Tocopherols, tocotrienols, and gamma-oryzanol contents in Japonica and Indica subspecies of rice (Oryza sativa L.) cultivated in Brazil

Whole rice contains several fat-soluble phytochemicals such. as tocopherols, tocotrienols, and gamma-oryzanol which have been reported to possess beneficial health properties. This study was conducted to determine whether brown rice belonging to indica and japonica subspecies were distinguishable from each other regarding the concentration of these compounds by analyzing 32 genotypes. The fat-soluble compounds were analyzed by normal-phase HPLC in a single run. The variability of the compounds analyzed was high, but the mean content of gamma-oryzanol across all samples was significantly higher (P < 0.01) in japonica (246.3 mg/kg) than in indica rice (190.1 mg/kg). Similar differences were found for total vitamin E contents which were 24.2 mg/kg injaponica and 17.1 mg/kg in indica rice, respectively. In japonica rice, alpha-tocopherol, alpha-tocotrienol, and gamma-tocotrienol were the most abundant homologs, while in indica rice the most abundant were gamma-tocotrienol, alpha-tocopherol, and alpha-tocotrienol. A significant Pearson coefficient (0.80, P < 0.001) between alpha-tocopherol and alpha-tocotrienol levels was found, independent of the subspecies. Both compounds were positively correlated to total tocols and gamma-oryzanol contents. Although more studies are needed to evaluate the interference of growing rice in different environments and multiple years, the present study provided information on natural variations of the vitamin E isomers and the gamma-oryzanol contents in different rice genotypes.

Field evidence for mechanical transmission of rabbit haemorrhagic disease virus (RHDV) by flies (Diptera : Calliphoridae) among wild rabbits in Australia

Field collected flies were screened for the presence of rabbit haemorrhagic disease virus (RHDV) by applying reverse transcriptase PCR (RT-PCR) in which primers specific to the capsid protein of the virus were used. The virus was detected in flies from locations where rabbit haemorrhagic disease (RHD) was reported and also soon after the release of RHDV in a 'clean' area. Oral and/or anal excretions of flies (flyspots) were found to contain viable virus and oral inoculation of rabbits revealed that a single flyspot was able to cause RHD. We conclude that flyspots are a major potential source of the virus for oral or conjunctival transmission of the virus to rabbits. (C) 1998 Elsevier Science B.V. All rights reserved.

Sequence analysis of rabbit haemorrhagic disease virus (RHDV) in Australia: alterations after its release

Liver samples from rabbits killed by RHDV, collected from five States in Australia in 1996 and 1997 were analysed by RT-PCR. A 398 bp fragment of the capsid protein (VP60) gene was amplified by PCR and directly sequenced. The alignment of the nucleotide and amino acid sequences and their comparison with the original strain of the virus released in Australia indicated genetic changes after two years have been small with 98.2% to 100% identity. The constructed phylogenetic tree suggests slight differences in nucleotide substitutions in various States but there is no clear evidence of clustering of sequences according to their geographic origin. In practical terms, sequencing of viral RNA provides a means of testing the efficacy of further releases and subsequent spread of the virus if such a strategy is employed as a means of enhancing RHD as a biological control of the wild rabbit in Australia.

Characterisation of Australian isolates of Fusarium oxysporum f. sp cubense by DNA fingerprinting analysis

Genetic variation among Australian isolates of the fungus Fusarium oxysporum f. sp. cubense (Foc), which causes Fusarium wilt in banana, was examined using DNA amplification fingerprinting (DAF). Ninety-four isolates which represented Races 1, 2, 3, and 4, and vegetative compatibility groups (VCGs) 0120, 0124, 0125, 0128, 0129, 01211, 01213/16, and 01220 were analysed. The genetic relatedness among isolates within each VCG, and between the 8 different VCGs of Foc present in Australia was determined. The DNA fingerprint patterns were VCG-specific, with each VCG representing a unique genotype. The genetic similarity among isolates within each VCG ranged from 97% to 100%. Among the different VCGs of Foc, 3 major clusters were distinguished which corresponded with race. All Race 1 and 2 isolates (VCGs 0124, 0125, 0128, and 01220) were closely related and clustered together, the Race 3 isolates from Heliconia clustered separately, and all Race 4 isolates (VCGs 0120, 0129, 01211, and 01213/16) clustered together. Fifteen isolates from Alstonville, NSW, were characterised because although they were classified as Race 2 based on their recovery from cooking banana cultivars, they belonged in VCG 0124, which had previously contained only Race 1 isolates. The occurrence of more than one race within a VCG means that vegetative compatibility grouping cannot be used to assign pathotype to pathogenic race as previously thought. It was possible to distinguish the Race 1 and Race 2 isolates within VCG 0124 using DNA fingerprinting, as each race produced a unique DNA fingerprint pattern. Among the Australian isolates, DNA fingerprinting analysis identified 9 different VCGs and genotypes of Foc.

Molecular characterization of potentially zoonotic isolates of Giardia duodenalis in horses

Giardia isolates from eight horses from New York State (NY), USA and two horses from Western Australia (WA) were genetically characterized at the SSU-rDNA and triose-phosphate isomerase (TPI) genes. Phylogenetic analysis of the TPI gene provided strong support for the placement of both isolates of Giardia from horses in WA and a single isolate from a horse in NY within the assemblage AI genotype of G. duodenalis. Another two isolates from horses in NY placed within the assemblage All genotype of G. duodenalis. Phylogenetic analysis of the TPI gene also provided strong bootstrap support for the placement of four G. duodenalis isolates from horses in NY into a potentially host-specific sub-assemblage of assemblage BIV. The results of this study are consistent with previous studies showing that assemblages AI and AII of G. duodenalis provide the greatest potential zoonotic risk to humans. Horses may therefore constitute a potential source for human infection of Giardia either directly or via watersheds. (c) 2005 Elsevier B.V. All rights reserved.

A controlled trial comparing the efficacy of rice-based and hypotonic glucose oral rehydration solutions in infants and young children with gastroenteritis

A prospective randomized trial was conducted to compare the efficacy of a rice-based oral rehydration solution (ORS) with glucose ORS in infants and children under 5 years of age with acute diarrhoea and mild to moderate dehydration (<10%). One hundred children presenting to a large metropolitan teaching hospital were eligible for entry to the study and were randomized to receive rice ORS or glucose ORS. Outcome measures were stool output (SO), duration of illness (DD) and recovery time to introduction of other fluids (RTF) and diet (RTD). Significant differences were found for all outcome measures in favour of the rice ORS group. Mean SO was lower (160 vs 213 mt; P<0.02), mean DD was reduced (17.3 vs 24.3 h; P = 0.03) and median RTF was decreased (12.7 vs 18.1 h; P< 0.001) in the rice ORS group compared with the glucose ORS group. The median rime to introduction of diet and mean length of hospital stay showed similar significant reductions. Our study has shown rice ORS to be an acceptable alternative to glucose ORS in young children and have shown that it is significantly more effective in reducing the course of diarrhoeal illness and the time taken to return to normal drinking and eating habits.

Molecular characterization of the Hepatitis B virus genotypes in Colombia: A Bayesian inference on the genotype F

Hepatitis B is a worldwide health problem affecting about 2 billion people and more than 350 million are chronic carriers of the virus. Nine HBV genotypes (A to I) have been described. The geographical distribution of HBV genotypes is not completely understood due to the limited number of samples from some parts of the world. One such example is Colombia, in which few studies have described the HBV genotypes. In this study, we characterized HBV genotypes in 143 HBsAg-positive volunteer blood donors from Colombia. A fragment of 1306 bp partially comprising HBsAg and the DNA polymerase coding regions (S/POL) was amplified and sequenced. Bayesian phylogenetic analyses were conducted using the Markov Chain Monte Carlo (MCMC) approach to obtain the maximum clade credibility (MCC) tree using BEAST v.1.5.3. Of all samples, 68 were positive and 52 were successfully sequenced. Genotype F was the most prevalent in this population (77%) - subgenotypes F3 (75%) and Fib (2%). Genotype G (7.7%) and subgenotype A2 (15.3%) were also found. Genotype G sequence analysis suggests distinct introductions of this genotype in the country. Furthermore, we estimated the time of the most recent common ancestor (TMRCA) for each HBV/F subgenotype and also for Colombian F3 sequences using two different datasets: (i) 77 sequences comprising 1306 bp of S/POL region and (ii) 283 sequences comprising 681 bp of S/POL region. We also used two other previously estimated evolutionary rates: (i) 2.60 x 10(-4) s/s/y and (ii) 1.5 x 10(-5) s/s/y. Here we report the HBV genotypes circulating in Colombia and estimated the TMRCA for the four different subgenotypes of genotype F. (C) 2010 Elsevier B.V. All rights reserved.

Dynamics of Hepatitis D (delta) virus genotype 3 in the Amazon region of South America

Hepatitis delta virus (HDV) is widely distributed and associated with fulminant hepatitis epidemics in areas with high prevalence of HBV. Several studies performed in the 1980s showed data on HDV infection in South America, but there are no studies on the viral dynamics of this virus. The aim of this study was to conduct an evolutionary analysis of hepatitis delta genotype 3 (HDV/3) prevalent in South America: estimate its nucleotide substitution rate, determine the time of most recent ancestor (TMRCA) and characterize the epidemic history and evolutionary dynamics. Furthermore, we characterized the presence of HBV/HDV infection in seven samples collected from patients who died due to fulminant hepatitis from Amazon region in Colombia and included them in the evolutionary analysis. This is the first study reporting HBV and HDV sequences from the Amazon region of Colombia. Of the seven Colombian patients, five were positive for HBV-DNA and HDV-RNA. Of them, two samples were successfully sequenced for HBV (subgenotypes F3 and Fib) and the five samples HDV positive were classified as HDV/3. By using all HDV/3 available reference sequences with sampling dates (n = 36), we estimated the HDV/3 substitution rate in 1.07 x 10(-3) substitutions per site per year (s/s/y), which resulted in a time to the most recent common ancestor (TMRCA) of 85 years. Also, it was determined that HDV/3 spread exponentially from early 1950s to the 1970s in South America. This work discusses for the first time the viral dynamics for the HDV/3 circulating in South America. We suggest that the measures implemented to control HBV transmission resulted in the control of HDV/3 spreading in South America, especially after the important raise in this infection associated with a huge mortality during the 1950s up to the 1970s. The differences found among HDV/3 and the other HDV genotypes concerning its diversity raises the hypothesis of a different origin and/or a different transmission route. (C) 2011 Elsevier B.V. All rights reserved.

Insulin-like growth factor-I induced and constitutive arginase activity differs among isolates of Leishmania derived from patients with diverse clinical forms of Leishmania braziliensis infection

Arginase activity has been related to leishmaniasis development, thus we studied the constitutive and insulin-like growth factor (IGF) I-induced arginase activity of Leishmania (Viannia) braziliensis isolates from patients with different clinical forms of American tegumentary leishmaniasis (ATL). Isolates from mucosal leishmaniasis presented higher basal levels of arginase activity than isolates from other clinical forms of ATL. Isolates from disseminated leishmaniasis that present mucosal lesion in some cases reached the arginase activity similar to that of isolates from mucosal leishmaniasis upon IGF-I stimulation. Differences in arginase activity may influence disease outcomes such as evolution to mucosal lesion in patients with L (V.) braziliensis infection. (C) 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients

Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe One was Bug, which amplified a sequence from the B1 gene The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were positive results were observed in 33.6% (50) patients The sensitivities were 98% for cnPCR (B22/B23), and 86 and 98% for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66 4% patients. The specificities were 97% for cnPCR and qrtPCR (B1Tg). and 88.8% for RETg These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers However, B1Tg PCR had good specificity, but lower sensitivity Among the patients, 20 had blood and CSF collected simultaneously Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive), (iii) positive molecular diagnosis with CSF and negative with blood, and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

Liver HLA-G expression is associated with multiple clinical and histopathological forms of chronic hepatitis B virus infection

As the mechanisms leading to the persistence of hepatitis B virus (HBV) infection are poorly understood and as the histocompatibility leucocyte antigen (HLA)-G is well described as a tolerogenic molecule, we evaluated HLA-G expression in 74 specimens of HBV liver biopsies and in 10 specimens obtained from previously healthy cadaver liver donors. HBV specimens were reviewed and classified by the METAVIR score, and HLA-G expression was assessed by immunohistochemistry. No HLA-G expression was observed in control hepatocytes. In contrast, 57 (77%) of 74 HBV specimens showed soluble and membrane-bound HLA-G expression in hepatocytes, biliary epithelial cells or both. No associations between the intensity of HLA-G expression and patient age or gender, HBeAg status, severity of liver fibrosis, and grade of histological findings were observed. Although significance was not reached (P = 0.180), patients exhibiting HLA-G expression presented a higher median HBV DNA viral load (105 copies/mL) than those who did not express HLA-G (103.7 copies/mL). These results indicate that HLA-G is expressed in most cases of chronic HBV infection in all stages and may play a role in the persistency of HBV infection.

Genetic diversity among Toxoplasma gondii isolates of small ruminants from Brazil: Novel genotypes revealed

Recent studies indicated that Toxoplasma gondii isolates of many domestic animal hosts from Brazil are genetically and biologically different from those in USA and Europe. Despite of high pathogenicity of this parasite to small ruminants, the epidemiology and genetic diversity of T. gondii in these animals are not well understood in Brazil. In this study, a total of 28 T. gondii samples (16 isolates from sheep in Sao Paulo state, and 12 isolates from goats in the states of Sao Paulo and Rio Grande do Norte) were genotyped using genetic markers SAG1, SAG2, SAG3, BTUB, CRAG, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven genotypes were identified from these T. gondii isolates. Eight isolates (4 from sheep and 4 from goats) were grouped into the common clonal type Brl lineage. One sheep isolate was grouped to the type BrIII lineage. Five isolates grouped to three previously identified genotypes in Brazil, and 13 isolates grouped to six novel genotypes. Mixed genotype was found in one isolate from goat in Sao Paulo. No classical clonal Type I. II or III isolates were found, confirming previous reports that these clonal lineages are rare in Brazil. The allele types at the CS3 locus are strongly linked to mouse virulence of the parasite. The results of this study indicate that even though a large number of T. gondii genotypes have been identified from a variety of animal hosts in Brazil, high percentage of new genotypes are continuously identified from different animal species, suggesting extremely high diversity of T. gondii in the population. (C) 2010 Elsevier B.V. All rights reserved.

Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus

The 12 cysteine residues in the flavivirus NS1 protein are strictly conserved, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. In this study, we examined the intramolecular disulfide bond arrangement of NS1 of Murray Valley encephalitis virus and elucidated three of the six cysteine-pairing arrangements. Disulfide linkages were identified by separating tryptic-digested NS1 by reverse-phase high pressure liquid chromatography and analysing the resulting peptide peaks by protein sequencing, amino acid analysis and/or electrospray mass spectrometry. The pairing arrangements between the six amino-terminal cysteines were identified as follows: Cys(4)-Cys(15), Cys(55)-Cys(143) and Cys(179)-Cys(223). Although the pairing arrangements between the six carboxyterminal cysteines were not determined, we were able to eliminate several cysteine-pairing combinations. Furthermore, we demonstrated that all three putative N-linked glycosylation sites of NS1 are utilized and that the Asn(207) glycosylation site contains a mannose-rich glycan.

Geographic variation of notified Ross River virus infections in Queensland, Australia, 1985-1996

The spatial and temporal variations of Ross River virus infections reported in Queensland, Australia, between 1985 and 1996 were studied by using the Geographic Information System. The notified cases of Ross River virus infection came from 489 localities between 1985 and 1988, 805 between 1989 and 1992, and 1,157 between 1993 and 1996 (X (2)((df = 2)) = 680.9; P < 0.001). There was a marked increase in the number of localities where the cases were reported by 65 percent for the period of 1989-1992 and 137 percent for 1993-1996, compared with that for 1985-1988. The geographic distribution of the notified Ross River virus cases has expanded in Queensland over recent years. As Ross River virus disease has impacted considerably on tourism and industry, as well as on residents of affected areas, more research is required to explore the causes of the geographic expansion of the notified Ross River virus infections.

Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T cells for nasopharyngeal carcinoma

Tumor cells from NPC patients are regularly and latently infected with EBV. To examine whether the virus serves as target for immune intervention of the cancer, we determined levels of EBV-specific CTLp in peripheral blood from NPC patients, long-term survivors of the cancer and healthy subjects. CTLp levels of test subjects varied between 3-3,000/10(6) PBMCs. The plasma EBV burden increased when the CTLp level fell below 150, whereas the EBV burden of PBMCs was not correlated with CTLp level. Compared with healthy carriers, CTLp levels of patients were lower and varied over a wider range, between 3-1,500/10(6) PBMCs. The quantitative immune deficit was probably attributed to the tumor because, first, CTLp in survivors was restored to levels similar to those in healthy carriers after the tumor had been successfully treated. Second, the CTLp level changed as disease progressed, being lower in local disease, increased in locoregional disease and decreased again when the tumor metastasized. Based on these findings, 4 patients with advanced disease were infused with 5 x 10(7)-3 x 10(8) autologous EBV CTLs. The treatment was safe and unaccompanied by inflammatory or other complications, but whether it improved tumor control could not be discerned from the large tumor bulk. Nevertheless, the treatment regularly increased CTLp levels of patients, maintained it at higher levels for protracted periods and, in 3 patients, restored host surveillance of EBV replication, reducing the plasma EBV burden. Taken together, these results provided a rationale to further explore EBV as a target of immune intervention of NPC. (C) 2001 Wiley-Liss, Inc.