Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

Necrotizing enteritis associated with Clostridium perfringensType B in chinchillas (Chinchilla lanigera)

Four 3-4 month-old chinchillas (Chinchilla lanigera) from a commercial flock of 395 chinchillas, were found dead with evidence of previous diarrhea and prolapsed rectum. A fifth 8 month-old chinchilla died 8 hours after being found recumbent, apathetic, diarrheic and with a prolapsed rectum. Two chinchillas were necropsied and observed gross lesions consisted of extensive hemorrhagic enteritis, mild pulmonary edema and enlarged and yellow liver; this latter finding was particularly prominent in the chinchilla presenting longer clinical course. Histologically there was necrotizing enteritis associated with abundant bacterial rods aggregates in the intestinal surface epithelium and within the lamina propria. In the lungs there were small amounts of pink proteinaceous material (edema) in the interstitium and marked vacuolar hepatocellullar degeneration (lipidosis) in the liver. Anaerobic cultures from the intestinal contents of one of the affected chinchillas yielded Clostridium perfringens. Genotyping of this C. perfringens isolate was achieved by multiplex polymerase chain reaction (mPCR) as C. perfringenstype B due to detection of alpha, beta and epsilon-toxin genes. These findings suggest C. perfringens type B as an important cause of sudden or acute death in chinchillas.

Antimicrobial resistance of Staphylococcus spp. from small ruminant mastitis in Brazil

The study aimed to determine the antimicrobial resistance patterns and to identify molecular resistance markers in Staphylococcus spp. (n=210) isolated from small ruminant mastitis in Brazil. The antimicrobial resistance patterns were evaluated by the disk diffusion test and by detection of the presence of mecA, blaZ, ermA, ermB, ermC and msrA genes by PCR. The efflux pump test was performed using ethidium bromide and biofilm production was determined by Congo red agar test along with PCR for detection of the icaD gene. The isolates were most resistant to amoxicillin (50.0%), streptomycin (42.8%), tetracycline (40.4%), lincomycin (39.0%) and erythromycin (33.8%). Pan-susceptibility to all tested drugs was observed in 71 (33.8%) isolates and 41 Staphylococcus isolates were positive for the efflux pump. Although phenotypic resistance to oxacillin was observed in 12.8% of the isolates, none harbored the mecA gene. However, 45.7% of the isolates harbored blaZ indicating that beta-lactamase production was the main mechanism associated with staphylococci resistance to beta-lactams in the present study. The other determinants of resistance to antimicrobial agents ermA, ermB, ermC, and msrA were observed in 1.4%, 10.4%, 16.2%, and 0.9% of the isolates, respectively. In addition, the icaD gen was detected in 32.9% of the isolates. Seventy three isolates (54 from goats and 19 from sheep) were negative for all resistance genes tested and 69 isolates presented two or more resistance genes. Association among blaZ, ermA, ermB, ermC and efflux pump were observed in 17 isolates, 14 of which originated from goats and three from sheep. The data obtained in this study show the resistance of the isolates to beta-lactamics, which may be associated with the use of antimicrobial drugs without veterinary control.

Some virulence genes of Escherichia coli isolated from cloacal swabs of healthy Alagoas Curassows (Pauxi mitu) in Brazil

Birds of the Cracidae family (curassows, guans, and chachalacas) are endemic of the Neotropics and 50 species are currently classified. Brazil has 22 species, seven of which are considered threatened. The Alagoas Curassow (Pauxi mitu) species is considered extinct in the wild; but about 120 birds are alive in captivity. Conservation of this species depends entirely on correct management. Health reports of both wildlife and captive curassows are rare. In this study the presence of Escherichia coli was evaluated in 23 healthy Alagoas Curassows from two private breeding centres. E. coli was isolated from cloacal swabs, and the presence of genes encoding cytotoxic necrotising factor 1 (cnf1), alpha-haemolysin (hly), aerobactin (iuc), serum resistance (iss) and the following adhesions: S fimbriae (sfa), pili associated with pyelonephritis (pap) and temperature-sensitive haemagglutinin (tsh) were investigated. E. coli was isolated from 78.3% (18/23) of the birds, and the percentage of curassows colonized by E. coli was similar between the two facilities. From the 22 E. coli isolates, 15 (68.2%) were positive for at least one virulence factor by PCR, and the most frequently found gene was iss (50%). No curassows had clinical signs of disease. Nevertheless, the presence of some E. coli strains may be a concern to the wildlife in captivity. Additional health surveillance studies are essential to guarantee successful conservation programmes for threatened cracids in Brazil.

Occurrence of enterotoxin-encoding genes in Staphylococcus aureus causing mastitis in lactating goats

Staphylococcal enterotoxins are the leading cause of human food poisoning worldwide. Staphylococcus spp. are the main mastitis-causing agents in goats and frequently found in high counts in goat milk. This study aimed to investigate the occurrence of enterotoxin-encoding genes in Staphylococcus aureus associated with mastitis in lactating goats in Paraiba State, Brazil. Milk samples (n=2024) were collected from 393 farms. Staphylococcus aureus was isolated in 55 milk samples. Classical (sea, seb, sec, sed, see) and novel (seg, seh, sei) enterotoxin-encoding genes were investigated by means of polymerase chain reaction (PCR). From thirty-six tested isolates, enterotoxin-encoding genes were detected in 7 (19.5%) S. aureus. The gene encoding enterotoxin C (seC) was identified in six isolates, while seiwas observed in only one isolate. The genes sea, seb, sed, see, seg and seh were not observed amongst the S. aureus investigated in this study. In summary, S. aureus causing mastitis in goats can harbor enterotoxin-encoding genes and seC was the most frequent gene observed amongst the investigated isolates. This finding is important for surveillance purposes, since enterotoxin C should be investigated in human staphylococcal food poisoning outbreaks caused by consumption of goat milk and dairy products.

Biofilm formation by Rhodococcus equi and putative association with macrolide resistance

Abstract: Rhodococcus equi is a facultative intracellular pathogen, which cause severe pyogranulomatous pneumonia in foals and tuberculosis-like lesions in humans. Its ability to form biofilm was described in strains isolated from chronic diseases associated to treatment failures in humans. This study aimed to verify the biofilm formation by 113 R. equi isolated from equine samples (clinical and fecal) using two different methods (biofilm-culturing with and without additional glucose and epifluorescence microscopy). We also aimed to determine the efficacy of azithromycin, clarithromycin and erythromycin on R. equi in established biofilm. We found 80.5% (26/41) and 63% (58/72) biofilm-positive isolates, in fecal and clinical samples, respectively. The additional glucose increased the biofilm formation by R. equi fecal samples, but not by clinical samples. The antimicrobials tested herein were not able to eradicate R. equi in biofilm even at higher concentrations. This is the first study showing the biofilm formation by R. equi isolated from equine samples. Our findings indicate that R. equi biofilm-producers may be more resistant to the antimicrobials evaluated. Further studies are warranted to test this hypothesis.

Genetic control of characters associated with bean golden mosaic geminivirus resistance in Phaseolus vulgaris L.

Bean golden mosaic is the most important viral disease of the bean crop (Phaseolus vulgaris L.) in Latin America. The genetics of resistance to a Brazilian strain of bean golden mosaic virus (BGMV), was studied in a 4 x 4 diallel cross without reciprocals, among the parental genotypes DOR 303, EMGOPA 201 Ouro, Carnaval, and Redlands Greenleaf C. Seedlings of the four parents, six F1 hybrids, 12 backcrosses, and F2 generations for each combination were inoculated on the eighth day after sowing by exposure to a viruliferous whitefly (Bemisia tabaci Genn.) population for 24 h, in a glasshouse, prior to transplantation to field conditions. The full set of two parents, F1, F2 and respective backcrosses for each combination was considered to be a family. Data were recorded and analyzed for foliar yellowing, plant dwarfing, and pod malformation, using a randomized block design, with two replications. Weighted generation mean analysis was performed for each of the six families. An additive gene action model was significant for the three characteristics evaluated. On the other hand, non-additive gene action had greater absolute value in most cases. Resistance to foliar yellowing conferred by genes from DRO 303 was highly heritable and was expressed equally well in the different genetic backgrounds evaluated. Such resistance may be oligogenic. Broad- and narrow-sense heritabilities were relatively high for all response traits. The three traits studied were all positively correlated, indicating that they can be simultaneously selected for enhancement. The highest correlation coefficient was obtained for dwarfing x pod malformation.

Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes

The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-g. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.

alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aa)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

The Agamma-195 (C->G) mutation in hereditary persistence of fetal hemoglobin is not associated with activation of a reporter gene in vitro

Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression.

Molecular identification of Sicilian (dß)º-thalassemia associated with ß-thalassemia and hemoglobin S in Brazil

We describe the clinical and molecular characteristics of two unrelated Brazilian families with an association of the Sicilian form of (dß)º-thalassemia with hemoglobin S and ß-thalassemia. Direct sequencing of the ß-globin gene showed only the hemoglobin S mutation in patient 1 and the ß-thalassemia IVS1-110 in patient 2. The other allele was deleted in both patients and PCR of DNA samples of the breakpoint region of both patients showed a band of approximately 1,150 bp, expected to be observed in the DNA of carriers of Sicilian (dß)º-thalassemia. The nucleotide sequence of this fragment confirmed the Sicilian deletion. There are few reports concerning the Hb S/(dß)º-thalassemia association and patient 2 is the first reported case of Sicilian type of (dß)º-thalassemia in association with ß-thalassemia documented at the molecular level.

ß-Spectrin São PauloII, a novel frameshift mutation of the ß-spectrin gene associated with hereditary spherocytosis and instability of the mutant mRNA

Hereditary spherocytosis (HS) is a common inherited anemia characterized by the presence of spherocytic red cells. Defects in several membrane protein genes have been involved in the pathogenesis of HS. ß-Spectrin-related HS seems to be common. We report here a new mutation in the ß-spectrin gene coding region in a patient with hereditary spherocytosis. The patient presented acanthocytosis and spectrin deficiency and, at the DNA level, a novel frameshift mutation leading to HS, i.e., a C deletion at codon 1392 (ß-spectrin São PauloII), exon 20. The mRNA encoding ß-spectrin São PauloII was very unstable and the mutant protein was not detected in the membrane or in other cellular compartments. It is interesting to note that frameshift mutations of the ß-spectrin gene at the 3' end allow the insertion of the mutant protein in the red cell membrane, leading to a defect in the auto-association of the spectrin dimers and consequent elliptocytosis. On the other hand, ß-spectrin São PauloII protein was absent in the red cell membrane, leading to spectrin deficiency, HS and the presence of acanthocytes.

Lipoprotein lipase PvuII polymorphism is associated with variations in serum lipid levels in non-diabetic pregnant women

The aim of the present study was to determine if there is an association between the single nucleotide polymorphisms (SNPs) of the lipoprotein lipase (LPL) and apolipoprotein E (apo E) genes and the serum lipid profile in pregnancy and puerperium. Non-diabetic women of European descent in the third semester of pregnancy (N = 120) were selected. Those with diseases or other condition that could modify their lipid profile were excluded from the study (N = 32). Serum lipids were measured by routine laboratory procedures and genomic DNA was extracted by a salting out method. LPL (PvuII and HindIII) and apo E (HhaI) SNPs were detected by the polymerase chain reaction and restriction fragment length polymorphism. Categorical and continuous variables were compared by the chi-square test and Student t-test or ANOVA, respectively. Women carrying the LPL P1P1 genotype had higher serum LDL cholesterol (N = 21; 155 ± 45 mg/dL) than women carrying the P1P2/P2P2 genotypes (N = 67; 133 ± 45 mg/dL; P = 0.032). During the puerperium period, serum levels of triglycerides and VLDL cholesterol were significantly reduced in women carrying the P1P1 (73%, P = 0.006) and P1P2 (51%, P = 0.002) genotypes but not in women carrying the P2P2 genotype (23%, P > 0.05). On the other hand, serum concentrations of lipids did not differ between the LPL HindIII and apo E genotypes during pregnancy and after delivery. We conclude that LPL PvuII SNP is associated with variations in serum lipids during pregnancy and the puerperal period in non-diabetic women.

Methylation status of ANAPC1, CDKN2A and TP53 promoter genes in individuals with gastric cancer

Gastric cancer is the forth most frequent malignancy and the second most common cause of cancer death worldwide. DNA methylation is the most studied epigenetic alteration, occurring through a methyl radical addition to the cytosine base adjacent to guanine. Many tumor genes are inactivated by DNA methylation in gastric cancer. We evaluated the DNA methylation status of ANAPC1, CDKN2A and TP53 by methylation-specific PCR in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosa in individuals from Northern Brazil. All gastric cancer samples were advanced stage adenocarcinomas. Gastric samples were surgically obtained at the João de Barros Barreto University Hospital, State of Pará, and were stored at -80°C before DNA extraction. Patients had never been submitted to chemotherapy or radiotherapy, nor did they have any other diagnosed cancer. None of the gastric cancer samples presented methylated DNA sequences for ANAPC1 and TP53. CDKN2A methylation was not detected in any normal gastric mucosa; however, the CDKN2A promoter was methylated in 30.4% of gastric cancer samples, with 35% methylation in diffuse-type and 26.9% in intestinal-type cancers. CDKN2A methylation was associated with the carcinogenesis process for ~30% diffuse-type and intestinal-type compared to non-neoplastic samples. Thus, ANAPC1 and TP53 methylation was probably not implicated in gastric carcinogenesis in our samples. CDKN2A can be implicated in the carcinogenesis process of only a subset of gastric neoplasias.

Obesity induces upregulation of genes involved in myocardial Ca2+ handling

Obesity is a complex multifactorial disorder that is often associated with cardiovascular diseases. Research on experimental models has suggested that cardiac dysfunction in obesity might be related to alterations in myocardial intracellular calcium (Ca2+) handling. However, information about the expression of Ca2+-related genes that lead to this abnormality is scarce. We evaluated the effects of obesity induced by a high-fat diet in the expression of Ca2+-related genes, focusing the L-type Ca2+ channel (Cacna1c), sarcolemmal Na+/Ca2+ exchanger (NCX), sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), ryanodine receptor (RyR2), and phospholamban (PLB) mRNA in rat myocardium. Male 30-day-old Wistar rats were fed a standard (control) or high-fat diet (obese) for 15 weeks. Obesity was defined as increased percent of body fat in carcass. The mRNA expression of Ca2+-related genes in the left ventricle was measured by RT-PCR. Compared with control rats, the obese rats had increased percent of body fat, area under the curve for glucose, and leptin and insulin plasma concentrations. Obesity also caused an increase in the levels of SERCA2a, RyR2 and PLB mRNA (P < 0.05) but did not modify the mRNA levels of Cacna1c and NCX. These findings show that obesity induced by high-fat diet causes cardiac upregulation of Ca2+ transport_related genes in the sarcoplasmic reticulum.