Monitoring and molecular characterization of group Drotavirus in Brazilian poultry farms

Abstract: Rotaviruses are etiological agents of diarrhea both in humans and in several animal species. Data on avian Group D rotaviruses (RVD) are scarce, especially in Brazil. We detected RVD in 4 pools of intestinal contents of broilers, layer and broiler breeders out of a total of 111 pools from 8 Brazilian states, representing an occurrence of 3.6%, by a specific RVD RT-PCR targeting the VP6 gene. Phylogenetic tree confirmed that the Brazilian strains belong to group D and 3 of the sequences were identical in terms of amino acid whereas one showed 99.5% identity with the others. The sequences described in this study are similar to other sequences previously detected in Brazil, confirming the conserved nature of the VP6 protein.

Chronic lactose intake modifies the gastric emptying of monosaccharides but not of disaccharides in weanling rats

Ninety-six weanling male Wistar rats were fed for four weeks one of two different chows: a normal rat chow containing 55.5% (w/w) starch (control group, N = 48) or a rat chow in which starch was partially replaced by lactose, in such a way that the experimental group (N = 48) received 35.5% (w/w) starch and 20% (w/w) lactose. The gastric emptying of fluid was then studied by measuring the gastric retention of four test meals containing lactose (5% or 10%, w/v) or glucose + galactose (5% or 10%, w/v). Homogenates of the small intestine were assayed for lactase activity. The gastric retention values were obtained 15 min after orogastric infusion of the liquid meals. The median values for gastric retention of the 5% lactose solutions were 37.7% for the control group and 37.0% for the experimental group (P>0.02). For the 10% lactose solution the median values were 51.2% and 47.9% (P>0.02) for the control and experimental groups, respectively. However, for the 2.5% glucose + 2.5% galactose meal the median gastric retention was lower (P<0.02) in the group fed a lactose-enriched chow (38.5%) than in the control group (41.6%). For the 5% glucose + 5% galactose solution the median values were not statistically different between groups, 65.0% for the control group and 58.8% for the experimental group. The median values of the specific lactase activity in the small intestine homogenate was 0.74 U/g in the control group and 0.91 U/g in the experimental group. These values were not statistically different (P>0.05). These results suggest that the prolonged ingestion of lactose by young adult rats changes the gastric emptying of a solution containing 5% monosaccharides. This adaptation may reflect the desensitization of intestinal nutrient receptors, possibly by an osmotic effect of lactose present in the chow.

Effect of pyloroplasty and fundectomy on the delay of gastric emptying and gastrointestinal transit of liquid elicited by acute blood volume expansion in awake rats

We evaluated the effects of fundectomy and pyloroplasty on the delay of gastric emptying (GE) and gastrointestinal (GI) transit of liquid due to blood volume (BV) expansion in awake rats. Male Wistar rats (N = 76, 180-250 g) were first submitted to fundectomy (N = 26), Heinecke-Mikulicz pyloroplasty (N = 25) or SHAM laparotomy (N = 25). After 6 days, the left external jugular vein was cannulated and the animals were fasted for 24 h with water ad libitum. The test meal was administered intragastrically (1.5 ml of a phenol red solution, 0.5 mg/ml in 5% glucose) to normovolemic control animals and to animals submitted to BV expansion (Ringer-bicarbonate, iv infusion, 1 ml/min, volume up to 5% body weight). BV expansion decreased GE and GI transit rates in SHAM laparotomized animals by 52 and 35.9% (P<0.05). Fundectomy increased GE and GI transit rates by 61.1 and 67.7% (P<0.05) and prevented the effect of expansion on GE but not on GI transit (13.9% reduction, P<0.05). Pyloroplasty also increased GE and GI transit rates by 33.9 and 44.8% (P<0.05) but did not prevent the effect of expansion on GE or GI transit (50.7 and 21.1% reduction, P<0.05). Subdiaphragmatic vagotomy blocked the effect of expansion on GE and GI transit in both SHAM laparotomized animals and animals submitted to pyloroplasty. In conclusion 1) the proximal stomach is involved in the GE delay due to BV expansion but is not essential for the establishment of a delay in GI transit, which suggests the activation of intestinal resistances, 2) pyloric modulation was not apparent, and 3) vagal pathways are involved

Use of nucleotides in weanling rats with diarrhea induced by a lactose overload: effect on the evolution of diarrhea and weight and on the histopathology of intestine, liver and spleen

Until recently, dietary sources of nucleotides were thought not to be essential for good nutrition. Certain states with higher metabolic demands may require larger amounts that cannot be provided by endogenous production. The objective of the present study was to determine the action of nucleotides on the recovery from lactose-induced diarrhea in weaned rats. Thirty-six weanling Fisher rats were divided into two groups. Group 1 received a standard diet and group 2 received a diet containing lactose in place of starch. On the 10th day, six animals per group were sacrificed for histopathological evaluation. The remaining animals were divided into two other subgroups, each with 6 animals, receiving a control diet, a control diet with nucleotides (0.05% adenosine monophosphate, 0.05% guanosine monophosphate, 0.05% cytidine monophosphate, 0.05% uridine monophosphate and 0.05% inosine monophosphate), a diet with lactose, and a diet with lactose and nucleotides. On the 32nd day of the experiment all animals were sacrificed. Animals with diarrhea weighed less than animals without diarrhea. The introduction of nucleotides did not lead to weight gain. Mean diet consumption was lower in the group that continued to ingest lactose, with the group receiving lactose plus nucleotides showing a lower mean consumption. Animals receiving lactose had inflammatory reaction and deposits of periodic acid-Schiff-positive material in intestinal, hepatic and splenic tissues. The introduction of nucleotides led to an improvement of the intestinal inflammatory reaction. In lactose-induced diarrhea, when the stimulus is maintained - lactose overload - the nucleotides have a limited action on the weight gain and on recovery of intestinal morphology, although they have a protective effect on hepatic injury and improve the inflammatory response.

Effect of a selective nonsteroidal anti-inflammatory inhibitor of cyclooxygenase-2 on the small bowel of rats

The pathogenesis of nonsteroidal anti-inflammatory drug (NSAID) enteropathy is a complex process involving the uncoupling of mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase (COX). Rofecoxib, a selective inhibitor of COX-2, has shown less gastric damage, but the same beneficial effect is not clear in the case of the small bowel. Fifty-seven male Wistar rats (250-350 g) were divided into three groups (N = 19 each) to evaluate the effect of this NSAID on the rat intestine. The groups received 2.5 mg/kg rofecoxib, 7.5 mg/kg indomethacin or water with 5% DMSO (control) given as a single dose by gavage 24 h before the beginning of the experiment. A macroscopic score was used to quantify intestinal lesions and intestinal permeability was measured using [51Cr]-ethylenediaminetetraacetic acid ([51Cr]-EDTA). The extent of intestinal lesion, indicated by a macroscopic score, was significantly lower when rofecoxib was administered compared to indomethacin (rofecoxib = 0.0 vs indomethacin = 63.6 ± 25.9; P < 0.05) and did not differ from control. The intestinal permeability to [51Cr]-EDTA was significantly increased after indomethacin (control = 1.82 ± 0.4 vs indomethacin = 9.12 ± 0.8%; P < 0.0001), but not after rofecoxib, whose effect did not differ significantly from control (control = 1.82 ± 0.4 vs rofecoxib = 2.17 ± 0.4%; ns), but was significantly different from indomethacin (indomethacin = 9.12 ± 0.8 vs rofecoxib = 2.17 ± 0.4%; P < 0.001). In conclusion, the present data show that rofecoxib is safer than indomethacin in rats because it does not induce macroscopic intestinal damage or increased intestinal permeability.

The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.

Enhanced expression of Ang-(1-7) during pregnancy

Pregnancy is a physiological condition characterized by a progressive increase of the different components of the renin-angiotensin system (RAS). The physiological consequences of the stimulated RAS in normal pregnancy are incompletely understood, and even less understood is the question of how this system may be altered and contribute to the hypertensive disorders of pregnancy. Findings from our group have provided novel insights into how the RAS may contribute to the physiological condition of pregnancy by showing that pregnancy increases the expression of both the vasodilator heptapeptide of the RAS, angiotensin-(1-7) [Ang-(1-7)], and of a newly cloned angiotensin converting enzyme (ACE) homolog, ACE2, that shows high catalytic efficiency for Ang II metabolism to Ang-(1-7). The discovery of ACE2 adds a new dimension to the complexity of the RAS by providing a new arm that may counter-regulate the activity of the vasoconstrictor component, while amplifying the vasodilator component. The studies reviewed in this article demonstrate that Ang-(1-7) increases in plasma and urine of normal pregnant women. In preeclamptic subjects we showed that plasma Ang-(1-7) was suppressed as compared to the levels found in normal pregnancy. In addition, kidney and urinary levels of Ang-(1-7) were increased in pregnant rats coinciding with the enhanced detection and expression of ACE2. These findings support the concept that in normal pregnancy enhanced ACE2 may counteract the elevation in tissue and circulating Ang II by increasing the rate of conversion to Ang-(1-7). These findings provide a basis for the physiological role of Ang-(1-7) and ACE2 during pregnancy.

Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil

Cystic fibrosis (CF) is the most common genetic disease among Caucasians and is rare among sub-Saharan Africans. The Brazilian population is not ethnically homogeneous but it is the result of three-way ethnic admixture of Europeans, Africans and Amerindians in varying proportions, depending on the region. In the present study, we investigated 33 patients who had been diagnosed and are currently under treatment for CF at the University Hospital João de Barros Barreto, Belém, Pará State. The molecular analysis for G542X, G551D and R553X mutations was performed by PCR followed by RFLP using BstNI, HincII and MboI, respectively, in polyacrylamide gel eletrophoresis and stained with AgNO3. ThedeltaF508 mutation (a deletion of 3 bp) was only analyzed by polyacrylamide gel electrophoresis and stained with AgNO3. Each sample was analyzed for regions of interest in the CFTR gene using amplified by PCR and specific primers. The deltaF508 and G551D mutations presented frequencies of 22.7 and 3%, respectively. In 74.3% of the remaining patients, none of the mutations investigated was found. The present study characterized in a sample of patients with an established clinical diagnosis of CF (asthma, repeated bronchopneumonia, disorders of nutritional status, etc.) the most frequent mutation ( deltaF508) in the North region of Brazil and is also the first report of the G551D mutation. In spite of the wide spectrum of CF mutations and the heterogeneous ethnic origin of the Amazon population, the molecular diagnosis is a helpful additional tool for the diagnosis and treatment of CF patients.

The validity and 4-year test-retest reliability of the Brazilian version of the Eating Attitudes Test-26

In a cross-sectional study conducted four years ago to assess the validity of the Brazilian version of the Eating Attitudes Test-26 (EAT-26) for the identification of abnormal eating behaviors in a population of young females in Southern Brazil, 56 women presented abnormal eating behavior as indicated by the EAT-26 and the Edinburgh Bulimic Investigation Test. They were each matched for age and neighborhood to two normal controls (N = 112) and were re-assessed four years later with the two screening questionnaires plus the Composite International Diagnostic Interview (CIDI). The EAT results were then compared to diagnoses originating from the CIDI. To evaluate the temporal stability of the two screening questionnaires, a test-retest design was applied to estimate kappa coefficients for individual items. Given the prevalence of eating disorders of 6.2%, the CIDI psychiatry interview was applied to 161 women. Of these, 0.6% exhibited anorexia nervosa and 5.6%, bulimia nervosa (10 positive cases). The validity coefficients of the EAT were: 40% sensitivity, 84% specificity, and 14% positive predictive value. Cronbach's coefficient was 0.75. For each EAT item, the kappa index was not higher than 0.344 and the correlation coefficient was lower than 0.488. We conclude that the EAT-26 exhibited low validity coefficients for sensitivity and positive predictive value, and showed a poor temporal stability. It is reasonable to assume that these results were not influenced by the low prevalence of eating disorders in the community. Thus, the results cast doubts on the ability of the EAT-26 test to identify cases of abnormal eating behaviors in this population.

Effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and on atherogenesis in apolipoprotein E knock-out mice

Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. Several studies have reported a decrease in serum cholesterol during the consumption of large doses of fermented dairy products or lactobacillus strains. The proposed mechanism for this effect is the removal or assimilation of intestinal cholesterol by the bacteria, reducing cholesterol absorption. Although this effect was demonstrated in vitro, its relevance in vivo is still controversial. Furthermore, few studies have investigated the role of lactobacilli in atherogenesis. The aim of the present study was to determine the effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and the possible hypocholesterolemic and antiatherogenic action of these bacteria using atherosclerosis-prone apolipoprotein E (apo E) knock-out (KO) mice. For this purpose, Swiss/NIH germ-free mice were monoassociated with L. delbrueckii and fed a hypercholesterolemic diet for four weeks. In addition, apo E KO mice were fed a normal chow diet and treated with L. delbrueckii for 6 weeks. There was a reduction in cholesterol excretion in germ-free mice, which was not associated with changes in blood or liver cholesterol concentration. In apo E KO mice, no effect of L. delbrueckii was detected in blood, liver or fecal cholesterol. The atherosclerotic lesion in the aorta was also similar in mice receiving or not these bacteria. In conclusion, these results suggest that, although L. delbrueckii treatment was able to reduce cholesterol excretion in germ-free mice, no hypocholesterolemic or antiatherogenic effect was observed in apo E KO mice.

Lipolysis of emulsion models of triglyceride-rich lipoproteins is altered in male patients with abdominal aorta aneurysm

Disorders of the lipid metabolism may play a role in the genesis of abdominal aorta aneurysm. The present study examined the intravascular catabolism of chylomicrons, the lipoproteins that carry the dietary lipids absorbed by the intestine in the circulation in patients with abdominal aorta aneurysm. Thirteen male patients (72 ± 5 years) with abdominal aorta aneurysm with normal plasma lipid profile and 13 healthy male control subjects (73 ± 5 years) participated in the study. The method of chylomicron-like emulsions was used to evaluate this metabolism. The emulsion labeled with 14C-cholesteryl oleate and ³H-triolein was injected intravenously in both groups. Blood samples were taken at regular intervals over 60 min to determine the decay curves. The fractional clearance rate (FCR) of the radioactive labels was calculated by compartmental analysis. The FCR of the emulsion with ³H-triolein was smaller in the aortic aneurysm patients than in controls (0.025 ± 0.017 vs 0.039 ± 0.019 min-1; P < 0.05), but the FCR of14C-cholesteryl oleate of both groups did not differ. In conclusion, as indicated by the triglyceride FCR, chylomicron lipolysis is diminished in male patients with aortic aneurysm, whereas the remnant removal which is traced by the cholesteryl oleate FCR is not altered. The results suggest that defects in the chylomicron metabolism may represent a risk factor for development of abdominal aortic aneurysm.

Comparison of the effects of lactated Ringer solution with and without hydroxyethyl starch fluid resuscitation on gut edema during severe splanchnic ischemia

The type of fluid used during resuscitation may have an important impact on tissue edema. We evaluated the impact of two different regimens of fluid resuscitation on hemodynamics and on lung and intestinal edema during splanchnic hypoperfusion in rabbits. The study included 16 female New Zealand rabbits (2.9 to 3.3 kg body weight, aged 8 to 12 months) with splanchnic ischemia induced by ligation of the superior mesenteric artery. The animals were randomized into two experimental groups: group I (N = 9) received 12 mL·kg-1·h-1 lactated Ringer solution and 20 mL/kg 6% hydroxyethyl starch solution; group II (N = 7) received 36 mL·kg-1·h-1 lactated Ringer solution and 20 mL/kg 0.9% saline. A segment from the ileum was isolated to be perfused. A tonometric catheter was placed in a second gut segment. Superior mesenteric artery (Q SMA) and aortic (Qaorta) flows were measured using ultrasonic flow probes. After 4 h of fluid resuscitation, tissue specimens were immediately removed for estimations of gut and lung edema. There were no differences in global and regional perfusion variables, lung wet-to-dry weight ratios and oxygenation indices between groups. Gut wet-to-dry weight ratio was significantly lower in the crystalloid/colloid-treated group (4.9 ± 1.5) than in the crystalloid-treated group (7.3 ± 2.4) (P < 0.05). In this model of intestinal ischemia, fluid resuscitation with crystalloids caused more gut edema than a combination of crystalloids and colloids.

Gut permeability to lactulose and mannitol differs in treated Crohn's disease and celiac disease patients and healthy subjects

The gut barrier monitors and protects the gastrointestinal tract from challenges such as microorganisms, toxins and proteins that could act as antigens. There is evidence that gut barrier dysfunction may act as a primary disease mechanism in intestinal disorders. The aim of the present study was to evaluate the barrier function towards sugars after the appropriate treatment of celiac disease and Crohn's disease patients and compare the results with those obtained with healthy subjects. Fifteen healthy volunteers, 22 celiac disease patients after 1 year of a gluten-free diet, and 31 Crohn's disease patients in remission were submitted to an intestinal permeability test with 6.0 g lactulose and 3.0 g mannitol. Six-hour urinary lactulose excretion in Crohn's disease patients was significantly higher than in both celiac disease patients (0.42 vs 0.15%) and healthy controls (0.42 vs 0.07%). Urinary lactulose excretion was significantly higher in celiac disease patients than in healthy controls (0.15 vs 0.07%). Urinary mannitol excretion in Crohn's disease patients was the same as healthy controls (21 vs 21%) and these values were significantly higher than in celiac disease patients (10.9%). The lactulose/mannitol ratio was significantly higher in Crohn's disease patients in comparison to celiac disease patients (0.021 vs 0.013) and healthy controls (0.021 vs 0.003) and this ratio was also significantly higher in celiac disease patients compared to healthy controls (0.013 vs 0.003). In spite of treatment, differences in sugar permeability were observed in both disease groups. These differences in the behavior of the sugar probes probably reflect different mechanisms for the alterations of intestinal permeability.

"Petit suisse" cheese from kefir: an alternative dessert with microorganisms of probiotic activity

"Petit Suisse" is a creamy cheese. Kefir is a symbiotic mixture of lactic acid bacteria and yeasts with probiotic activity including immunomodulation and balance of intestinal microflora. The present study aims to develop "Petit Suisse" cheese from kefir. Kefir grains were grown in pasteurized cow milk, and after the separation of kefir the serum was discarded and the "Petit Suisse" cheese was prepared using strawberry, mangaba, herbs, and dried tomatoes. The acceptance of the different preparations was evaluated using a nine-point hedonic scale followed by ANOVA. The sweet and salty products were compared by the Student's t-test. Purchase intent was evaluated by the means test and frequency distribution. All products were well accepted by the judges. The product was characterized by low yield, but it can be prepared at home at low cost. The nutritional composition analyses and the variety of flavors as well as the range of age of the judges are alternatives for further studies.

Infection by Cryptosporidium sp. in immunocompromised haematological patients

The frequency of intestinal infection by Cryptosporidium sp. was determined in 60 patients, attended at the Haematological and Haemotherapeutical Service of "Santa Casa de Misericórdia" of São Paulo, suffering lymphoproliferative diseases (Group 1). As control group (Group 2) 59 persons without haematological diseases, but with the same life time and living at the same place of that of haematological patients, had been examined. The stool parasitological tests performed disclosed Cryptosporidium sp. oocysts in six (10%) individuals belonging to Group 1, whereas, in Group 2, nobody showed infection by this coccidian. Among the patients infected by Cryptosporidium sp. only one showed diarrhoeal faeces.