Genetic diversity of the species Debaryomyces hansenii and the use of chromosome polymorphism for typing of strains isolated from surface-ripened cheeses.

AIMS: To investigate the genetic diversity among strains of Debaryomyces hansenii and further to evaluate chromosome polymorphism determined by pulse-field gel electrophoresis (PFGE) as a tool for strain typing. METHODS AND RESULTS: In total 56 isolates of D. hansenii were analysed by PFGE. The isolates included type strains and other strains obtained from culture collections as well as strains collected during production of Danish surface-ripened cheeses. By use of the PFGE technique the number and size of the chromosomal bands were calculated and the total genome size estimated. The number of chromosomal bands observed was found to vary from five to 10. The most common chromosome number was found to be six and for strains with six chromosomes the total genome size was found to vary from 9.4 to 12.6 Mb. The chromosome numbers for the type strain of each variety of D. hansenii (D. hansenii var. hansenii and D. hansenii var. fabryi) appeared to be six and seven respectively. By use of the PFGE technique it was possible to differentiate between all the investigated CBS strains and the vast majority of the dairy isolates. The dairy isolates that were found to have identical profiles (three of 56 isolates) were all isolated during production of one batch of surface-ripened cheeses and are likely to be the same strain isolated several times during cheese production. Further it was shown that the PFGE analysis did not result in a division of the two D. hansenii varieties, i.e. D. hansenii var. fabryi and D. hansenii var. hansenii into separate groups. CONCLUSION: The present study shows that the chromosomal arrangement of D. hansenii strains is heterogenic and does have a distinct chromosome polymorphism. Further the PFGE technique was proved to have a high discriminative power for strain typing of D. hansenii. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained add to the first knowledge on the genetic diversity of the species D. hansenii. Further the distinct chromosome polymorphism of D. hansenii strains as shown in this study makes the PFGE technique a useful tool for strain typing of D. hansenii, e.g. during cheese production.

Microbial succession of Debaryomyces hansenii strains during the production of Danish surfaced-ripened cheeses.

Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].

The effect of concurrent infections with Pasteurella multocida and Ascaridia galli on free range chickens.

Pasteurella multocida and Ascaridia galli are observed with high prevalences in free range chickens in Denmark, but the impact is unknown. A study was carried out to examine the interaction between A. galli and P. multocida in chickens and the impact on production. Five groups, each with 20 18-week-old Lohmann Brown chickens were infected. Group 1 was orally infected with 1000+/-50 embryonated A. galli eggs. Group 2 received 10(4) cfu P. multocida intratracheally. Group 3 was infected with A. galli and subsequently with P. multocida. Group 4 was infected with P. multocida followed by A. galli. Group 5 was the control. The study ran for 11 weeks where clinical manifestations, weight gain and egg production were recorded. Excretion of P. multocida was determined on individual basis and blood smears were made for differential counts. At the end of the study pathological lesions and the number of adult worms, larvae and eggs in the faeces were recorded. The birds were more severely affected when infected with both pathogens compared to single infections with A. galli or P. multocida, respectively. A lower weight gain and egg production was observed with dual infections. A. galli infection followed by a secondary P. multocida infection resulted in more birds with pathological lesions and continued P. multocida excretion. In conclusion a negative interaction between A. galli and P. multocida was observed and it is postulated that free range chickens are at higher risk of being subjected to outbreaks of fowl cholera when they are infected with A. galli.

DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses.

The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses. In total 60 isolates were included. Of these, 40 strains of the following species, Debaryomyces hansenii var. hansenii, D. hansenii var. fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D. hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses. ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections. Ten strains of each variety of D. hansenii and five strains of each of the above mentioned species were analysed. For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed. Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses. ITS-PCR RFLP was investigated for the purpose of strain typing of D. hansenii. Ten CBS strains of each variety of D. hansenii were analysed. Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains. This enzyme divided the 20 strains in three groups. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D. hansenii showed an identity of 99.84%, corresponding to a difference in one basepair. Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D. hansenii. MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D. hansenii on the 20 CBS strains of D. hansenii. The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D. hansenii at subspecies level. The 20 dairy isolates showed a lower genetic variability than the CBS strains as they were divided into eight groups. Cluster analysis of the 20 CBS strains and the 20 dairy isolates based on their mtDNA restriction profiles showed (max. similarity level = 52%) that the dairy isolates only clustered with the CBS strains of D. hansenii var. hansenii. For some of the dairies more than one strain of D. hansenii were found to be involved in the ripening process, indicating that the method could be useful for subspecies typing and investigation of the microbial succession between strains of D. hansenii during the ripening process of surface ripened cheeses.

Specific detection of Pasteurella multocida in chickens with fowl cholera and in pig lung tissues using fluorescent rRNA in situ hybridization.

A Pasteurella multocida species-specific oligonucleotide probe, pmhyb449, targeting 16S rRNA was designed and evaluated by whole-cell hybridization against 22 selected reference strains in animal tissues. It differentiated P. multocida from other bacterial species of the families Pasteurellaceae and Enterobacteriaceae and also from divergent species of the order Cytophagales (except biovar 2 strains of Pasteurella avium and Pasteurella canis, which have high 16S rRNA similarity to P. multocida). The potential of the probe for specific identification and differentiation of P. multocida was further detected in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues P. multocida cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood vessels (wall and lumen). In pig lung, postmortem-injected P. multocida was detected in the alveoli (lumen and wall), and in both animals the bacterial cells were seen in the bronchi. The results showed that with the oligonucleotide probe pmhyb449, fluorescent in situ hybridization is a suitable and fast method for specific detection of P. multocida in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to P. multocida.

High prevalence of Helicobacter pylori in the Alaska native population and association with low serum ferritin levels in young adults.

Iron deficiency anemia is a common public health problem in the Alaska Native population. Yet, a clear etiology has eluded researchers for decades. Previous studies suggested a link between Helicobacter pylori infection, gastrointestinal blood loss due to hemorrhagic gastritis, and generalized iron deficiency anemia in adult Alaska Natives. Therefore, we examined the association between the prevalence of H. pylori-specific immunoglobulin G (IgG) and serum ferritin levels, a marker of iron deficiency. A random sample of 2,080 serum samples from Alaska Native residents drawn between 1980 and 1986 from residents in 13 regions was selected, and the samples were stratified by age, sex, and region. Overall, 75% were positive for H. pylori-specific IgG. The rate of H. pylori seropositivity increased with age; by age 14 years, 78% of the residents were positive. There were no gender differences in H. pylori seropositivity. However, marked regional differences were observed. Serum ferritin levels of <12 ng/ml were found most commonly among persons <20 years of age and among women of childbearing age. A significant association between low serum ferritin levels and prevalence of H. pylori-specific IgG was found, particularly for people aged less than 20 years. H. pylori may be a factor contributing to the iron deficiency anemia in the Alaska Native population.

Phenotypic and genetic diversity of Saccharomyces contaminants isolated from lager breweries and their phylogenetic relationship with brewing yeasts.

A taxonomic study was carried out for isolates of Saccharomyces spp. identified as contaminants ("wild yeast") in 24 different lager breweries. With reference to the current taxonomy all isolates were found to belong to the Saccharomyces sensu stricto complex and 58% of the isolates were further identified as S. cerevisiae, 26% as S. pastorianus and 3% as S. bayanus. The remaining isolates (13%) could not be identified to the species level based on their phenotypic characteristics. However, some of these isolates were identified as S. cerevisiae by HaeIII restriction digest of PCR-amplified intergenic transcribed spacer (ITS) regions. Chromosome length polymorphism (CLP) was evident among the Saccharomyces brewing contaminants with chromosome profiles typical of Saccharomyces sensu stricto. Based upon cluster analysis of their chromosome profiles the majority of the brewing contaminants could be grouped as either S. cerevisiae or S. pastorianus/S. bayanus. Further, the technique was able to differentiate between almost all brewing contaminants and to separate them from any specific lager brewing yeast. The diversity of the Saccharomyces brewing contaminants clearly demonstrated by their CLP was further reflected by MAL genotyping. For the majority of the isolates more than two MAL loci were found with MAL1, MAL2 MAL3, MAL4 and MAL11, MAL31, MAL41 as the dominant genotypes. For all isolates MAL11 and MAL31 were found whereas MAL61 only was found for one isolate. The high number of MAL loci found in the SaccharomYces brewing contaminants indicate their adaptation to a maltose-enriched environment.

Immunogenicity of a heptavalent pneumococcal conjugate vaccine in Apache and Navajo Indian, Alaska native, and non-native American children aged <2 years.

High rates of invasive pneumococcal disease have been described among infants living in various Native American communities. In this study, we evaluated the immunogenicity of a 7-valent pneumococcal vaccine consisting of serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F covalently linked to the outer membrane protein complex of Neisseria meningitidis in Apache and Navajo Indian, Alaska Native, and non-Native American children. The vaccine was administered at ages 2, 4, and 6 months; a booster dose was given at age 15 months. Levels of serotype-specific immunoglobulin G (IgG) were measured by a standardized enzyme-linked immunosorbent assay. The responses after 3 primary doses of vaccine were similar in all 3 groups of children, except for those to serotypes 14 and 23F. One month after the booster dose, geometric mean concentrations (GMCs) of serotype-specific IgG antibodies increased significantly in all 3 groups of children, compared with GMCs of IgG antibodies to pneumococcal serotypes before the booster dose.

Invasive Haemophilus influenzae disease in Alaskan residents aged 10 years and older before and after infant vaccination programs.

CONTEXT: The introduction of Haemophilus influenzae type b (Hib) vaccination of children has led to a decline in incidence of Hib disease in young Alaskan children. However, the impact of vaccination on unimmunized Alaskan adolescents and adults has not been studied. OBJECTIVE: To characterize trends in incidence of and mortality due to invasive H influenzae disease in Alaskan residents aged 10 years and older prior to and after the introduction of a statewide Hib infant vaccination program. DESIGN AND SETTING: Population-based, descriptive correlational study conducted 1980-1996 in Alaska. SUBJECTS: One hundred twenty-nine individuals (31 Alaska Natives and 98 nonnative Alaska residents) aged 10 years and older in whom H influenzae was cultured from a normally sterile site. MAIN OUTCOME MEASURES: Incidence of H influenzae infection before (1980-1990) vs after (1991-1996) vaccination program initiation; serotype, biotype, and beta-lactamase production of isolates. RESULTS: The overall annual incidence of invasive H influenzae in those aged 10 years and older declined 33%, from 2.1 per 100,000 persons per year to 1.4 per 100,000 persons per year (P=. 03) after initiation of statewide infant Hib vaccination programs in 1991. This reduction appeared to be the result of a decrease in serotype b disease (82%; P<.001). Infection with other H influenzae serotypes and nontypeable strains increased from 0.5 per 100,000 persons per year to 1.1 per 100,000 persons per year (P=.01). Incidence declined from 4.2 per 100,000 persons per year to 1.2 per 100,000 persons per year in Alaska Natives (P=.005) and from 1.7 per 100,000 persons per year to 1.4 per 100,000 persons per year in nonnative Alaska residents (P=.37). Pneumonia (43%), sepsis (26%), and meningitis (16%) were the most common clinical presentations. Alcohol/drug abuse was comorbid in 15% of patients, while 13% of patients were pregnant women. beta-Lactamase production occurred in 35% of isolates and was stable throughout the surveillance. The overall case-fatality rate was 15%. CONCLUSION: The overall statewide incidence of invasive H influenzae infections in unimmunized persons aged 10 years and older decreased after the initiation of an infant Hib vaccine program, perhaps by decreasing Hib carriage in child reservoirs. An increase in non-serotype b strains was observed. This trend justifies the need for continued surveillance of invasive disease caused by H influenzae. JAMA. 2000.

Bronchiectasis in Alaska Native children: causes and clinical courses.

Although bronchiectasis has become a rare condition in U.S. children, it is still commonly diagnosed in Alaska Native children in the Yukon Kuskokwim Delta. The prevalence of bronchiectasis has not decreased in persons born during the 1980s as compared with those born in the 1940s. We reviewed case histories of 46 children with bronchiectasis. We observed that recurrent pneumonia was the major preceding medical condition in 85% of patients. There was an association between the lobes affected by pneumonia and the lobes affected by bronchiectasis. Eight (17%) patients had surgical resection of involved lobes. We conclude that the continued high prevalence of bronchiectasis appears to be related to extremely high rates of infant and childhood pneumonia. Pediatr Pulmonol. 2000;29:182-187. Published 2000 Wiley-Liss, Inc.

Severe respiratory syncytial virus disease in Alaska native children. RSV Alaska Study Group.

Hospitalization rates for respiratory syncytial virus (RSV) infection range from 1 to 20/1000 infants. To determine the rate and severity of RSV infections requiring hospitalization for infants in the Yukon-Kuskokwim (YK) Delta of Alaska, a 3-year prospective surveillance study was conducted. The annual rate of RSV hospitalization for YK Delta infants <1 year of age was 53-249/1000. RSV infection was the most frequent cause of infant hospitalization. RSV disease severity did not differ among non-high-risk infants in the YK Delta and at Johns Hopkins Hospital (JHH). On average, 1/125 infants born in the YK Delta required mechanical ventilation for RSV infection. During the peak season, approximately $1034/child <3 years of age was spent on RSV hospitalization in the YK Delta. In YK Delta infants

RSV-associated hospitalizations in Alaska Native infants.

PURPOSE: Retrospective reviews for 1986-1992 suggested that Alaska Native children experience high rates of respiratory syncytial virus (RSV)-associated hospitalization; however, the epidemiology of RSV infections has been poorly characterized. METHODS: A prospective hospital-based surveillance study was undertaken to determine rates of RSV-associated hospitalization in Alaska Native children < 36 months from the Yukon-Kuskokwim Delta. RESULTS: During the first study year, October 1993 to September 1994, there were 40 RSV cases (hospitalization rate, 53/1,000 infants < 1 year of age); however, during the second year, October 1994 to September 1995, there were 251 RSV cases (hospitalization rate, 294/1,000 infants). An unusually high proportion, 12%, of RSV cases were < 1 month of age. Disease severity was higher for children with a history of prematurity, heart, or lung disease (p = .001, X2 analysis). Of 255 cell cultures during 1994-1995, 190 were RSV-positive, 11 were positive for influenza, 4 for adenovirus, and 1 for parainfluenza. This study demonstrates wide seasonal variation in a population with an extremely high RSV hospitalization rate; increased disease severity associated with young age and pre-existing medical conditions; and co-circulation of RSV with other viruses.

Nutritional rickets among breast-fed black and Alaska Native children.

Although nutritional rickets remains a problem primarily in developing countries, children in northern climates in developed countries may also be at risk. We reviewed the case histories of five children diagnosed in Alaska during 1993-96. Three of the children were black and two Alaska Native. Their ages ranged from 11 to 20 months and they presented during January, April, and September. All of the children were breast-fed but only two received their milk intake exclusively from breast milk. The presenting complaint included abnormal gait in two children and seizures, bowed legs, and growth delay in one child each. All five children demonstrated a decrease in their height-for-age percentile. The most common physical finding was a rachitic rosary which was present in four children. In Alaska, all black and Alaska Native children (and other more pigmented children) less than two years of age who receive all or part of their milk intake from breast milk should receive vitamin D supplementation regardless of the time of year.

Pervasive occult gastrointestinal bleeding in an Alaska native population with prevalent iron deficiency. Role of Helicobacter pylori gastritis.

OBJECTIVE: To confirm prevalent iron deficiency among Yupik Eskimos living in Alaska and to explore the frequency of and potential lesions accounting for occult gastrointestinal bleeding. DESIGN: Descriptive survey. SETTING: Rural Arctic community. SUBJECTS: A total of 140 adult volunteers from 3 villages in the Yukon-Kuskokwim Delta region of western Alaska. MAIN OUTCOME MEASURES: Daily iron intake, hematologic and biochemical indexes of iron status, fecal hemoglobin levels, stool parasites, and endoscopic findings. RESULTS: While dietary iron intake by Yupiks was similar to that of a reference population, iron deficiency prevalence was increased 13-fold in Yupik men and 4-fold in Yupik women. Fecal hemoglobin levels were elevated in 90% of subjects contrasted with only 4% of a reference group; median levels were 5.9 and 0.5 mg of hemoglobin per gram of stool, respectively. Among 70 Yupik subjects with elevated fecal hemoglobin levels who had endoscopy performed, 68 (97%) had an abnormal gastric appearance consisting of erythema, mucosal thickening, diffuse mucosal hemorrhages, erosions, or ulcerations. Gastric biopsies revealed chronic active gastritis with associated Helicobacter pylori in 68 (99%) of 69. No other hemorrhagic gastrointestinal disease was detected. CONCLUSIONS: Based on this study sample, occult gastrointestinal bleeding appears to be pervasive in the Yupik population and likely underlies the prevalent iron deficiency. An atypical hemorrhagic gastritis associated with H pylori infection is present almost universally and may represent the bleeding source.

Iron deficiency anemia among Alaska Natives may be due to fecal loss rather than inadequate intake.

To define more fully the nature of a persistently high prevalence of iron deficiency anemia observed among Alaska Native children, we examined dietary iron intake, hemoglobin concentrations, and storage iron (serum ferritin) based on multiple cross-sectional surveys of Alaska Natives between 1983 and 1989. Approximately 30 to 50% of the children studied < 12 y of age had depleted iron stores. Anemia and depleted iron stores also were prevalent among adult men and women, about twice as prevalent as in the U.S. population based on the Second National Health and Nutrition Examination Survey (NHANES II). The higher rate of iron deficiency, occurring even when the dietary assessment found Alaska Native iron intake to be higher than the U.S. average with an ample intake of food high in bioavailable iron, suggests blood loss as a possible cause of the unusual pattern of iron deficiency observed. In a pilot study of stool blood loss in two villages, 65% of the samples had a significantly elevated stool heme concentration. Further investigation of iron deficiency due to gastrointestinal blood loss for the Alaska Native is warranted.

Vitamin A therapy for children with respiratory syncytial virus infection: a multicenter trial in the United States.

BACKGROUND: High dose vitamin A therapy is effective in reducing morbidity and mortality associated with measles infection. Children with acute respiratory syncytial virus (RSV) infection have low serum vitamin A concentrations. METHODS: We performed a multicenter, randomized, placebo-controlled trial of high dose vitamin A therapy among 239 children 1 month to 6 years of age to determine whether high dose vitamin A therapy would reduce morbidity associated with RSV infection. RESULTS: There were no differences between the vitamin A and placebo recipients for most clinical outcomes; however, vitamin A recipients had-longer hospital stays than placebo recipients (5.0 days vs. 4.4 days, P = 0.01) after enrollment. This effect was significant for children who were older than 1 year (who also had received the highest doses of vitamin A), particularly among those at low risk for complications of RSV infection and those enrolled during the second study season. Serum retinol levels at enrollment were inversely correlated with severity of illness. CONCLUSIONS: We found no evidence of a beneficial effect of vitamin A for the treatment of RSV infection in children in the United States. There may be groups of children for which vitamin A has an adverse effect, resulting in longer hospital stays.

Tobacco erases 30 years of progress: preliminary analysis of the effect of tobacco smoking on Alaska Native birth weight.

OBJECTIVE: Investigate the relationship between tobacco and/or alcohol use and Alaska Native birth weight. METHODS: Data on weight, tobacco smoking and alcohol use among Alaska Natives were abstracted from 1989-91 Indian Health Service natality records based on birth certificates. RESULTS: Birth certificate data were available for 9,175 live births. Single live births were analyzed for 8,994 Alaska Natives. In women with no tobacco smoking the mean birth weight of their infants was 3,571 g; 1-5 cigarettes/day 3,429 g; 6-10 cigarettes/day 3,332 g (p < .05); and > 10 cigarettes/day 3,260 g (p < .05). Infants of Alaska Natives who reported no alcohol and no tobacco use had a mean birth weight of 3,579 g; alcohol use but no tobacco use 3,452 g; no alcohol but tobacco use 3,388 g; and both alcohol and tobacco use 3,281 g. (p < .001). CONCLUSIONS: The mean birth weight of infants born to Alaska Native women with the highest use of tobacco were reduced by over 300 g compared to non-smoking Alaska Native women. Mean infant birth weight of tobacco smoking Yup'ik women in 1989-91 were reduced by over 400 g, comparable to weights reported in the 1960s.

Hospitalizations for respiratory syncytial virus infection in Alaska Native children.

To characterize the epidemiology of Alaska Native children hospitalized for respiratory syncytial virus infections, we reviewed records of hospitalizations during the winter seasons of 1991 to 1992 and 1992 to 1993 at a hospital in Anchorage and a rural hospital in the Yukon Kuskokwim Delta (YKD) region of southwestern Alaska. The median age of hospitalization for respiratory syncytial virus infection was 2 months of age for YKD residents and 4.5 months for Anchorage residents. Sixteen percent of the hospitalized YKD children were less than 1 month of age, whereas the same was true for only 3% of the Anchorage children. Eight percent of the YKD patients required mechanical ventilation, whereas none of the Anchorage patients required ventilation. The median hospital stay was 4.8 days for YKD patients and 3.2 days for Anchorage patients. Hospitalization rates for infants less than 1 year of age were 33/1000 for Alaska Natives in Anchorage and 100/1000 for those in the YKD region. The extremely high hospitalization rate, especially among very young infants in the rural YKD region, points to a need for early preventive efforts.

Growth and hematological changes in the Eskimo children of Wainwright, Alaska: 1968 to 1977.

The growth and hematological status of all 152 Eskimo children of Wainwright, AK observed in 1977 are reported and compared with the respective status of the children living there in 1968. Fifty-two children (34%) in the 1977 study were also in the 1968 study. Comparison of height data observed in 1977 with those observed in 1968 reveals that male children less than 11 yr of age and female children ages 3 to 9 yr showed a significant improvement in stature (p less than 0.05). At the same time, an improvement in nutrition was observed among the children in 1977 as evidenced by the marked decrease in anemia for children under 6 yr (p less than 0.001). These changes in growth and hematological status have occurred during a time in which health services were improved, a supplemental nutrition program was instituted, and subsistence food gathering remained high. These changes underscore the contribution of environmental factors rather than of genetic variability to the growth potential of these children.