Consumption of a sourdough-leavened croissant enriched with a blend of fibers influences fasting blood glucose in a randomized controlled trial in healthy subjects.

BACKGROUND: An incorrect lifestyle, including diet, is responsible for the worldwide dramatic increase in obesity and type 2-diabetes. Increasing dietary fiber consumption may lead to health benefits and reformulation of bakery products may be a strategy to globally improve the diet. OBJECTIVE: This study aimed to assess the impact of a two-week breakfast consumption with a sourdough-leavened croissant containing a blend of dietary fiber from 10 sources (4.8g/100g, FIBCRO), compared to a control croissant (dietary fibers 1.3g/100g, CONCRO) on daily energy intake, appetite, metabolic variables, and the gut microbiome. METHODS: Thirty-two healthy participants were randomly allocated in two groups consuming FIBCRO or CONCRO. Participants self-recorded their diet and appetite through 7-day weighted food diaries and visual analogue scales every day over the two weeks. At baseline and after the intervention, fasting blood and urine samples, and fecal samples were collected beside blood pressure, anthropometry, and body composition. Serum glucose, lipids, C-reactive protein, and insulin according to the official methods and serum dipeptidyl peptidase-4 (DPPIV) activity by photometric method were measured. Polyphenols and urolithins in urines were analyzed by LC/MS/MS whereas gut microbiome in feces by shotgun metagenomics. RESULTS: FIBCRO consumption improved fasting blood glucose compared to CONCRO (mean changes from baseline -2.0 mg/dL in FIBCRO vs +3.1 mg/dL in CONCRO, p=0.022), also reducing serum DPPIV activity by 1.7 IU/L (p=0.01) and increasing urinary excretion of urolithin A-sulfate by 6.9 ng/mg creatinine (p=0.04) compared to baseline. No further changes in any of monitored variables or in the gut microbiome were detected. CONCLUSIONS: Results suggested that a two-week consumption of a sourdough croissant claimed as "source of dietary fiber" improved fasting glycemia compared to a conventional sourdough croissant in healthy subjects. The reduced serum DPPIV activity and increased bioavailability of urolithin likely contributed to determine that effect independently from gut microbiome changes. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE WHERE IT WAS OBTAINED: NCT04999280 (https://clinicaltrials.gov/).

Milk protein digestion and the gut microbiome influence gastrointestinal discomfort after cow milk consumption in healthy subjects.

Many healthy people suffer from milk-related gastrointestinal discomfort (GID) despite not being lactose intolerant; the mechanisms underpinning such condition are unknown. This study aimed to explore milk protein digestion and related physiological responses (primary outcome), gut microbiome and gut permeability in 19 lactose-tolerant healthy nonhabitual milk consumers [NHMCs] reporting GID after consuming cow milk compared to 20 habitual milk consumers [HMCs] without GID. NHMCs and HMCs participated in a milk-load (250 mL) test, underwent blood sample collection at 6 time points over 6 h after milk consumption and collected urine samples and GID self-reports over 24 h. We measured the concentrations of 31 milk-derived bioactive peptides (BAPs), 20 amino acids, 4 hormones, 5 endocannabinoid system mediators, glucose and the dipeptidyl peptidase-IV (DPPIV) activity in blood and indoxyl sulfate in urine samples. Subjects also participated in a gut permeability test and delivered feces sample for gut microbiome analysis. Results showed that, compared to HMCs, milk consumption in NHMCs, along with GID, elicited a slower and lower increase in circulating BAPs, lower responses of ghrelin, insulin, and anandamide, a higher glucose response and serum DPPIV activity. The gut permeability of the two groups was similar, while the habitual diet, which was lower in dairy products and higher in the dietary-fibre-to-protein ratio in NHMCs, possibly shaped the gut microbiome; NHMCs exhibited lower abundance of Bifidobacteria, higher abundance of Prevotella and lower abundance of protease-coding genes, which may have reduced protein digestion, as evidenced by lower urinary excretion of indoxyl sulfate. In conclusion, the findings showed that a less efficient digestion of milk proteins, supported by a lower proteolytic capability of the gut microbiome, may explain GID in healthy people after milk consumption.

The Vaginal Microbiome: A Long Urogenital Colonization Throughout Woman Life.

Vaginal microbial niche is a dynamic ecosystem, composed by more than 200 bacterial species which are influenced by genes, ethnic background and environmental-behavioral factors. Several lines of evidence have well documented that vaginal microbiome constantly changes over the course of woman's life, so to exert an important impact on woman quality of life, from newborn to post-menopausal ages. This review aims at analyzing the role of vaginal microbiome in the maintenance of woman's homeostasis and at tracking critical changes that commonly occur across woman's lifetime. The role of hormone replacement therapy in the modulation of vaginal microbiome composition and in the improvement of vaginal wellness in postmenopausal women with decreasing levels of circulating estrogen is discussed.

Diet influences the functions of the human intestinal microbiome.

Gut microbes programme their metabolism to suit intestinal conditions and convert dietary components into a panel of small molecules that ultimately affect host physiology. To unveil what is behind the effects of key dietary components on microbial functions and the way they modulate host-microbe interaction, we used for the first time a multi-omic approach that goes behind the mere gut phylogenetic composition and provides an overall picture of the functional repertoire in 27 fecal samples from omnivorous, vegan and vegetarian volunteers. Based on our data, vegan and vegetarian diets were associated to the highest abundance of microbial genes/proteins responsible for cell motility, carbohydrate- and protein-hydrolyzing enzymes, transport systems and the synthesis of essential amino acids and vitamins. A positive correlation was observed when intake of fiber and the relative fecal abundance of flagellin were compared. Microbial cells and flagellin extracted from fecal samples of 61 healthy donors modulated the viability of the human (HT29) colon carcinoma cells and the host response through the stimulation of the expression of Toll-like receptor 5, lectin RegIIIα and three interleukins (IL-8, IL-22 and IL-23). Our findings concretize a further and relevant milestone on how the diet may prevent/mitigate disease risk.

The fate of cigarette butts in different environments: Decay rate, chemical changes and ecotoxicity revealed by a 5-years decomposition experiment.

Cigarette butts (CBs) are the most common litter item on Earth but no long-term studies evaluate their fate and ecological effects. Here, the role of nitrogen (N) availability and microbiome composition on CBs decomposition were investigated by a 5-years experiment carried out without soil, in park grassland and sand dune. During decomposition, CBs chemical changes was assessed by both 13C CPMAS NMR and LC-MS, physical structure by scanning electron microscope and ecotoxicity by Aliivibrio fischeri and Raphidocelis subcapitata. Microbiota was investigated by high-throughput sequencing of bacterial and eukaryotic rRNA gene markers. CBs followed a three-step decomposition process: at the early stage (∼30 days) CBs lost ∼15.2% of their mass. During the subsequent two years CBs decomposed very slowly, taking thereafter different trajectories depending on N availability and microbiome composition. Without soil CBs showed minor chemical and morphological changes. Over grassland soil a consistent N transfer occurs that, after de-acetylation, promote CBs transformation into an amorphous material rich in aliphatic compounds. In sand dune we found a rich fungal microbiota able to decompose CBs, even before the occurrence of de-acetylation. CBs ecotoxicity was highest immediately after smoking. However, for R. subcapitata toxicity remained high after two and five years of decomposition.

Attenuated Lactococcus lactis and Surface Bacteria as Tools for Conditioning the Microbiota and Driving the Ripening of Semisoft Caciotta Cheese.

This study aimed at establishing the effects of attenuated starters and surface bacteria on various features of caciotta cheese. The cheese undergoes a ripening period during which the house microbiota contaminates the surface. Conventional cheese (the control cheese [CC]) is made using only primary starters. Primary starters and attenuated (i.e., unable to grow and synthesize lactic acid) Lactococcus lactis (Lc. lactis) subsp. lactis were used to produce caciotta cheese without (ATT cheese) or with an inoculum of surface bacteria: (i) Leuconostoc lactis (Le. lactis) (LL cheese), (ii) Vibrio casei (VC cheese), (iii) Staphylococcus equorum (SE cheese), (iv) Brochothrix thermosphacta (BX cheese), and (v) a mixture of all four (MIX cheese). Attenuated Lc. lactis increased microbial diversity during cheese ripening. At the core, attenuated starter mainly increased indigenous lactococci and Lactobacillus delbrueckii group bacteria. At the surface, the main effect was on Macrococcus caseolyticus Autochthonous Le. lactis strains took advantage of the attenuated starter, becoming dominant. Adjunct Le. lactis positively affected Lactobacillus sakei group bacteria on the LL cheese surface. Adjunct V. casei, S. equorum, and B. thermosphacta did not become dominant. Surfaces of VC, SE, and BX cheeses mainly harbored Staphylococcus succinus Peptidase activities were higher in cheeses made with attenuated starter than in CC, which had the lowest concentration of free amino acids. Based on the enzymatic activities of adjunct Le. lactis, LL and MIX cheeses exhibited the highest glutamate dehydrogenase, cystathionine-γ-lyase, and esterase activities. As shown by multivariate statistical analyses, LL and MIX cheeses showed the highest similarity for microbiological and biochemical features. LL and MIX cheeses received the highest scores for overall sensory acceptability.IMPORTANCE This study provides in-depth knowledge of the effects of attenuated starters and surface bacterial strains on the microbiota and related metabolic activities during cheese ripening. The use of attenuated Lc. lactis strongly impacted the microbiota assembly of caciotta cheese. This led to improved biochemical and sensory features compared to conventional cheese. Among surface bacterial strains, Le. lactis played a key role in the metabolic activities involved in cheese ripening. This resulted in an improvement of the sensory quality of caciotta cheese. The use of attenuated lactic acid bacteria and the surface adjunct Le. lactis could be a useful biotechnology to improve the flavor formation of caciotta cheese.

Coffee prevents fatty liver disease induced by a high-fat diet by modulating pathways of the gut-liver axis.

Coffee consumption is inversely associated with the risk of non-alcoholic fatty liver disease (NAFLD). A gap in the literature still exists concerning the intestinal mechanisms that are involved in the protective effect of coffee consumption towards NAFLD. In this study, twenty-four C57BL/6J mice were divided into three groups each receiving a standard diet, a high-fat diet (HFD) or an HFD plus decaffeinated coffee (HFD+COFFEE) for 12 weeks. Coffee supplementation reduced HFD-induced liver macrovesicular steatosis (P < 0·01) and serum cholesterol (P < 0·001), alanine aminotransferase and glucose (P < 0·05). Accordingly, liver PPAR- α (P < 0·05) and acyl-CoA oxidase-1 (P < 0·05) as well as duodenal ATP-binding cassette (ABC) subfamily A1 (ABCA1) and subfamily G1 (ABCG1) (P < 0·05) mRNA expressions increased with coffee consumption. Compared with HFD animals, HFD+COFFEE mice had more undigested lipids in the caecal content and higher free fatty acid receptor-1 mRNA expression in the duodenum and colon. Furthermore, they showed an up-regulation of duodenal and colonic zonulin-1 (P < 0·05), duodenal claudin (P < 0·05) and duodenal peptide YY (P < 0·05) mRNA as well as a higher abundance of Alcaligenaceae in the faeces (P < 0·05). HFD+COFFEE mice had an energy intake comparable with HFD-fed mice but starting from the eighth intervention week they gained significantly less weight over time. Data altogether showed that coffee supplementation prevented HFD-induced NAFLD in mice by reducing hepatic fat deposition and metabolic derangement through modification of pathways underpinning liver fat oxidation, intestinal cholesterol efflux, energy metabolism and gut permeability. The hepatic and metabolic benefits induced by coffee were accompanied by changes in the gut microbiota.

Gut microbiota signatures in cystic fibrosis: Loss of host CFTR function drives the microbiota enterophenotype.

BACKGROUND: Cystic fibrosis (CF) is a disorder affecting the respiratory, digestive, reproductive systems and sweat glands. This lethal hereditary disease has known or suspected links to the dysbiosis gut microbiota. High-throughput meta-omics-based approaches may assist in unveiling this complex network of symbiosis modifications. OBJECTIVES: The aim of this study was to provide a predictive and functional model of the gut microbiota enterophenotype of pediatric patients affected by CF under clinical stability. METHODS: Thirty-one fecal samples were collected from CF patients and healthy children (HC) (age range, 1-6 years) and analysed using targeted-metagenomics and metabolomics to characterize the ecology and metabolism of CF-linked gut microbiota. The multidimensional data were low fused and processed by chemometric classification analysis. RESULTS: The fused metagenomics and metabolomics based gut microbiota profile was characterized by a high abundance of Propionibacterium, Staphylococcus and Clostridiaceae, including Clostridium difficile, and a low abundance of Eggerthella, Eubacterium, Ruminococcus, Dorea, Faecalibacterium prausnitzii, and Lachnospiraceae, associated with overexpression of 4-aminobutyrate (GABA), choline, ethanol, propylbutyrate, and pyridine and low levels of sarcosine, 4-methylphenol, uracil, glucose, acetate, phenol, benzaldehyde, and methylacetate. The CF gut microbiota pattern revealed an enterophenotype intrinsically linked to disease, regardless of age, and with dysbiosis uninduced by reduced pancreatic function and only partially related to oral antibiotic administration or lung colonization/infection. CONCLUSIONS: All together, the results obtained suggest that the gut microbiota enterophenotypes of CF, together with endogenous and bacterial CF biomarkers, are direct expression of functional alterations at the intestinal level. Hence, it's possible to infer that CFTR impairment causes the gut ecosystem imbalance.This new understanding of CF host-gut microbiota interactions may be helpful to rationalize novel clinical interventions to improve the affected children's nutritional status and intestinal function.

Profiling white wine seed vinegar bacterial diversity through viable counting, metagenomic sequencing and PCR-DGGE.

The production of traditional vinegar is usually carried out using the so-called "seed vinegar" or "mother of vinegar" that is composed of an undefined and complex pool of microorganisms deriving from a previous vinegar production. To date, there have been relatively few studies on the microbiota of seed vinegars. The present study was carried out to discover the bacterial biota of seed vinegar samples used in the homemade production of local vinegars obtained from the acetic fermentation of white wine. The seed vinegar samples were subjected to viable counting and advanced molecular analyses, namely, Illumina sequencing and PCR-DGGE. The adopted polyphasic approach allowed the bacterial diversity of the analyzed samples to be profiled, thus revealing the presence of acetic acid bacteria ascribed to the genera Acetobacter, Gluconacetobacter, Gluconobacter and Komagataeibacter. Moreover, other microbial genera as Pseudomonas, Bacillus and Clostridium were abundantly found in almost all the samples, together with other minority genera. The results of viable counting confirmed the well-acknowledged limitations inherent with acetic acid bacteria recovery on plate growth media. The overall results confirmed that seed vinegars have a complex and heterogeneous biodiversity, thus encouraging their exploitation for the isolation and future technological characterization of cultures to be selected for the manufacture of mixed starter cultures.

Different temperatures select distinctive acetic acid bacteria species and promotes organic acids production during Kombucha tea fermentation.

Kombucha is a traditional beverage produced by tea fermentation, carried out by a symbiotic consortium of bacteria and yeasts. Acetic Acid Bacteria (AAB) usually dominate the bacterial community of Kombucha, driving the fermentative process. The consumption of this beverage was often associated to beneficial effects for the health, due to its antioxidant and detoxifying properties. We characterized bacterial populations of Kombucha tea fermented at 20 or 30 °C by using culture-dependent and -independent methods and monitored the concentration of gluconic and glucuronic acids, as well as of total polyphenols. We found significant differences in the microbiota at the two temperatures. Moreover, different species of Gluconacetobacter were selected, leading to a differential abundance of gluconic and glucuronic acids.

A Metagenomic and in Silico Functional Prediction of Gut Microbiota Profiles May Concur in Discovering New Cystic Fibrosis Patient-Targeted Probiotics.

Cystic fibrosis (CF) is a life-limiting hereditary disorder that results in aberrant mucosa in the lungs and digestive tract, chronic respiratory infections, chronic inflammation, and the need for repeated antibiotic treatments. Probiotics have been demonstrated to improve the quality of life of CF patients. We investigated the distribution of gut microbiota (GM) bacteria to identify new potential probiotics for CF patients on the basis of GM patterns. Fecal samples of 28 CF patients and 31 healthy controls (HC) were collected and analyzed by 16S rRNA-based pyrosequencing analysis of GM, to produce CF-HC paired maps of the distribution of operational taxonomic units (OTUs), and by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) for Kyoto Encyclopedia of Genes and Genomes (KEGG) biomarker prediction. The maps were scanned to highlight the distribution of bacteria commonly claimed as probiotics, such as bifidobacteria and lactobacilli, and of butyrate-producing colon bacteria, such as Eubacterium spp. and Faecalibacterium prausnitzii. The analyses highlighted 24 OTUs eligible as putative probiotics. Eleven and nine species were prevalently associated with the GM of CF and HC subjects, respectively. Their KEGG prediction provided differential CF and HC pathways, indeed associated with health-promoting biochemical activities in the latter case. GM profiling and KEGG biomarkers concurred in the evaluation of nine bacterial species as novel putative probiotics that could be investigated for the nutritional management of CF patients.

Whole-grain wheat consumption reduces inflammation in a randomized controlled trial on overweight and obese subjects with unhealthy dietary and lifestyle behaviors: role of polyphenols bound to cereal dietary fiber.

BACKGROUND: Epidemiology associates whole-grain (WG) consumption with several health benefits. Mounting evidence suggests that WG wheat polyphenols play a role in mechanisms underlying health benefits. OBJECTIVE: The objective was to assess circulating concentration, excretion, and the physiologic role of WG wheat polyphenols in subjects with suboptimal dietary and lifestyle behaviors. DESIGN: A placebo-controlled, parallel-group randomized trial with 80 healthy overweight/obese subjects with low intake of fruit and vegetables and sedentary lifestyle was performed. Participants replaced precise portions of refined wheat (RW) with a fixed amount of selected WG wheat or RW products for 8 wk. At baseline and every 4 wk, blood, urine, feces, and anthropometric and body composition measures were collected. Profiles of phenolic acids in biological samples, plasma markers of metabolic disease and inflammation, and fecal microbiota composition were assessed. RESULTS: WG consumption for 4-8 wk determined a 4-fold increase in serum dihydroferulic acid (DHFA) and a 2-fold increase in fecal ferulic acid (FA) compared with RW consumption (no changes). Similarly, urinary FA at 8 wk doubled the baseline concentration only in WG subjects. Concomitant reduction in plasma tumor necrosis factor-α (TNF-α) after 8 wk and increased interleukin (IL)-10 only after 4 wk with WG compared with RW (P = 0.04) were observed. No significant change in plasma metabolic disease markers over the study period was observed, but a trend toward lower plasma plasminogen activator inhibitor 1 with higher excretion of FA and DHFA in the WG group was found. Fecal FA was associated with baseline low Bifidobacteriales and Bacteroidetes abundances, whereas after WG consumption, it correlated with increased Bacteroidetes and Firmicutes but reduced Clostridium. TNF-α reduction correlated with increased Bacteroides and Lactobacillus. No effect of dietary interventions on anthropometric measurements and body composition was found. CONCLUSIONS: WG wheat consumption significantly increased excreted FA and circulating DHFA. Bacterial communities influenced fecal FA and were modified by WG wheat consumption. This trial was registered at clinicaltrials.gov as NCT01293175.

NaOH-debittering induces changes in bacterial ecology during table olives fermentation.

Limited information is available on the impact of the NaOH treatment on table olive fermentations, and for this reason a polyphasic approach has been adopted here to investigate its effect on the fermentation dynamics and bacterial biodiversity. The microbial counts of the main groups involved in the transformation have not shown any differences, apart from a more prompt start of the fermentation when the olives were subjected to the NaOH treatment. The data produced by culture-independent analyses highlighted that the fermentation of table olives not treated with NaOH is the result of the coexistence of two different ecosystems: the surface of the olives and the brines. A sodium hydroxide treatment not only eliminates this difference, but also affects the bacterial ecology of the olives to a great extent. As proved by high-throughput sequencing, the fermentation of the olives not treated with NaOH was characterized by the presence of halophilic bacteria, which were substituted by Lactobacillus at the later stages of the fermentation, while enterobacteria were dominant when the olives were treated with sodium hydroxide. Higher biodiversity was found for Lactobacillus plantarum isolated during untreated fermentation. Different biotypes were found on the olive surface and in the brines. When the debittering process was carried out, a decrease in the number of L. plantarum biotypes were observed and those originating from the surface of the olive did not differentiate from the ones present in the brines.

Differential protein expression patterns between planktonic and biofilm cells of Salmonella enterica serovar Enteritidis PT4 on stainless steel surface.

In the present study, the proteome of a strain of S. enterica serovar Enteritidis PT4, grown either as biofilm on stainless steel surface or as free-floating (planktonic) in Brain Heart (BH) broth, was investigated in order to detect the strong differences in whole-cell protein expression patterns between the two growth styles. The proteins extracted from both types of cells were subjected to 2-D PAGE, followed by in-gel tryptic digestion, extraction, subsequent MALDI-TOF mass spectrometry (MS) analysis and finally database searches for protein identification. Using this approach, 30 proteins were identified as differentially expressed between the two growth modes on an "on-off" basis, that is, proteins that were detected in one case but not in the other. In particular, 20 and 10 proteins were identified in biofilm and planktonic-grown cells, respectively. The group of proteins whose expression was visible only during biofilm growth included proteins involved in global regulation and stress response (ArcA, BtuE, Dps, OsmY, SspA, TrxA, YbbN and YhbO), nutrient transport (Crr, DppA, Fur and SufC), degradation and energy metabolism (GcvT, GpmA, RibB), detoxification (SseA and YibF), DNA metabolism (SSB), curli production (CsgF), and murein synthesis (MipA). To summarize, this study demonstrates that biofilm growth of S. Enteritidis causes distinct changes in protein expression and offers valuable new data regarding some of the proteins presumably involved in this process. The putative role of these proteins in the maintenance of a biofilm community in Salmonella and other bacteria is discussed.

Diversity of Salmonella spp. serovars isolated from the intestines of water buffalo calves with gastroenteritis.

BACKGROUND: Salmonellosis in water buffalo (Bubalus bubalis) calves is a widespread disease characterized by severe gastrointestinal lesions, profuse diarrhea and severe dehydration, occasionally exhibiting a systemic course. Several Salmonella serovars seem to be able to infect water buffalo, but Salmonella isolates collected from this animal species have been poorly characterized. In the present study, the prevalence of Salmonella spp. in water buffalo calves affected by lethal gastroenteritis was assessed, and a polyphasic characterization of isolated strains of S. Typhimurium was performed. RESULTS: The microbiological analysis of the intestinal contents obtained from 248 water buffalo calves affected by lethal gastroenteritis exhibited a significant prevalence of Salmonella spp. (25%), characterized by different serovars, most frequently Typhimurium (21%), Muenster (11%), and Give (11%). The 13 S. Typhimurium isolates were all associated with enterocolitis characterized by severe damage of the intestine, and only sporadically isolated with another possible causative agent responsible for gastroenteritis, such as Cryptosporidium spp., Rotavirus or Clostridium perfringens. Other Salmonella isolates were mostly isolated from minor intestinal lesions, and often (78% of cases) isolated with other microorganisms, mainly toxinogenic Escherichia coli (35%), Cryptosporidium spp. (20%) and Rotavirus (10%). The S. Typhimurium strains were characterized by phage typing and further genotyped by polymerase chain reaction (PCR) detection of 24 virulence genes. The isolates exhibited nine different phage types and 10 different genetic profiles. Three monophasic S. Typhimurium (B:4,12:i:-) isolates were also found and characterized, displaying three different phage types and three different virulotypes. The molecular characterization was extended to the 7 S. Muenster and 7 S. Give isolates collected, indicating the existence of different virulotypes also within these serovars. Three representative strains of S. Typhimurium were tested in vivo in a mouse model of mixed infection. The most pathogenic strain was characterized by a high number of virulence factors and the presence of the locus agfA, coding for a thin aggregative fimbria. CONCLUSIONS: These results provide evidence that Salmonella is frequently associated with gastroenteritis in water buffalo calves, particularly S. Typhimurium. Moreover, the variety in the number and distribution of different virulence markers among the collected S. Typhimurium strains suggests that within this serovar there are different pathotypes potentially responsible for different clinical syndromes.

Genetic resistance to Brucella abortus in the water buffalo (Bubalus bubalis).

Brucellosis is a costly disease of water buffaloes (Bubalus bubalis). Latent infections and prolonged incubation of the pathogen limit the efficacy of programs based on the eradication of infected animals. We exploited genetic selection for disease resistance as an approach to the control of water buffalo brucellosis. We tested 231 water buffalo cows for the presence of anti-Brucella abortus antibodies (by the agglutination and complement fixation tests) and the Nramp1 genotype (by PCR-denaturing gradient gel electrophoresis). When the 231 animals (58 cases and 173 controls) were divided into infected (seropositive) and noninfected (seronegative) groups and the Nramp1 genotypes were compared, the seropositive subjects were 52 out of 167 (31%) in the Nramp1A+ (Nramp1AA or Nramp1AB) group and 6 out of 64 (9.4%) in the Nramp1A- (Nramp1BB) group (odds ratio, 4.37; 95% confidence limits, 1.87 to 10.19; chi2, 11.65 for 1 degree of freedom). Monocytes from Nramp1BB subjects displayed significantly (P < 0.01) higher levels of Nramp1 mRNA than Nramp1AA subjects and also a significantly (P < 0.01) higher ability in controlling the intracellular replication of several Brucella species in vitro. Thus, selection for the Nramp1BB genotype can become a valuable tool for the control of water buffalo brucellosis in the areas where the disease is endemic.