Detection and Molecular Characterization of 'Candidatus Liberibacter asiaticus' and Citrus Tristeza Virus Associated with Citrus Decline in Bhutan.

Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of 'Candidatus Liberibacter asiaticus' (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of 'Ca. L. asiaticus' was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The 'Ca. L. asiaticus' isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of 'Ca. L. asiaticus' and CTV isolates and their association with citrus decline in Bhutan.

Internet Addiction among Secondary School Adolescents: A Mixed Methods Study.

INTRODUCTION: Excessive use of internet can lead to "Internet Addiction". A sharp rise in the number of internet users among adolescents in Bhutan have been observed lately which could have potential side-effects on their health. The aim of this study was to find the prevalence of internet addiction and psychological co-morbidities among the secondary school-going adolescents. METHODS: This is a descriptive cross-sectional quan-qual sequential mixed method design with a quantitative component and qualitative component. The study was conducted between 1st May and 30st November 2017 conducted in twelve schools across Bhutan after taking ethical clearance was from Research Ethics Board of Health of Bhutan. Data were doubleentered in EpiData Entry, validated and imported into STATA version 12.0 for analysis. Point estimate at 95% CI was calculated along with frequency and proportion for binary data. RESULTS: Among 721 adolescents from twelve selected schools, prevalence of internet addiction was 248 (34.440%) at , 95% Confidence Interval (31-38%). Out of A total of 586 (81%) preferred smartphone for internet use with 388 (54%) actually using it. Depression and anxietywere the associated psychological co-morbidities seen along with internet addiction. Internet addiction was high among those using internet at home, using smartphone for internet access, social networking and night time internet use were associated with internet addiction. Boredom, stress/anxiety and peer pressure were triggers of internet use. Internet addiction also affected academic performance, social interactions and sleep. CONCLUSIONS: Prevalence of internet addiction among adolescents in Bhutan is high comparing to previous standard data which requires immediate attention.

Detection of Echinococcus granulosus and Echinococcus ortleppi in Bhutan.

In this pilot study, fecal samples were collected from community dogs around slaughterhouses and from the city of Thimphu (n=138) as well as from carnivores in the forest area around a farm in Bhutan (n=28). Samples were analyzed microscopically for the presence of taeniid eggs by the floatation and sieving method. Further molecular analyses of 20 samples of community dogs positive for taeniid eggs confirmed 10 Echinococcus granulosus sensu lato and one Taenia hydatigena case. From 14 environmental fecal samples from the forest area positive for taeniid eggs, one contained E. granulosus s.l., six T. hydatigena and one Taenia taeniaeformis DNA. In the remaining samples considered positive for taeniid eggs, no molecular confirmation could be achieved. Additionally, Echinococcus cysts were collected from locally slaughtered cattle and imported cattle organs. Seven Echinococcus cysts (one fertile) from the local animals and 35 (four fertile) from imported cattle organs were confirmed as E. granulosus (G1-3) by PCR/sequencing. One Echinococcus cyst each from a local animal and from an imported cattle organ (both fertile) were confirmed to be Echinococcus ortleppi (G5). Sterile Echinococcus cysts were also collected from local yaks (n=10), and all revealed to be E. granulosus (G1-G3). Hospital records of cystic echinococcosis in humans and the presence of Echinococcus spp. in dogs and ungulates indicate the existence of local transmission for both E. ortleppi and E. granulosus in Bhutan.