Dr Jochen Schulz
email: jochen.schulz@tiho-hannover.de

Sebastiaan Van Hoorebeke

Birthe Hald

Jörg Hartung

Filip Van Immerseel

Ines Radtke

Susanne Kabell

Jeroen Dewulf

The dynamics of Salmonella occurrence in commercial laying hen flocks throughout a laying period

Keywords: Salmonella, occurence, laying hen flocks, laying period

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Longitudinal studies can help to increase our knowledge about the dynamics of the occurrence of Salmonella in the course of a laying period. The total of 41 laying hen flocks, 18 in Belgium, 6 in Denmark and 17 in Germany were followed during an entire laying period. Samples taken from the empty cleaned and disinfected poultry houses were all negative for Salmonella. After hens arrived on the farms five pooled faecal samples, one pooled dust sample and 40 cloacal swabs (Belgium and Germany) or 40 swabs from fresh droppings (Denmark) were taken four times from 18 flocks, three times from 21 flocks and two times from 2 flocks in the course of the laying period. Ten flocks (two Belgian and eight German flocks) were tested up to three times positive for Salmonella. Forty three out of 50 positive samples contained Salmonella Enteritidis phage type 4 (29 isolates) or phage type 8 (14 isolates). The probability of subsequent Salmonella positive findings increased significantly in Salmonella positive flocks (p < 0.05, odds ratio = 6.4). However, the probability of finding Salmonella did not depend on the time of sampling in the laying period or the season.

Introduction

Salmonellosis belongs to the most frequently reported zoonotic food-borne diseases worldwide. It is known since many years that contaminated eggs and egg products are important vehicles for the transmission of these organisms to the consumer in the European Union and in the United States [START_REF] Patrick | Salmonella Enteritidis Infections, United States, 1985-1999[END_REF]European Food Safety Authority, 2009).

Salmonella can be found either on the surface of the eggshell or in the egg content. The eggshell can become contaminated as a result of infection of the oviduct or by colonization of the intestine of a hen [START_REF] De Reu | Bacterial contamination of table eggs and the influence of housing system[END_REF]. Also the presence of Salmonella in faeces can lead to eggshell contamination [START_REF] Gast | Isolation of Salmonella Enterititidis from internal organs of experimentally infected hens[END_REF]. Salmonella in the egg content is resulting from an infection of the reproductive organ or is a consequence of the migration of these bacteria through the egg shell and membranes [START_REF] Gantois | Mechanisms of egg contamination by Salmonella Enteritidis[END_REF]. The most common Salmonella serovar in eggs and egg products is Salmonella Enteritidis which is also the most frequently reported serovar in Salmonella infections in humans in the European Union and in the United states [START_REF] Braden | Salmonella enterica serotype Enteritidis and eggs: A national epidemic in the United States[END_REF]European Food Safety Authority, 2009). Case-control studies and outbreak investigations have demonstrated the association between human cases and the presence of Salmonella Enteritidis in laying hen flocks [START_REF] Altekruse | A comparison of Salmonella Enteritidis phage types from egg-associated outbreaks and implicated laying flocks[END_REF][START_REF] Rampling | Salmonella Enteritidis five years on[END_REF][START_REF] Trepka | An increase in sporadic and outbreak-associated Salmonella Enteritidis Infections in Wisconsins: The role of eggs[END_REF].

Many studies have investigated the transmission routes and identified risk factors for

Salmonella infections in laying hen flocks [START_REF] Davies | Observations on Salmonella contamination of commercial laying farms before and after cleaning and disinfection[END_REF][START_REF] Gradel | Monitoring the efficacy of steam and formaldehyde treatment of naturally Salmonella-infected layer houses[END_REF]Mollenhorst, 2005;[START_REF] Humphrey | Are happy chickens safer chickens? Poultry welfare and disease susceptibility[END_REF]Carrique-Mas et al., 2008;[START_REF] Namata | Salmonella in Belgian laying hens: an identification of risk factors[END_REF][START_REF] Van Hoorebeke | Determination of the within and between flock prevalence and identification of risk factors for Salmonella infections in laying hen flocks housed in conventional and alternative systems[END_REF]. However, studies over longer time periods describing the dynamics of the within flock Salmonella prevalence during a production period are still rare. This may be caused by high costs of such complex field studies and simply by the difficulty to convince [START_REF] Wales | A longitudinal study of environmental Salmonella contamination in caged and free-range layer flocks[END_REF]. Therefore, in the framework of the EU project SAFEHOUSE a longitudinal study was carried out in laying hen flocks on different farms in Belgium (BE), Denmark (DK) and Germany (DE). The flocks were sampled several times in the course of one laying period in order to describe the dynamics of Salmonella occurrence throughout a production period under field conditions.

Materials and Methods

Sampled flocks. In total 41 flocks were included in the study: 18 flocks from BE (1 enriched cage system, 4 battery cage, 5 free range, 4 barn and 4 organic systems), 6 flocks from DK (1 free range system, 3 battery cage and 2 barn system) and 17 flocks from DE (4 battery cage, 5 free range, 5 barn and 3 small colony keeping systems). A flock was defined according to regulation (EC) 2160/2003 of the European Parliament and of the Council [START_REF] Anon | Regulation (EC) No 2160/2003 of the European Parliament and of the Council of November 2003 on the control of Salmonella and other specific foodborne zoonotic agents[END_REF]. That means all hens of the same health status kept on the same premises or in the same enclosure and constituting a single epidemiological unit. In case of housed hens this includes all birds sharing the same airspace. Only flocks larger than 1000 birds were included. On farms with several flocks (overall 24 farms and 21 of them with flocks of different ages) one flock was selected randomly for sampling. The housing systems were categorised according to the EU directive 2002/4/EC and council regulations (EC) No 1804/1999 [START_REF] Anon | Council regulation (EC) No 1804/1999 of 19 July 1999 supplementing Regulation (EEC) No 2092/91 on organic production of agricultural products and indications referring thereto on agricultural products and foodstuffs to include livestock production[END_REF][START_REF] Anon | Comission Directive 2002/4/EC of 30 January 2002 on the registration of establishment keeping laying hens covered by Council Directive 1999/74/EC[END_REF].

An exception was the German small colony keeping system which is defined by the German authorities (German Federal Ministry of Justice, 2006). This system resembles the EU furnished cage but offers the hens more space and extra perches at different heights. were divided in six 10-week intervals for data analysis. Table 1 shows the number of flocks sampled in each country during these intervals.

Number and sample type taken on-farm. One pooled dust sample, five nest-box samples or 5 cage samples were taken in the empty and disinfected houses. The dust was collected from 20 different locations in the animal house. The dusty material was transferred by a gloved hand or sterile brush into a sterile bag. The minimum amount was 25 g dust per pooled sample. If no suitable surfaces with dust were present in the cleaned houses, surfaces from 20 locations were swabbed with sterile swabs, which were placed in Ames medium (Oxoid, Deutschland GmbH). Swabs and Ames medium were also used to swab 5 randomly selected nest-boxes except in battery cages which do not provide nest-boxes. In the latter case the grids of 5 randomly selected cages were swabbed.

From each laying hen flock 5 nest-box samples or 5 cage samples and 1 pooled dust sample were taken per visit as described above. Furthermore 5 pooled faecal samples and individual cloacal swabs from 40 randomly selected laying hens (in BE and DE) were sampled at each farm visit. In Denmark, the 40 swabs were obtained from 40 randomly selected fresh faecal droppings because farmers did not allow sampling their hens. Depending on the housing system, the pooled faecal samples were taken as follows:

• Cage systems and small colony keeping systems: 5 samples of mixed fresh faeces were either taken from dropping belts, scrapers or deep pits depending on the type of cage (conventional battery cage, furnished cage, small colony keeping system). Each pooled faecal sample consisted of approximately 250 g.

• Barn, free-range and organic systems: for each of the 5 pooled faecal samples 60 piles of fresh faeces were collected from the floor and the slats. Each pooled faecal sample consisted of approximately 250 g.

The pooled faecal samples were put in a sterile box. New gloves were used for collecting each pooled faecal sample.

Cloacal swabs were taken by inserting a cotton-tipped swab approximately 5 cm into the cloaca, taking care to avoid contact with the surrounding feathers and skin. Afterwards, the swabs were stored in tubes containing Ames medium. A sample size of 40 swabs allows detecting a within-flock prevalence of 7% at a level of confidence of 95% in a flock with more than 1000 hens (WinEpiscope 2.0). All samples were placed in leak-proof bags and transported in container boxes to the laboratory under ambient temperatures. The samples were analysed either immediately after arrival at the laboratory or stored at 4°C for analysis within the next 24 hours. were mixed in a stomacher bag for 1 minute. All samples were incubated for 18 ± 2 h at 37 ± 1 °C. Three droplets of each pre-enrichment culture were dropped on the surface of a modified semi-solid Rappaport-Vassiliadis (MSRV) (Difco; Becton Dickinson) agar plate containing 0.01 g l -1 novobiocine. The plates were incubated for 2 × 24 ± 3 h at 41.5 ± 1°C.

Material of suspected white cultures from the borderlines of the growth zones were transferred on Brilliant Green Agar (BGA; Oxoid) and Xylose Lysine Deoxycholate agar (XLD; Oxoid), followed by incubation for 24 ± 2 h at 37 ± 1°C. Presumed Salmonella colonies on BGA and XLD were biochemically confirmed using urea agar, triple sugar iron agar and lysine-decarboxylase broth. Serotyping of Salmonella-isolates according to the White-Kauffmann-Le Minor scheme [START_REF] Grimont | Antigenic Formulae of the Salmonella Serovars[END_REF] was performed in the national Reference Laboratories for Salmonella in each participating country. Phage typing of S.

Enteritidis was carried out as described by [START_REF] Ward | A phage-typing scheme for Salmonella enteritidis[END_REF].

Data processing and analysis. In order to get reliable information on the sampled flocks (e.g. number and age of hens in a flock) and the farms (e.g. total number of flocks and hens, age of different flocks, cleaning measures and disinfection procedures) a questionnaire was developed and filled in by interviewing the farmer at one of the farm visits as described by [START_REF] Van Hoorebeke | Determination of the within and between flock prevalence and identification of risk factors for Salmonella infections in laying hen flocks housed in conventional and alternative systems[END_REF]. Answers in the questionnaires were coded and transferred into an excel spread sheet (Microsoft Office Excel 2003, Microsoft Cooperation). The variable "period of sampling" was divided into six 10-weeks intervals. To define the category "season", the meteorological seasons for Europe (spring begins on the first of March, summer on the first of June, autumn on the first of September and winter on the first of December) were chosen. The effect of the period of sampling and the season on the within flock prevalence was analysed using logistic regression as was the effect of a previous sample result

Results

Salmonella detection.

In none of the animal houses members of Salmonella were found after cleaning and disinfecting prior to stocking. Ten out of the 41 sampled flocks were tested once, twice or three times positive for Salmonella in the course of one laying period.

Two Belgian and eight German flocks were Salmonella-positive across five different housing systems. Table 2 gives an overview of the kind of positive samplings, the number of positive samples, sampling intervals and detected serovars (including S. Enteritidis phage types) in the positive tested flocks. Six of these flocks (no. 7, 8, 25, 26, 31 and 41) were housed in all-in and all-out systems, two flocks (no. 27 and 32) originate from farms with hens of different ages in different houses and two flocks (no. 33 and 40) were kept in multiage systems. Two flocks (no. 25 and 41) hosted different serovars. The most prevalent isolate was S. Enteritidis found in 44 (88%) of the 50 positive tested samples (29 isolates of phage type 4, 14 isolates of phage type 8, phage type of 1 isolate could not be identified).

The serovars S. Infantis, S. London and one unidentified serotype from serogroup B were isolated from 6 samples (12%). Table 2 shows Statistical analysis. Observed Salmonella prevalence throughout the production period is given in Figure 1. Although differences are obvious between the different sampling intervals, these were not statistically significant. No significant effect of the season was found either.

However, the risk to find Salmonella in a flock increased significantly (p < 0.01) (odds ratio = 6.4, 95% confidence interval: 2.051 -19.745) when Salmonella was found in the previous sampling of the same flock.

Discussion

The occurrence of Salmonella was observed in Belgian and German laying hen flocks but not in Danish flocks what can be explained by the relatively small number of Danish flocks included into the study and the well-known low Salmonella prevalence in Danish laying hen flocks [START_REF] Wegener | Salmonella Control Programs in Denmark[END_REF][START_REF] Food | Preliminary report on the analysis of the baseline study on the prevalence of Salmonella in Laying Hen Flocks of Gallus Gallus[END_REF]. Therefore swab samples taken from fresh droppings instead of cloacal swabs in Denmark had most likely no considerable impact on the overall results.

Ten out of the 41 sampled flocks were tested one, two or three times Salmonellapositive in the course of the investigated laying period by using cloacal swabs, dust samples and samples of faeces. Taking cloacal swabs was less useful to detect Salmonella. This was probably influenced by the relatively low within flock prevalence of shedding hens (< 7%) even in a Salmonella contaminated environment. Sampling the environment was rather successful and especially the combination of faeces and dust samplings seems to be more effective than using a single sampling technique. These findings support the recommendation to sample both faeces and dust to increase the sensitivity of Salmonella detection in laying Davies, 2008). Although non-shedding hens with infected organs cannot be detected with the applied methods [START_REF] Van Hoorebeke | Faecal sampling underestimates the actual prevalence of Salmonella in laying hen flocks[END_REF], the use of more sensitive environmental samplings can help identify flocks at risk of producing S.

Enteritidis-contaminated eggs [START_REF] Henzler | Management and environmental risk factors for Salmonella enteritidis contamination of eggs[END_REF].

A Salmonella positive finding in a flock significantly increased the probability of subsequent positive results in the same flock. In these cases generally the same serovars and phage types were detected. Especially the same phage types in environmental samples and in cloacal swabs of the same flock demonstrate the close relationship between the infected animals and the environment which seems to play an important role as a reservoir.

Furthermore, in most of the positive tested flocks the infection was already observed in the early stage of the laying period. Although the samplings of the cleaned and disinfected laying hen houses were negative, undetected residual contamination may have contributed to an initial infection of hens. Especially in cage and multi-age systems, which represent seven (70%) of the positive tested flocks in this study, an effective cleaning and disinfection is difficult to perform in practice and may even fail [START_REF] Davies | Observation on Salmonella contamination of eggs from infected commercial laying hen flocks where vaccination for Salmonella enterica serovar Enteritidis had been used[END_REF][START_REF] Wales | A longitudinal study of environmental Salmonella contamination in caged and free-range layer flocks[END_REF]. Another source for infections in the early laying period may be living vectors such as rodents and arthropods. This was not specifically investigated in this study. However, it is known that they can contribute to the horizontal transmission of infectious agents including Salmonella [START_REF] Gast | Airborne transmission of Salmonella Enteritidis infection between groups of chicks in controlled-environment isolation cabinets[END_REF]Carrique-Mas et al., 2008). These vectors may also be responsible for infections even on farms with all-in all-out management because they can stay on the farm after depopulation and re-enter into the new flocks [START_REF] Hald | Typhaea stercorea (Coleoptera: Mycetophagidae), a carrier of Salmonella enterica serovar Infantis in a Danish broiler house[END_REF]. Beside horizontal transmission also vertical transmission may have contributed to the Salmonella presence in the investigated flocks. Salmonella infections in parent breeding flocks are described as an existing problem in Germany (European Food Safety Authority, 2009).

Although vaccination is compulsory in German parent breeding flocks, this route of Salmonella Enteritidis and may transmit this organism to their descendants [START_REF] Davies | Observation on Salmonella contamination of eggs from infected commercial laying hen flocks where vaccination for Salmonella enterica serovar Enteritidis had been used[END_REF].

The presented results show no significant differences concerning the Salmonella prevalence in the different sampling intervals. These findings contradict somehow other studies which described an increase of infected flocks with increasing age of the hens. A higher susceptibility of older hens to infections, molting and an accumulation of Salmonella in the environment were suggested as factors which lead to an increase of Salmonella positive flocks and an increase of Salmonella positive samples in ageing flocks respectively [START_REF] Schlosser | The Salmonella enterica serovar Enteritidis pilot project[END_REF][START_REF] Garber | Salmonella enterica Serotype enteritidis in table egg layer house environments and in mice in US layer houses and associated risk factors[END_REF][START_REF] Van De Giessen | Surveillance of Salmonella spp. and Campylobacter spp. in poultry production flocks in the Netherlands[END_REF][START_REF] Wales | A longitudinal study of environmental Salmonella contamination in caged and free-range layer flocks[END_REF]. In the present study induced molting was not practiced on the sampled farms and no higher numbers of Salmonella positive faecal, dust and cloacal swab samples were observed towards the end of the laying period. These findings may partly be influenced by cleaning procedures and the removal of faeces during the production cycle [START_REF] Wales | A longitudinal study of environmental Salmonella contamination in caged and free-range layer flocks[END_REF]. Hence, differences in the farm management and in the design of studies make it difficult to compare the results of this study and previous investigations. However, an early Salmonella contamination in the laying period did apparently not result in an increased number of shedding hens. In contrast to other opinions, it seems that an increasing number of shedding hens towards the end of a laying period does not hold true for infected flocks in general. This may be typical in particular for early infected flocks due to the decline of shedding hens within a few weeks after infection [START_REF] De | Effect of the housing system on shedding and colonization of gut and internal organs of laying hens with Salmonella Enteritidis[END_REF] and should be considered in future longitudinal studies by taking samples in the very early stage of lay (in the first three weeks after housing).

Seasonal effects may also influence the detection of Salmonella in laying hen flocks in longitudinal studies [START_REF] Wales | A longitudinal study of environmental Salmonella contamination in caged and free-range layer flocks[END_REF]. Therefore the season was included in the logistic regression model but no significant seasonal effects were observed. The discussion about 12 seasonal effects is controversial and it seems that not the changing climatic conditions itself but rather their consequences for the management or the presence of living vectors are influencing Salmonella detection and transmission (Carrique-Mas et al., 2008;[START_REF] Van Hoorebeke | Determination of the within and between flock prevalence and identification of risk factors for Salmonella infections in laying hen flocks housed in conventional and alternative systems[END_REF].

The dynamics of Salmonella infection in laying hen flocks is very complex. It is strongly depending on the farm management (e.g. cleaning and disinfection procedures), the immune status of the birds, the presence of living vectors as carriers and an accumulation of Salmonella in the flock environment. This study indicates that early infections of laying hen flocks with the important human pathogen S. Enteritidis (PT4 and PT8) can lead to a continuous infection of hens and to a persistent contamination of their environment. This may increase also the risk off egg contaminations. Therefore more stringent action should be taken both to eliminate Salmonella from breeding and production flocks by applying comprehensive bio-security measures. Special regards should be paid to avoid Salmonella introduction in laying hen flocks at any stage. This will be a considerable contribution to decrease the risk for the consumer.

  egg products have been recognised since many years as an important source of Salmonella infections in humans in the European Union and in the United States.

  part in long lasting investigations

  In DK flocks were sampled from October 2007 until March 2009. First samples were taken from the empty and disinfected animal houses one week before the hens arrived at the farm. The age at placement varied between 15 and 22 weeks. In three flocks no samples were taken from empty houses because hens of different ages were housed in the same premise and the animal houses were never empty. In total 139 samplings were carried out at different times during one egg-laying period. Flocks were sampled three or four times at different intervals with the exception of two flocks which were sampled only twice. Between the different countries sampling intervals varied because of different logistics. The laying periods

  Bacteriological analysis of samples. All samples were analyzed according to ISO 6579:2002/Amd 1:2007 Annex D: Detection of Salmonella spp. in animal faeces and in environmental samples from the primary production stage. From each of the pooled faecal and mixed dust sample, 25 g was added to 225 ml of buffered peptone water (BPW) (Oxoid, Basingstoke, Hampshire, UK). All cloacal swabs, swabs from nest-boxes and from dusty placed in 9 ml of BPW. Pooled faecal and mixed dust samples

  production round on the next sample result. For statistical analysis the software SPSS 17.0 (SPSS Inc., Chicago, IL) was used.

  that Salmonella were detected 18 times in 10 flocks by using three kinds of sampling techniques. Already 17 positive samplings in 10 flocks were obtained by environmental samplings (pooled faecal and pooled dust samples). The number of positive flocks (pooled faecal = 7, pooled dust = 6, cloacal swabs = 3) and samplings (pooled faecal = 12, pooled dust = 10, cloacal swabs = 5) decreases when only a single sampling technique is taken into account.

  be neglected because even vaccinated hens can become infected with

Figure 1 .

 1 Figure Legends

E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp Avian Pathology

Acknowledgements

The study was funded by the European Community (Project FOOD-CT-2006-035547). The authors would like to thank all farmers participating in the study for very good cooperation.

F o r P e e r R e v i e w O n l y


MD Eric Benefice
email: eric.benefice@ird.fr

Pierre Lévi
email: pierre.levi@ird.fr

Progressive growth deterioration in a context of nutritional transition: a case study from Vientiane (Lao PDR)

Keywords: Stunting, Food Diversity Score, urbanization, sex difference Progressive growth deterioration Stunting, Food Diversity Score, urbanization, sex differences

Background

The high rates of rapid urban and economic growth occurring in Asia are bringing about parallel changes in both food consumption patterns and nutritional status.

Aim

The aim of this study is to examine the impact of these changes on the nutritional and health status of mothers and their offspring in Vientiane, Lao PDR.

Methodology

Over two consecutive years, a follow-up study of 150 infant-mother pairs living at three different levels of urbanization was performed in Vientiane. The mothers completed a questionnaire on their eating habits. Clinical examinations and anthropometric measurements were also carried out.

Results

The results showed that, in general, the dietary energy content was low (providing only 83% of the energy requirement) and there were deficiencies in calcium, vitamin A, Folate folates and iron. The main energy source was rice (providing 40.9 %) while 40% of the protein was provided by meat with fish providing 19.8%. The differences observed in the food contribution to energy intakes and in food diversity varied with the level of urbanization. The prevalence of stunting (13.9%; CI 10.0 ~ 18.6%) was less than that reported at the country level. Stunting was related to age, the sex of the child, and the mother's physique and varied according to the level of urbanization.

Conclusion

The level of urbanization in Vientiane influences the pace of the ongoing process of nutritional transition.

Introduction

The changes in the eating habits and nutritional status that have occurred in the developing world over the last two decades are generally termed "nutritional transition". Nutritional transition is characterized by an alteration in eating habits (the increased consumption of refined foods and the abandonment of the traditional starchy and high-fibre diet) and a decline in the level of physical activity. The present worldwide epidemic of overweight and obese individuals is presumed as being the consequence of this nutritional transition in those countries experiencing fast rates of economic growth and urbanization [START_REF] Popkin | The nutrition transition: worldwide obesity dynamics and their determinants[END_REF].

In Asia, it is certain that epidemics of overweight individuals have developed in parallel with the speed of industrialization and urbanization [START_REF] Misra | The metabolic syndrome in South Asians: epidemiology, determinants, and prevention[END_REF][START_REF] Zhai | Prospective study on nutrition transition in China[END_REF][START_REF] Misra | Nutrition transition in India: Secular trends in dietary intake and their relationship to diet-related non-communicable diseases[END_REF]. Not only are affluent upper and middle social classes affected, but also poor people living in urban slums or in economically backward rural areas can be exposed [START_REF] Misra | The metabolic syndrome in South Asians: epidemiology, determinants, and prevention[END_REF]. Paradoxically, several regions affected by the epidemics in overweight adults, are at the same time hit by child undernutrition (malnutrition) [START_REF] Khan | Double burden of malnutrition: the Vietnamese perspective[END_REF]. It is a fact that malnutrition is still widespread in Asia. About 100 million pre-school children were estimated as suffering from malnourishment in 2010 [START_REF] De Onis | Prevalence and trends of stunting among preschool children, 1990-2020[END_REF]. The most prevalent form, that of chronic malnutrition, manifests itself as growth retardation or stunting [START_REF] Waterlow | Classification and definition of protein-calorie malnutrition[END_REF]. One of the most worrying problems of stunting is the trans-generational consequences [START_REF] Ozaltin | Association of maternal stature with offspring mortality, underweight, and stunting in low-to middle-income countries[END_REF] . This means that stunted girls become short adult women who, in turn, give birth to low birth weight and small for gestational age babies that will in turn be affected by growth retardation [START_REF] Ferreira | Short stature of mothers from an area endemic for undernutrition is associated with obesity, hypertension and stunted children: a population-based study in the semi-arid region of Alagoas, Northeast Brazil[END_REF][START_REF] Dewey | Long-term consequences of stunting in early life[END_REF]. However, the situation has changed since the turn of the century and the future looks brighter. The nutritional situation in Asia has improved dramatically in that the prevalence of stunting in pre-school aged children has decreased from 52% to 34% in two decades [START_REF] De Onis | Is malnutrition declining? An analysis of changes in levels of child malnutrition since 1980[END_REF]. This is confirmed by a more recent evaluation which reported a further spectacular reduction from 49% to 28% in 2010 [START_REF] De Onis | Prevalence and trends of stunting among preschool children, 1990-2020[END_REF]).

However advances are not uniform and there still exist great differences both between and within countries.

The probable link between two events, the decline in malnutrition and the occurrence of nutritional transition is an important issue in the better understanding of the natural history of malnutrition. The dilemma is how to take advantage of the impact of economic improvements on health in order to reduce child malnutrition without the disadvantage of later paying an outrageous price in terms of adult chronic diseases. Analyzing the nutritional transition phenomenon at its very beginning could help solve this dilemma. The present situation in the Lao People's Democratic Republic (Lao PDR) may contribute to this analysis. Lao PDR is the least populated country in South-East Asia (with about 6 million inhabitants) and one of the less economically developed. However, important economic advances have recently taken place. Lao PDR is no longer considered as a "less economically developed country" by the World Bank, but is now classified as a "lower middle income" country. Nevertheless, according to the World Food Programme (WFP 2011), economic growth has not led to a parallel improvement in the nutritional status. Food insecurity is still a challenge in rural and mountainous areas (WFP 2011) with great disparities existing between the northern highlands where stunting is very frequent (58%) and the urban areas of the Mekong plain, were the prevalence is much lower (35%) [START_REF] Anonym | Lao PDR Multiple Indicators Survey[END_REF].

In this paper, data on the nutritional status and eating habits of children and their mothers living in the districts of Vientiane (Capital City) at different levels of urbanization are presented. The objectives of the present paper are 1) to examine the relationships between urban development, nutritional status and food eating patterns and 2) to look at the association between maternal status and growth of the offspring.

Methods

Study site and sampling

The metropolitan area of Vientiane is loosely urbanized and comprises 148 administrative units named ban or villages. Thirteen human and environmental indicators [START_REF] Vallée | Urbanisation et santé à Vientiane (Laos): Les disparités spatiales de santé dans la ville Thèse présentée pour obtenir le grade de Docteur en Géographie Humaine de l[END_REF] together with aerial photographs and the most recent census data (of 1995 and 2005) were used to classify the different zones of the city according to their "level of urbanization". These indicators were representative of both environmental and demographic characteristics (house construction; demographic density; public infrastructure; markets; water supply; electricity supply; schools; hospitals; shops; adequate sewerage; people whose main activity was agriculture; distance to main road; distance to city center…) and represent a gradient of urbanization ranging from the more densely built-up and well equipped areas to those suburbs where agricultural activities are still pursued. Three levels of urbanization (or urbanization strata) were thus identified using a hierarchical forward classification procedure : 1) the central zone (strata 1); 2) the first urbanized belt (strata 2): and the second urbanized belt (strata 3) [START_REF] Vallée | Urbanisation et santé à Vientiane (Laos): Les disparités spatiales de santé dans la ville Thèse présentée pour obtenir le grade de Docteur en Géographie Humaine de l[END_REF].

Sampling followed a two-step procedure. First, nine villages (ban) were randomly drawn from each stratum (making 27 villages in total) and then 1,000 households were randomly drawn from these for a demographic, educational and economic follow-up [START_REF] Lévi | Suivi démographique et de scolarisation à Vientiane : 1. Rapport d'enquête : 2. Rapport annexe de présentation des questionnaires 2006-2008. Présentation des principaux résultats d'un suivi de population en milieu urbain, dans une capitale asiatique[END_REF]) Finally, within this sample, the youngest women, either pregnant or having just given birth at the beginning of the survey, were selected for an in-depth growth and nutrition survey. A total of 157 (150) mother-infant pairs evenly distributed in the 3 strata (strata 1: 37; strata 2: 61; strata 3: 59), were followed-up over two consecutive years. Mean family size was 6.4 individuals per unit and in our sample, no difference was found between urbanization levels.

Dietary intakes

Dietary intakes were studied in two different ways. First, a qualitative evaluation was performed using a food frequency questionnaire and applied to mothers. This allowed the building of a food diversity score (FDS) derived from the WFP procedure based on a list of food consumed in the household over the previous seven days [START_REF] Kennedy | Proxy measures of household food consumption for food security assessment and surveillance: comparison of the household dietary diversity and food consumption scores[END_REF]). To do this, different weight values were assigned to eight food groups: Group 1: Cereals, tubers and roots (weight = 2); Group 2: meat and fish, (weight = 3); Group 3: milk (weight = 4); Group 4: oil/fat, (weight = 0.5); Group 5: fruit (weight = 1); Group 6: vegetables (weight = 1); Group 7: pulses (weight = 3); Group 8: sugar, (weight = 0.5); and condiments (weight = 0). These food items were the same as those in the food composition table used for the 24 hour dietary recalls as described below.

Second, mothers responded to a 24 hour dietary recall. They were asked to enumerate each meal eaten for breakfast, lunch, dinner and in-between meals on the day preceding the survey.

Then they had to list the foods eaten in each meal, together with the cooking methods. The quantities consumed were roughly estimated using common household measures (bowl, glasses, and plates). Portions were then recorded as normal, smaller than normal or larger than normal. Data were computed with Nutrisurvey software (http://www.nutrisurvey.de/ Canadian file of nutrients (http://www.hc-sc.gc.ca/fn-an/nutrition/fiche-nutri-data/indexfra.php). The final file had 252 entries and the analysis of more than 100 nutrients was possible. In practice we analyzed just energy and 23 nutrients. Nutrient requirements were based on recommendations of the German Society for Nutrition (DGE) and the Joint WHO/FAO Committees for Energy and Nutrients. Calculation was done for a 25 -30 year old woman engaging in light to medium physical activity.

Nutritional status

The nutritional status was determined on the basis of anthropometric measurements.Measurements were taken according to recommended international techniques [START_REF] Lohman | Anthropometric standardization reference manual[END_REF]) . The weight (kg) was recorded with an electronic Seca ® medical scale Anthropometric indices of nutritional status were also calculated using the new WHO reference data set (http://www.who.int/childgrowth/en/).

The following nutritional indices were calculated: height-for-age (H-age, indicator of chronic malnutrition), weight-for-age (W-age, indicator of global malnutrition) and weight-for-height (W-H, indicator of acute malnutrition). Values were expressed as z-scores from the references.

Finally, the body mass index (BMI, kg/m 2 ) was computed as the weight divided by the square of height and used as an index of corpulence in adult women.

Statistical analysis

Data were entered and double-checked using Epi Info software. NCSS statistical software was employed (http://www.ncss.com/). All continuous variables were checked for the normality of the distribution. Appropriate transformations were done if necessary. Pearson correlation coefficients to test the association between variables were calculated. We performed general linear models of analysis of variance designed to take into account the visit effect (repetition of measurements from one year to another) in comparing values of continuous variables between different urban strata. Associations between growth indices and mother characteristics were studied through a multiple regression procedure. A hierarchical model was run with a forward selection of variables. Main effects and two-way interaction terms were included. Normality of residual distribution and multi-collinearity between variables were checked.

Ethical consideration

After formal administrative authorizations had been received from the provincial director of public health, the health directors of each district and the "ban" leaders, the objective of the study was explained in general to each mother individually in the presence of her husband.

Then the mothers were asked to sign an informed consent form for themselves and on behalf

Results

Differences in food eating patterns

In general, Lao women had three main meals during the day punctuated by two snacks (mid morning and afternoon). There was a great diversity of foods and recipes. The most frequently consumed food was sticky rice (Oryza sativa var. glutinosa). In addition, women ate non glutinous rice and a variety of dishes made with rice flour: noodle, vermicelli, and ravioli.

Chicken and pork consumption was common while beef was eaten less often. Fruits and vegetables were eaten every day. The fish eaten most often were from paddy fields or fish farms and were usually common carp (Cyprinus carpio carpio) or Tilapia (Cichlidae). Wild fish from the Mekong River, though still abundant, were consumed less often because they were more expensive.

Daily intakes of macronutrients are presented in Table I. The daily energy intake of these young women was rather low. They ate about 92 g of protein which is more than enough to fulfill the estimated daily requirement of 47 g. Table II presents the percentage of the fulfillment of the women's needs. The total energy intake was insufficient by 15% as was the intake of poly unsaturated fatty acids (20%) , vitamin A (45%) , total folic acid (50%), calcium (70%) and iron (25%). Using a general linear model of variance to take into account the year of visit effect, no relationship was determined between the differences in the fulfillment of energy and nutrients requirements and the levels of urbanization.

Cereals were the most important contributor to the overall energy content of the diet (mean: 40.9%; exact confidence interval at 95% (CI 95% ): 39.6 ~ 42.3%) ; meat was the second (22.6%; CI 95% : 21.3 ~23.9%); fish the third (7.3%; CI 95% : 6.4~8.2%); then fruit (6.6%; CI 95% : 5.9~7.3%); fat (6.7%; CI 95% : 6.0~7.4%) and vegetables (5.5%; CI 95% : 5.0~6.0%). Relative weights of the contribution of the food groups changed in terms of the protein intakes: Meat comprised 48% (CI 95% : 45.6~50.4%) of the protein intake; fish 19.8% (CI 95% : 17.6~22.0%); cereals 15.3% (CI 95% : 14.5~16.2%); and vegetables 6.6% (CI 95% : 6.0 ~ 7.2%). All these variables were normally distributed. The relationship between the contribution of the food groups to the energy content of the diet and the level of urbanization was analyzed using a general linear model of analysis of variance (Table III). Women living in the central area (strata 1) tended to eat fewer cereals and fish, and more meat and milk than did those living at the periphery (strata 2 and 3). There were no differences for the other food groups.

The median of the food diversity score (FDS) calculated for one week was equal to 49.5 (95% CI: 46~53.5). There existed significant differences according to the level of urbanization (F = 3.8; p <0.02): FDS was significantly lower in the second urbanized belt (strata 2 median: 43) than in the other two belts (strata 1 median: 51.7; strata 3, median: 52.5).

Status of mothers

A demographic study revealed that a process of demographic transition was already ongoing in Vientiane (with a decrease in both infant and maternal mortality to 24/1000 and 183/100 000 respectively, and low fecundity with a median descent of 3.1 children in 45 year old women). There was also a high percentage in the percentage of The decrease was even higher in women who had attended primary school (to 89%) [START_REF] Lévi | Suivi démographique et de scolarisation à Vientiane : 1. Rapport d'enquête : 2. Rapport annexe de présentation des questionnaires 2006-2008. Présentation des principaux résultats d'un suivi de population en milieu urbain, dans une capitale asiatique[END_REF].

In general, in our study, the mothers were young (mean age: 28.2 years ± 5.0) and in apparent good health at the time of the visit. Table IV reports their physical characteristics. When compared to international references the women were shorter than average with their median heights for standing and sitting below the 5 th percentile of the reference [START_REF] Hamill | NCHS growth curves for children[END_REF].

However, their weight, arm circumference and triceps skinfolds were slightly over the 25 th percentile of the reference data [START_REF] Hamill | NCHS growth curves for children[END_REF][START_REF] Johnson | Basic data on anthropometry and angular measurements of the hip and knee joints, for selected groups 1-74 years of age, United States, 1971-1975[END_REF]. They were generally well proportioned: according to their BMI values (WHO 1995), 70.3% were normal; 9.3% were thin (BMI < 18.5 kg/m 2 ) and 20.5% were overweight (BMI > 25 kg/m 2 ) but none were obese.

Their body mass (weight: r=0.25, p< 0.0001) and fat mass (skinfold thickness: r=0.34, p < 0.0001) increased significantly with age. Anthropometric measurements were not related to food intakes nor were they related to the level of urbanization.

The mean blood pressures of the women were 120.7 ± 12.7 mmHg for systolic pressure and 75.7 ± 8.3 for diastolic pressure. A total of 14.5% (Interval of confidence, IC 95% : 10.5 ~19.3) had systolic blood pressures > 140 mmHg and 4.8% (IC 95% : 2.6 ~8.1) with diastolic blood pressures > 90 mmHg. These numbers are considered as the entry points to level 1 of hyper blood pressure. Systolic pressure correlated with BMI (r = 0.22, p < 0.001) but not with age and physique. Diastolic pressure correlated with age (r=0.15, p < 0.01) and BMI (r=0.28, p< 0.001) but not with physique. There was no significant relationship between blood pressure and dietary intakes and no relationship with the level of urbanization.

Status of children

Birth weight were measured at the hospitals were mothers gave birth and retrieved from children health records. The mean birth weight was 3.08 ± 0.45 kg (median: 3.1 kg; 25 th percentile: 2.8 kg and 75 th percentile: 3.4 kg). At birth, girls were heavier than boys after adjusting for the year of visit effect (girls 3.15 ± 0.49 kg versus boys 2.9 ± 0.45 kg, F=7.5, p < 0.006). There was no relationship with the level of urbanization. About 10% of babies were of low birth weight (LBW) with weights lower than 2.5 kg (10.4%; IC 95% 7.0 ~ 14.7%). There were no differences in prevalence of LBW in relation to sex or urbanization strata.

The prevalence of malnourished children remained steady during the study with wasting (Weight for height <-2 z-scores) being 7.3% (IC 95% : 4.5 ~11.0%) and stunting (Height for age <-2 z-scores) 14.2% (IC 95% : 10.2 ~18.8%). The prevalence of thinness (Weight for age <-2 z-scores) was 15.2% (IC 95% : 11.1 ~20.1%). There were no sex differences. The prevalence of stunting was not related to the level of urbanization.

Table V presents a comparison of the mean values of child nutrition indices during the two

visits, taking into account the year of visit and the sex effect, performed with a general model of variance analysis. The difference in child numbers is due to the inclusion of new born babies in the cohort in the second year of surveys. The W-H index remained steady from one visit to another while there was deterioration in the H-age index. It is noteworthy that there was also a significant difference according to sex (without any interaction between visits and sex) meaning that the progression of growth stunting was more severe for boys. W-age values tented to decrease with time but the differences were not significant. Boys had lower nutritional indices than girls. There was no urbanization effect on child nutritional indices.

Figure 1 illustrates the progressive worsening of growth retardation with age (F=3.4; p< 0.01) and the difference according to sex (F=4.3; p <0.04) after adjusting for the year of visit effect.

Physique was close to the median of the reference during the first six months of life, but thereafter there was a dramatic fall that ceased at around nine months in girls and leveled off thereafter. In boys, the deterioration was a continuing process.

Relationships between mothers and children

To further elucidate the relationships between child growth (H-age), age of mother, birth weight, maternal status and urbanization level a multiple regression analysis was run. The level of urbanization was added as categorical variable. Visit number was also entered to take into account the repetition of measurements. There was no multi-collinearity between variables. Residuals were normally distributed without significant serial correlations. Table VI displays the results. The model was pertinent (F=12.4, p<0.0001) and explained 20% of the variability of H-age (R 2 =0.20). The most powerful determinant of H-age variability was the birth weight. H-age was also significantly and positively associated with the mother's stature and the sex (girls) of the child. Interestingly, living in strata 2, was negatively related to H-age.

Discussion

Two major findings emerge from this study: 1) dietary intakes were generally low and eating habits may vary according to the place of residence; 2) there existed a progressive stunting in growth of children related to maternal characteristics and sex.

Dietary intakes reported here were lower than those currently recommended for energy and some important nutrients (folic acid, calcium, iron). The main staple food was sticky rice (Oryza sativa var. glutinosa) which has a lower amylase content than normal white rice [START_REF] Roder | Glutinous rice and its importance for hill farmers in Laos[END_REF]. Weaknesses in the 24 hour recall, and in particular the memory bias that tends to over or underestimate the amounts of foods eaten are acknowledged. However, it has been shown that while variability of estimates is greater than the direct weighing method, the median values are similar [START_REF] Todd | Food intake measurement: problems and approaches[END_REF] . Besides, if the dietary recall can be repeated the accuracy of estimates is improved. This was the case here, where the questionnaire was twice answered with similar results. Without being able to ascertain the accuracy of the results, we nonetheless believe that the reported deficiency in energy and in some nutrient intakes is assuredly correct.

We have been unable to compare our results with other food consumption surveys in Vientiane or in other Lao cities since no such surveys have been published. An unpublished working paper written two decades ago reports estimates of energy intakes to be between 1,745 and 1,976 kcal per capita in Vientiane capital (reported in [START_REF] Kachondham | A new horizon: Addressing food and nutrition problems in the Lao people's Democratic Republic[END_REF]) . This figure is higher than ours. Protein intakes were lower: about 45g against 92g in the present study. Contributions of food groups were also different: cereals (mainly rice) provided 80% of the energy and 50% of the protein intake whereas in our study, rice made a smaller contribution to both energy (41%) and protein (15%) intake. This suggests that a noticeable change in eating habits may have occurred since then, especially in terms of a greater consumption of foods of animal origin.

Our data point to differences in food patterns related to urbanization levels: in the central belt (strata 1) women consumed more meat and milk but less cereals and fish than those in the outskirts (strata 2 and 3). This could be interpreted as evidence of a transitional process in central urbanized areas while peripheral zones retain many characteristics of a traditional Lao diet (rice and fish from the Mekong River or ponds). This finding could be further refined by information given by FDS scores. They are higher in the central (strata 1) and in the second urbanized belt (strata 3) than in first urbanized belt (strata 2). Interestingly our median FDS score was very close to that found in a national Lao survey conducted in 2005: 49.5 versus 51.1 [START_REF] Kennedy | Proxy measures of household food consumption for food security assessment and surveillance: comparison of the household dietary diversity and food consumption scores[END_REF] . The first urbanized belt (strata 2) appears to offer a less diverse diet than does either the central zone (strata 1) or the more agricultural zone (strata 3).

For this reason it could be hypothesized that it is more vulnerable than either strata 1 or 3. Urban space is not clearly delineated in Vientiane and there exists a continuity with rural landscapes [START_REF] Vallée | Urbanisation et santé à Vientiane (Laos): Les disparités spatiales de santé dans la ville Thèse présentée pour obtenir le grade de Docteur en Géographie Humaine de l[END_REF]. Urban agriculture is still a major and dynamic economic activity in Vientiane capital. It is not restricted to market gardening but also includes rice growing and fish farming. Such changes at a micro scale have been observed in rural Katu villages from southern Lao PDR and which resulted in a forced movement away from their traditional land [START_REF] Krahn | The dynamics of dietary change of transitional food systems in tropical forest areas of Southeast Asia. The contemporary and traditional food system of the Katu in the Sekong Province, Lao PDR Doktor der Ernährungs-und Haushaltswissenschaft[END_REF]). In the present study items like fruits, vegetables, legumes and fish contribute 23.8 ± 12.4% of energy and 32.9 ±19.7 % of protein requirements. These food items are thought to be healthier than meat, animal fat or sweetened drinks [START_REF] Delisle | Dietary patterns and quality in West-African immigrants in Madrid[END_REF]. Though a process of transition is certainly operating in Lao cities, it seems to be taking place slowlyat a slower rate, with subtle variations according to the place of residence and social background, than . This is in contrast to that reportedin the two neighboring countries of Vietnam [START_REF] Khan | Double burden of malnutrition: the Vietnamese perspective[END_REF] and Thailand [START_REF] Kosulwat | The nutrition and health transition in Thailand[END_REF]. Indeed, some findings (14.5% of women with high systolic blood pressure and 20.5% presenting as overweight) indicate that women from Vientiane are at risk of degenerative diseases associated with the nutritional transition phenomenon. These figures are close to those observed in the sister country of Cambodia [START_REF] King | Diabetes and associated disorders in Cambodia: two epidemiological surveys[END_REF].

This study points to a progressive deficit in height growth, more pronounced in boys than in girls in Lao PDR. In addition, regression analysis suggests that growing patterns varied according to the level of urbanization of the child's place of residence. The percentage of stunted children in our sample (14.2%) was about twice lower less than half than that reported at the national level for urban children (32.3%) or for children aged 1 to 2 years (48.7%) (Data from the 2006 National Survey; WHO Global Database on Child Growth and Malnutrition, July 2009). However wasting prevalence 7.3% in the present study was almost the same as that at the national level for urban children (6.9%). This indicates that the causes of wasting and stunting are different. It has been suggested that both conditions are the end result of different nutritional deficiencies [START_REF] Golden | The nature of nutritional deficiency in relation to growth failure and poverty[END_REF]. The prevalence of wasting has decreased uniformly throughout the country, probably as a direct consequence of global improvement in the health sector (better access to medical care and prevention of infectious diseases, better surveillance of pregnant and lactating women, better management of severe malnutrition) than observed elsewhere [START_REF] Paul | Reproductive health, and child health and nutrition in India: meeting the challenge[END_REF]. Stunting has decreased markedly in Lao cities but is still excessively high in rural areas (50 to 52%, according to the Lao National Survey 2006, WHO Global Database). In a recent survey in the same areas of Vientiane, using a representative sample of 0-5 year old children, a prevalence of 23% (20.3~25.2) was found. This was lower than that in the national survey but higher than our figure [START_REF] Vallée | Urbanisation et santé à Vientiane (Laos): Les disparités spatiales de santé dans la ville Thèse présentée pour obtenir le grade de Docteur en Géographie Humaine de l[END_REF]. Wasting prevalence was very similar to ours (7.7%). Discrepancies in the prevalence of stunting could be due to the age structure of the sample, suggesting a long lasting effect of stunting without catch up growth. In addition, a significant effect of the level of urbanization on the prevalence of stunting was observed [START_REF] Vallée | Urbanisation et santé à Vientiane (Laos): Les disparités spatiales de santé dans la ville Thèse présentée pour obtenir le grade de Docteur en Géographie Humaine de l[END_REF]. This Stunting was greater in strata 2 (21.1%) and 3 (27.5%) than in strata 1 (16.8%). While the smallness of our sample does not allow a firm conclusion, our findings are in line with this finding.

The apparent sex differences in growth deterioration have also been observed elsewhere. In a recent prospective cohort study performed in Thailand, it was shown that the incidence of stunting was significantly greater in boys than in girls [START_REF] Mongkolchati | Prevalence and incidence of child stunting from birth to two years of life in Thai children: based on the Prospective Cohort Study of Thai Children (PCTC)[END_REF] . Likewise, in Africa, examination of results of 16 Demographic and Health Surveys (DHS) across ten different countries, indicated that the prevalence of stunting was greater among boys than girls and that girls had greater mean z-scores than boys [START_REF] Wamani | Boys are more stunted than girls in sub-Saharan Africa: a meta-analysis of 16 demographic and health surveys[END_REF]. Aside from biological explanations, the authors proposed that boys were less robust and more vulnerable to health inequalities than were girls, especially those in the poorer socio-economic strata.

However, other studies have demonstrated the reverse. In India, a greater nutritional morbidity was observed in girls [START_REF] Bharati | Growth and nutritional status of pre-school children in India: rural-urban and gender differences[END_REF]. In a large survey covering 26 states and more than 25,000 children, it was reported that girls were more frequently underweight and stunted than were boys. The authors stated that this was almost certainly due to discrimination in terms of access to health care to the detriment of girls and not to any discrimination in the allocation of food. Thus, a cultural influence should not be overlooked. This could be the case here where women and girls have a highly valued position in Lao society.

It is acknowledged that stunting is a progressive process, beginning in-utero and preventing the children from achieving their full genetic potential for growth [START_REF] Dewey | Long-term consequences of stunting in early life[END_REF].

Several studies have already stressed an association between stunting during infancy and lower fine motor abilities (Chang 2010), motor performances and physical working capacity [START_REF] Benefice | Early nutritional history and motor performance of Senegalese children, 4-6 years of age[END_REF], poor psychological and emotional functioning [START_REF] Walker | Early childhood stunting is associated with poor psychological functioning in late adolescence and effects are reduced by psychosocial stimulation[END_REF], and low school achievement (Martorell and[START_REF] Martorell | Interrelationship between growth and development in low and middle income countries[END_REF]. A recent study conducted in Vientiane in children between 36 and 44 months, using the Denver Development Screening Test, demonstrated that there existed a significant association between growth retardation and language acquisition and social/personal skills [START_REF] Akkavong | Retard de croissance et développement psycho moteur des enfants de 36-44 mois en zone péri-urbaine de Vientiane. 4ème suivi de cohorte, année 2008[END_REF] . Slow child development could lead to suboptimal physical functioning during adulthood and eventually to low economic productivity [START_REF] Florencio | Short stature and food habits as determining factors for the low productivity of sugarcane labourers in the State of Alagoas, north-eastern Brazil[END_REF], closing the vicious circle linking malnutrition, poverty and the development of human capital [START_REF] Dufour | Living on the edge: dietary strategies of economically impoverished women in Cali, Colombia[END_REF][START_REF] Carba | Early childhood length-for-age is associated with the work status of Filipino young adults[END_REF]) [START_REF] Dufour | Living on the edge: dietary strategies of economically impoverished women in Cali, Colombia[END_REF][START_REF] Carba | Early childhood length-for-age is associated with the work status of Filipino young adults[END_REF]). In addition, our results indicated that height growth of children was significantly related to their mother's stature, proving the existence of an inter-generational effect. This confirm our fears about this issue [START_REF] Ozaltin | Association of maternal stature with offspring mortality, underweight, and stunting in low-to middle-income countries[END_REF].

For this reason, and though real progress has been realized in recent years, policies aiming at reducing growth retardation without a swing over to over nutrition and obesity should be implemented. These must emphasize the positive outcomes of the ongoing transition while tackling improved access to health care and education especially for poor women living in the less developed urban belts.

List of tables

List of figures

(

  Model 354) accurate to 10 g in infants and a classical electronic Seca ® scale accurate to 100 g in adults. Recumbent length was measured with a Seca ® measuring mat (Model 210) up to the age of 2 years. Standing height was measured with a Harpenden ® anthropometer in mothers and children older than 2 years.Biceps and triceps subcutaneous skinfolds (mm) were recorded on the right side after marking the site on the skin with a Holtain ® caliper. Two successive measurements were performed and the average was used for analysis. Arm circumference was measured at mid-distance between the olecranon and acromion. Arm muscle circumference (AMC, cm) was calculated using the formula of Gurney and Jelliffe[START_REF] Gurney | Arm anthropometry in nutritional assessment: nomogram for rapid calculation of muscle circumference and cross-sectional muscle and fat areas[END_REF]: AMC= arm circumference-π * (triceps skinfold). The same person performed all anthropometric measurements. Stature and subcutaneous skinfolds were measured twice at the beginning and at the end of the visit during the 2 first days of the studies. Intramesurer technical errors were in the lower range of published data (Lohman, Roche et al. 1988).

  of their offspring. The Dean of the department of post doctoral studies, Faculty of medicine gave his agreement after consulting the scientific committee. The Lao National Ethics Committee for Health Research (Council of Medical Sciences), Ministry of Health) gave their ethical approval.

Figure 1 :Figure 1 :

 11 Figure 1: Height-age values at different ages

  ). A food composition table was compiled from those tables already existing: Nutrisurvey data for Thailand and Germany; United States Department of Agriculture database (http://www.nal.usda.gov/fnic/foodcomp/search/), Food and Agriculture Organization: Food Composition Table for Asia (http://www.fao.org/docrep/003/X6878E/X6878E00.htm);

Table I

 I Daily intake of macronutrients by Lao womenTable II Fulfillment (percentages) of nutritional requirements by Lao women Table III Contribution of food group to energy content of the diet according to 3 urbanization levels Table IV Physical characteristics of women (n=152) Table V Evolution of Trend in nutritional indices of children during the survey Table VI Multiple regression analysis between H-age, child and mother characteristics and urban strata

Table I

 I Daily intake of macronutrients by Lao women

	Table II Fulfillment (percentages) of nutritional requirements by Lao women Table IV Physical characteristics of women (n=152)
			Mean		25th SD	50th Percentile 25	75th Median	Percentile 75
	Weight (kg)		52.2	Percentile 8.7		Percentile 46.6	Percentile 51.4	56.9
	Energy Height (cm)	Energy 152.8	Protein 70.9 4.8	Fat 149.7	CHO 85.3	94.6 152.3	155.7
	kcal Polyunsaturated fatty acids Sitting height (cm) 81.4	g	45.8 2.8	g	79.4		81.3	g	118.9 81.5	83.3
	Crude value Vit A Arm circ (cm)	1,620.7 1 25.2	92.6 3.0 31.7	47.8 23.2		205.1 53.6	24.9	91.0	26.9
	Vit B1 MAC (cm)	344.9 2 19.8	30.2 1.9 117.8	19.0 18.5	54.7 164.5	230.3 19.9	20.9
	Vit B2 Triceps skf (mm)	14.0		80.6 5.2		107.5 10.05		138.5 13.1	17.05
	Energy
	percentage Vit B6 Biceps skf (mm)	-	6.2	23.0 3.0 85.2	25.8 3.95	51.2 121.7	5.6	150.2	7.7
	Total folic acid BMI (kg/m 2 )	-	22.3	5.0	31.4 3.4	8.2 20.1		8.7 49.8	21.6	68.4	24.4
	Vit C				96.2			155.3		242.6
	1 mean, 2 standard deviation) Calcium		22.5				32.8		47.7
	Iron				58.0				76.1		96.4
	Zinc				108.3			144.1		189.9

Table V

 V Evolution Trends in of nutritional indices values of children during the survey

				H-age	H-age	W-age	W-age
	Visit	W-H boys W-H girls	boys	girls	boys	girls
	2008	-0.64	-0.41	-0.83	-0.58	-0.94	-0.63
	(n=122)	0.14	0.15	0.14	0.16	0.13	0.15
	2009	-0.56	-0.50	-1.26	-0.82	-1.03	-0.76
	(n=156)	0.12	0.14	0.13	0.15	0.12	0.14
	Visit effect	0.0 1		5.3		0.6
		ns 2		0.02		ns
	Sex effect	1.2		5.7		4.7
		ns		0.01		0.03
	1 F value

2 p value; ns: not significant

p value; ns: not significant