Social network analysis of the movement of poultry to and from live bird markets in Bali and Lombok, Indonesia.

Highly pathogenic avian influenza H5N1 has resulted in large losses to the Indonesian poultry sector. Evidence suggests that live bird markets (LBMs) play an important role in the epidemiology of the disease. Knowledge of the frequency and type of contact between the various poultry market players should allow animal health authorities to develop a better understanding of factors influencing virus transmission between Indonesian villages. A questionnaire-based cross-sectional survey was conducted in 17 LBMs on the neighbouring Indonesian islands of Bali and Lombok to investigate the movement patterns of poultry to and from markets. Using social network analyses, a network of contacts was created for each island from a total of 413 live poultry traders and 134 customers. Individual nodes with high degree and/or betweenness were identified in each network. The Lombok network was more dense and connected than the Bali network indicating that disease transmission would be more efficient in the Lombok network. Our findings indicate that whilst live poultry are typically transported over relatively short distances of approximately 10 km, it is not uncommon for traders and customers to travel in excess of 100 km to buy or sell poultry, which may facilitate the spread of disease over a large geographical area. This study highlights the different roles markets play in poultry movement networks and their potential for disease dissemination. The identification of highly influential market nodes allows authorities to target HPAI surveillance activities to locations where disease is more likely to be present, which is crucial in low-resource settings.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of ∼50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (∼100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.