a leading symptom. CT and MRI are essential in the diagnosis of sphenoid disease.

Familial Simpson-Golabi-Behmel syndrome: studies of X-chromosome inactivation and clinical phenotypes in two female individuals with GPC3 mutations.

Simpson-Golabi-Behmel syndrome (SGBS) is an overgrowth/multiple congenital anomalies syndrome with an X-linked inheritance. Most cases of SGBS are attributed to mutations in the glypican 3-gene (GPC3), which is highly expressed in the mesodermal embryonic tissues and involves in a local growth regulation. Typical clinical features include pre/postnatal overgrowth, developmental delay, macrocephaly, characteristic facies with prominent eyes and macroglossia, diaphragmatic hernia, congenital heart defects, kidney anomalies, and skeletal anomalies. Obligate carrier females with GPC3 mutations are usually asymptomatic or with mild symptoms. It is thought that skewed X-inactivation is the underlining mechanism for the female patients to present with findings of SGBS. We identified three siblings with typical SGBS (two male and one female cases) and their mother with very mild symptoms in a family carrying c.256C>T (p.Arg86X) mutation in GPC3. X-inactivation studies on the androgen-receptor gene (AR) and the Fragile XE (FRAXE) gene were performed with blood, buccal swabs, and fibroblasts in the carrier females. The studies with blood showed moderately skewed X-inactivation with paternal X-chromosome being preferentially inactivated (71-80% inactivated) in the female patient with SGBS and no skewing was shown in the mother with very mild symptoms. The X-inactivation studies in the mother showed inactivation of the X-chromosome with the mutation by 57%. This suggests that loss of the functional GPC3 protein by 43% is closed to the threshold to develop the SGBS phenotype. Studies with buccal swabs and fibroblasts failed to show different X-inactivation patterns between the two female individuals.

Alfalfa (Medicago sativa L.) shoot saponins: identification and bio-activity by the assessment of aphid feeding.

Biochemical components in alfalfa (Medicago sativa L.), such as saponins, can act as protecting factors against bio-stresses. Saponins are also antifeedants and show oral toxicity towards higher and lower animals. Changes in saponins, such as variation in the carbon skeleton, or hydrolysis of saponin glycosides and other conjugates, may change their biological effects. The aims of this research were to study saponin variation in different growth stages of alfalfa and to investigate the biological role of saponins in the spotted alfalfa aphid, Therioaphis maculata. Saponins from alfalfa shoots in different growth stages were extracted, chemically purified and analysed by TLC. Specific saponins such as soyasaponin1 from root and shoot and two bisdesmosides of medicagenic acid, one from shoot and another from root tissues, were identified using reference compounds allowing changes in saponin composition during plant development in different shoot tissues of alfalfa to be assessed. The response of the alfalfa aphid to feeding on alfalfa in different growth stages was studied. No significant difference in the survival of aphids, from neonate to adult, was observed, but due to the antibiotic effects of saponins, two differences were found in the onset of nymph production and cumulative nymph production. The results show that the saponin composition in alfalfa changes with plant development and this, in turn, can often negatively affect the development of specific insect pests such as the spotted alfalfa aphid, suggesting a possible biological role of alfalfa saponins.

Witches'-Broom Disease of Lime Affects Seed Germination and Seedling Growth But Is Not Seed Transmissible.

Witches'-broom disease of lime (WBDL), caused by 'Candidatus Phytoplasma aurantifolia', has devastated many Mexican lime orchards and is currently a threat to lime production in neighboring provinces of southern Iran. Several reports have suggested transmission and spread of WBDL phytoplasma through the seed of infected plants. In the present study, claims of seed transmission of this phytoplasma were examined. Fruit were collected from infected trees in the infested areas of Minab (Hormozgan Province) and from symptomless trees in noninfested areas. Lime seed from symptomless and witches'-broom-affected trees were sown in separate beds in an insect-proof screenhouse and the resulting seedlings were examined for phytoplasmal infection. Leaf, stem, and root samples were collected from both groups of seedlings every 3 months for 2 years and tested for WBDL phytoplasma using direct and nested polymerase chain reaction (PCR). Repeated PCR tests on the seedlings did not reveal the presence of phytoplasmal DNA. Likewise, symptoms of the disease were not observed on these seedlings after 2 years. PCR assays detected the phytoplasma in coats of some seed from infected trees; however, no excised embryos were positive for the phytoplasma. All positive PCR results were confirmed by restriction fragment length polymorphism assay. One-year-old seedlings derived from seed of noninfected plants appeared more vigorous in terms of height, number of leaves, and fresh weight of shoot compared with those from infected trees. The germination percentage, mean daily germination, peak value, and germination value were significantly higher for seed of fruit from noninfected trees and seed from fruit on asymptomatic branches of infected trees than those from fruit on symptomatic branches of infected trees.