MEGA-10

The research protocol was designed to guide collaborating groups in cities in developing and developed countries to use a standardized method to assess their community’s age-friendliness and identify areas for remedial action at the same time as they contributed to WHO’s objective of identifying the essential features that constitute an age-friendly city. The protocol had to be straightforward, require a minimum of material and technical resources, and be adaptable to varying cultural and economic contexts. The broad lines of the methodology were defined in consultation with a group of advisers who had expertise in policy, community action, or qualitative research, and who were familiar with the social context of developing as well as developed countries. The draft protocol was then reviewed and finalized at a workshop in Vancouver, Canada, in March 2006, attended by project leaders from most of the participating cities then enlisted. The “Vancouver protocol,”17 as it became known, was adopted in all cities that participated in the research.
With adaptations to accommodate communities in widely varying countries, a set of eight features of urban life was identified for examination in the Vancouver protocol based on the WHO concept of active aging as well as on the key features identified by existing elder-friendly community models. The topic areas explored in the focus groups were: outdoor spaces and public buildings, transportation, housing, social participation, respect and social inclusion, civic participation and employment, communication and information, community support, and health services. In semi-structured focus groups, participants were asked to identify the positive and negative features of the city in each of these eight major areas and to offer suggestions for improvement. To prepare participants for the discussions, local project leaders were encouraged to distribute the list of topic areas when participants were recruited.
In all, the focus group research was conducted in 33 cities1 situated in 22 countries of North and South America, Western Europe, Russia, the Eastern Mediterranean, Africa, the Indian sub-continent, Oceania, and the Pacific Rim. Of the 33 participating sites, 19 were in developing countries, and 14 were in industrialized countries. The cities represent the diversity of contemporary urban settings. There were seven mega-cities with over 10 million inhabitants (Mexico City, Moscow, New Delhi, Rio de Janeiro, Istanbul, Shanghai, and Tokyo) and large metropolises, such as Nairobi and London. Also included were smaller but regionally significant urban centers such as Geneva, Amman, Melbourne, Islamabad, Kingston, and Halifax, as well as towns located near metropolitan areas [e.g., Melville, adjacent to Perth, Australia; Saanich, near Buenos Aires, Argentina; and La Plata, close to Buenos Aires (Argentina)]. Project sites and leaders were recruited through informal networks of the WHO project leaders, formal representation to municipal or state governments, and promotion of the project at professional conferences. A grant from the Public Health Agency of Canada allowed WHO to award small research contracts to non-government organizations and research centers to enable the inclusion of several project sites in the developing world: Kingston, Montego Bay, Mexico City, San Jose, Rio de Janeiro, La Plata, Tripoli, and Nairobi. Also, Help the Aged UK contracted with HelpAge India to conduct the research in two sites in India: New Delhi and Udaipur.
Informed consent and local ethics review was mandatory, recognizing variations in local practices and legal requirements. A procedure for obtaining informed consent adapted from the Pan-American Health Organization SABE Survey (Survey on Health, Wellbeing and Aging in Latin America and the Caribbean)18 was proposed for study sites which had no accepted practices in place.

DNA and/or RNA extracted from freshly collected tissue fragments from the calves and the aborted fetus (Boom et al. 1990 (link)), associated with proteinase K (Ambion, Grand Island, NY, USA), and a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate method (Alfieri et al. 2006 (link)), was used in PCR assays designed to amplify specific amplicons of principal infectious disease agents of cattle. These PCR protocols targeted the ovine herpesvirus type 2 (OvHV-2) tegument protein gene (Baxter et al. 1993 (link)), the glycoprotein C gene of bovine herpesvirus-1 (BoHV-1) and −5 (Claus et al. 2005 (link)), the listeriolysin (hlyA) gene of Listeria monocytogenes (Wesley et al. 2002 (link)), the 16S rRNA gene of H. somni (Angen et al. 1998 (link)), and the 5′-UTR region of pestivirus (Vilček et al. 1994 (link)). Additionally, the feces from the calf with clinical diagnosis of enteritis were evaluated for the presence of the nucleoprotein gene of bovine coronavirus BCoV (Takiuchi et al. 2006 (link)) and G (VP7) and P (VP4) genes of bovine group A rotavirus, BoRV-A (Gouvea et al. 1990 (link); Gentsch et al. 1992 (link)).
Positive controls consisted of DNA/RNA from previous cases: OvHV-2 (Headley et al. 2012b ), L. monocytogenes (Headley et al. 2012a ), cell culture-adapted Los Angeles and AA Par strains of BoHV-1 and −5, respectively (Claus et al. 2005 (link)), BVDV (NADL strain)-infected Madin Darby bovine kidney cells, and BCoV (Takiuchi et al. 2006 (link)). No positive control was included in the H. somni PCR assays. Nuclease-free water (Invitrogen Corp. Carlsbad, CA, USA) was used as negative controls in all PCR assays. All PCR products were separated by electrophoresis in 2 % agarose gels, stained with ethidium bromide, and examined under ultraviolet light.
The amplified PCR products were then purified (illustra GFX PCR DNA and Gel Band Purification Kit, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and submitted for direct sequencing using the forward and reverse primers. The partial nucleotide sequences were initially compared by the BLAST (http://www.ncbi.nlm.nih.gov/BLAST) program with similar selected sequences deposited in GenBank. Phylogenetic tree and sequence alignments based on the 16S rRNA gene of the Pasteurellaceae family were then created by using MEGA 5.10 (Tamura et al. 2011 (link)), constructed by the neighbor-joining method, based on 1,000 bootstrapped data sets; distance values were calculated by using the Kimura 2 parameter model. Escherichia coli was used as the out-group to provide stability to the generated tree.