Direct detection of Salmonella from poultry samples by DNA isothermal amplification.

1. Salmonellosis is one of the most important diseases in public health and it is usually associated with poultry product consumption. This study aimed to validate rapid methods to detect Salmonella spp. from poultry samples. 2. A DNA isothermal amplification method, previously developed for other matrices, was applied for the specific detection of Salmonella spp. from various samples, including poultry tissues, drag and boot swabs, faeces and feed. A new procedure was validated with Salmonella spp. serotypes and isolates from other enteric bacterial species, as well as naturally contaminated poultry samples. 3. The study demonstrated the successful development and implementation of a procedure, including a DNA isothermal amplification method, for the detection of Salmonella spp. directly from tissues, drag and boot swabs, faeces and feed. The whole procedure can be performed in less than 24 hours and it has been successfully used in a veterinary diagnostic laboratory.

Molecular detection of Salmonella serovars Enteritidis, Heidelberg and Typhimurium directly from pre-enriched poultry samples.

1. Salmonella is one of the most important pathogens in public health and it is usually associated with food-borne diseases. Salmonella serovars Enteritidis and Typhimurium are widespread in the world with outbreaks frequently associated with consumption of poultry products; furthermore, there is an increasing public health concern with the wide dissemination of the serovar Heidelberg in poultry flocks. 2. The aim of the experiment was to develop and to validate rapid methods to detect Salmonella serovars Enteritidis, Typhimurium, and Heidelberg by real-time PCRs and test isolates from pre-enriched poultry samples. 3. Three real-time PCRs were developed and used in combination to detect the serovars Enteritidis, Typhimurium and Heidelberg. These assays were validated by the analysis of 126 Salmonella isolates, eight other enteric bacterial species and 34 naturally contaminated poultry samples after pre-enrichment with buffered peptone water (BPW). 4. Real-time PCRs detected the isolates of the most important poultry serovars (Enteritidis, Typhimurium and Heidelberg) with 100% inclusivity and exclusivity in each assay. The PCR identified monophasic variants of the serovars Typhimurium and Heidelberg. All PCRs were validated in detecting these specific serovars directly from pre-enriched poultry samples. The whole analytical procedure was performed in less than 24 h in a veterinary diagnostic laboratory.

Detection of genetically modified maize in processed products, dry grains, and corn ears intended for fresh consumption in South Brazil.

Conventional and genetically modified (GM) maize cultivars have been widely planted in Brazil to produce grains for processed food, feed, or to be consumed fresh as corn ears. This study used real-time PCR to detect GM maize in processed products and fresh commercial corn ears produced in the last two years in South Brazil. Eighteen conventional and GM maize cultivars were obtained from seed production companies and 50 commercial samples (including canned corn, corn flour, dry grains, and fresh corn ears) were purchased in small local stores and supermarkets. All samples were analyzed by real time TaqMan PCR to detect one constitutive maize gene (hmg) and three genetic regions present in GM plants (p-35S promoter, major gene cry 1A.105, and t-Nos terminator). Each commercial sample was classified as conventional or GM based on the PCR results. PCR targeting the hmg gene generated positive results from all DNA samples, which were further tested with the GM targets. These targets were not detected in the five conventional maize cultivars, but were detected in the GM seeds hosting these fragments. Analysis of processed foods identified four cultivars as conventional and six as GM, which were mostly correctly labeled. Seven (53.8%) dry grain samples were classified as conventional, while six (46.2%) were classified as GM. Three (11.1%) corn ear samples were identified as conventional, and the remaining 24 (88.9%) were GM maize. These results demonstrate the high frequency of GM maize in processed products, including fresh corn ears intended for consumption in South Brazil.

Prevalence and genotypic diversity of cervical human papillomavirus infection among women from an urban center in Brazil.

Human papillomavirus (HPV) infection is a common viral sexually transmitted infection and the main cause of cervical cancer in women worldwide. Epidemiological data on the prevalence of HPV in a given population is essential for the establishment of effective prevention strategies. The aim of this study was to determine HPV prevalence in women who attended a public health service within an urban center in Brazil. Cervical samples were collected from 337 women recruited from a primary public health care clinic in the city of Cruz Alta located in Rio Grande do Sul, the southernmost State of Brazil. Samples were analyzed for HPV DNA and with Pap smear screening tests. HPV was detected in 114 (34%) women. HPV type analysis revealed that 95 (83.3%) of those represented infections with a single genotype, while 19 (16.7%) were mixed genotype infections. High- and low-risk HPV genotypes were detected in 83 (72.8%) and 48 (42.1%) samples, respectively. Furthermore, a great diversity of HPV genotypes was observed (18 high-risk, 12 low-risk, and 1 indeterminate). The most commonly identified low-risk types were candHPV62 (7.9%) and 61 (5.3%), while the most common high-risk types were 16 and 33 (8.8% each). Abnormal cytology was observed in 10 (3.0%) women, 9 of which were infected with HPV. Of the remaining 327 women with normal cytology results, 107 (32.7%) were positive for HPV DNA. HPV infection was correlated with younger age (less than 40 years), a first Pap smear, and other vaginal infections.

Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds.

In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.

Epidemiologic correlates of antibody response to human papillomavirus among women at low risk of cervical cancer.

A population at low risk for developing cervical cancer in Southern Brazil was studied to identify the main determinants of serological response to human papillomavirus (HPV). Enzyme-linked immunosorbent assay tests were performed in 976 women to detect serum IgG antibodies against HPV 16 L1 virus-like particles (VLPs) and HPVs 16, 18, 6 and 11 L1 VLPs as a mixture of antigens. Women with four or more sexual partners were more likely to be seropositive than women with one partner (HPV 16 serology odds ratio [OR]=3.06, 95% confidence interval [CI]: 2.0-4.8; HPV 6/11/16/18 serology OR=4.64, 95% CI: 3.0-7.2). HPV DNA and both serological responses were associated. Those positives to HPV 16 serology were twice as likely to have a cytological diagnosis of squamous intraepithelial lesions (SILs) than seronegatives (OR=2.07; 95% CI: 1.0-4.5, and OR=1.73; 95% CI: 0.8-3.8). Seropositivity to HPV 16 and HPV 6/11/16/18 antigens seem to be better markers of past sexual activity than current HPV infection, and humoral response to HPV 16 or HPV 6/11/16/18 may not be a strong indicator of cervical lesions in populations at low risk for cervical lesions.

Molecular characterization of Spanish infectious bursal disease virus field isolates.

Nine Spanish isolates of infectious bursal disease virus (IBDV) were characterized and classified after reverse transcriptase-polymerase chain reaction of a 248-bp fragment of the VP2 gene hypervariable region and restriction fragment length polymorphism (RFLP). The restriction endonucleases (REs) used were BstNI, Sad, SspI, TaqI, DraI, and StyI. Sequencing of the amplified product and further comparison of these sequences with published sequence data from other IBDV strains were also performed. Very virulent and classic strains were identified. None of the strains identified had molecular characteristics similar to that of the American variant strains. Four very virulent strains (VG-248, 5939, 6145, and 7333) were digested by the TaqI, SspI, and StyI enzymes. The sequences of these strains were closely related to other European and Japanese very virulent IBDV (vvIBDV) strains. Strains VG-311, VG-262, and VG-208 were digested by the BstNI and Sad REs and were classified as classic strains. Strains VG-276 and VG-313 had unique RFLP patterns. VG-276 exhibited the SspI RE site, which has been reported as a characteristic of vvIBDV strains, whereas the VG-313 strain exhibited a Sad and StyI RE site indicative of the classic IBDV Edgar and 52-70 strains. However, nucleotide sequence analysis of the amplified hypervariable region strain VG-276 revealed a higher identity with the classic strains STC, 52/70, and 9109 IBDV strains, whereas strain VG-313 exhibited a higher identity with the vvIBDV strains.

Change of migration time and separation window accompanied by field-enhanced sample stacking in capillary zone electrophoresis.

When field-enhanced sample stacking was used in capillary zone electrophoresis (CZE) analysis of cations, the decrease of migration time and the reduction of separation window was observed with increase of sample plug length. A simple equation expressing the migration velocity in the stacking process was derived to explain the above phenomenon. From experiments and theoretical consideration, we confirmed that this effect was caused by the higher potential gradient and larger eletroosmotic flow (EOF) mobility at the sample plug than those at the supporting electrolyte. A mathematical model appropriate for the computer simulation of such a system was studied considering the experimental results, and it was concluded that electroosmotic velocity (v(eof)) should be introduced to the equation of continuity as a constant.

Operational modes for transient isotachophoretic preconcentration-capillary zone electrophoresis and detectable concentration of rare earth ions.

Operational modes for transient isotachophoretic preconcentration capillary zone electrophoresis (tr-ITP-CZE) were studied by using 5 microM and 0.5 microM rare earth mixtures as analytes in comparison with field-enhanced sample stacking. After examination of several operational modes for tr-ITP, it was found that tr-ITP effectively occured even if both the leading electrolyte and the terminating electrolyte were injected after the sample plug. This was explained as the result of a field-enhanced stacking for both sample components and the leading and terminating ions. The observed theoretical plate numbers were 4-20 times higher than those obtained by normal stacking; and the estimated low limit of detectable concentration of rare-earth elements (REE) was ca. 0.1 microM which was 2.5 times lower compared to normal stacking. For the 0.5 microM sample, a concentration factor of 20 000 could be achieved after only tr-ITP.

Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction.

We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.

Generation of an X-ray microbeam for spectromicroscopy at SPring-8 BL39XU.

A pair of elliptical mirrors (KB mirror) was designed and fabricated to realize an energy tunable x-ray microbeam for spectromicroscopy at SPring-8 BL39XU. As is commonly recognized, the obtainable beam size with the aspherical total reflection mirrors is strongly affected with the slope error of the mirror. Considering that the extremely high brilliance of the undulator radiation from the SPring-8, the small mirror size and the small mirror-to-focus distance were employed to minimize effects of the slope error. Preliminary evaluation of the KB mirror was carried out using 10 keV monochromatized undulator radiation. Alignment of the mirror was assisted by the beam monitor system composed of a scintillator and a CCD, and the beam size less than 5 microm can be easily achieved even when the source was fully used. The beam size obtained with this experiment was 2 x 4 microm2 with the photon flux of 1 x 10(10) photons/s. Smaller beam size may be expected with the use of intermediate slits. Characterization of trace elements with the spatial resolution will be carried out by using x-ray fluorescence (XRF) analysis and x-ray absorption fine structure (XAFS) measurements with XRF yield method.

[Anti-GD1b IgG antibody-related Guillain-Barré syndrome initially mimicking brainstem infarction].

We described a 58-year-old woman with Guillain-Barré syndrome, who initially showed rapid progression of brainstem infarction-like signs. She developed superficial sensory disturbance on the left side, dysarthria, and left-predominant limb weakness within a few hours. She showed bilateral extensor plantar responses and head CT scan detected no abnormality. It was difficult to be distinguished from brainstem infarction until symmetrical limb weakness and generalized areflexia appeared. Serum anti-GD1b IgG antibody with cross-reactivity with GM1b was detected. Cerebrospinal fluid examination revealed albuminocytologic dissociation on day 5. After 5 sessions of immunoadsorption therapy, her symptoms gradually lessened. Anti-GD1b antibody has been detected in patients with sensory ataxic neuropathy. Our patient, however, was characterized with early involvement of brainstem with ataxia of cerebellar type. Our case suggests that anti-GD1b antibody-associated neuropathy has a broad spectrum of clinical features, which are related to cross-reactivity of this antibody.

Isotachophoretic separation behavior of rare-earth EDTA chelates and analysis of minor rare-earth elements in an iron ore by bidirectional isotachophoresis-particle-induced X-ray emission.

Mobilities of 16 anions of rare-earth-EDTA 1:1 chelate (RE-EDTAs) were isotachophoretically measured by using two leading electrolytes (pH 3.6 and 6.0) in order to assess their separation behavior. The leading electrolyte was 20 mM hydrochloric acid. The pH of the solution was adjusted to 3.6 by adding beta-alanine and to 6.0 by adding histidine. The obtained mobilities were very close to each other in the range 20.1x10(-5)-21.9x10(-5) cm2 V(-1) s(-1) with the minimum mobilities for Pr-EDTA and Nd-EDTA for pH 3.6 and 6.0, respectively, and pH dependence was hardly observed. On the basis of the above knowledge. minor rare-earth elements in a standard iron ore sample were determined as RE-EDTAs by bidirectional isotachophoresis-particle-induced X-ray emission (PIXE), where the Fe(II) matrix digested by alkali fusion was separated as Fe(II)Phen3(2+) (phen = 1,10-phenanthroline). Since 5% of the total iron was still detected as Fe(III)EDTA- and might disturb PIXE analysis of RE-EDTA-, itaconic acid was used as the spacer for Fe(III)EDTA- and RE-EDTA-. The fractions of RE-EDTA- were successfully analyzed off-line by a multielemental analytical method, PIXE [analytical result (3.62% (w/w) as RE2O3]; the nominal value was 3.37% (w/w) as RExOy.

Molecular characterization of Brazilian infectious bursal disease viruses.

A reverse transcriptase-polymerase chain reaction (RT-PCR) procedure was used to amplify a VP2 gene fragment (248 bp) from infectious bursal disease virus (IBDV). The procedure allowed the detection of known IBDV strains from the United States, along with field isolates and commercial vaccines produced in Brazil. Amplified VP2 fragments were further characterized by restriction fragment length polymorphism (RFLP) analysis. From 55 Brazilian commercial flocks, 48 field samples were IBDV positive by RT-TCR. Vaccine RFLP patterns were found in 12 flocks, a pattern compatible with classic IBDV in one flock, four new patterns in 31 flocks, and a pattern compatible with very virulent (vv) IBDV in four flocks. Sequence analysis showed that the vvIBDV RFLP patterns were closely related to the vvIBDVs described in Europe and Asia. Phylogenetic analysis of the four new RFLP patterns showed that they were closely related to but distinct from other classic, variant, and vvIBDVs, suggesting a high prevalence of different IBDV strains in Brazilian commercial flocks.

[A case presenting with Raeder's syndrome-like symptoms due to vertebral artery aneurysm].

A 50-year-old man complained of headache around his left orbit, left frontal pain and paresthesia associated with left incomplete Horner syndrome. MRI demonstrated a mass at the level of medulla oblongata. Left vertebral angiogram revealed an aneurysm of left vertebral artery. Following the removal of the aneurysm, these Raeder's syndrome-like symptoms improved. Therefore, they were probably caused by a compression of the spinal tract of the trigeminal nerve and the central sympathetic tract by the aneurysm. This is the first report of Reader's syndrome-like symptoms caused by vertebral artery aneurysm, thus indicating that MRI and cerebral angiogram are necessary for differential diagnosis of this syndrome.

Standardization of capillary zone electropherograms obtained by using field enhanced sample stacking.

Migration times in a capillary zone electropherogram obtained by using the field enhanced sample stacking technique are strongly affected by the injected sample volume. That is, the migration times significantly decrease with the increase of the sample volume. To avoid inaccurate qualitative analysis due to the above phenomena, the time axis of the electropherograms was converted into an effective mobility axis using our conversion method taking account of the temperature increase in the separation tube and relaxation of the potential gradient of the separation field. After the conversion, accurate qualitative analysis was possible in spite of drastic change of the migration time, suggesting our conversion method could be successfully used for the standardization of electropherograms obtained even by using the stacking effect. The cause of the decrease of the migration time in the stacking process was briefly discussed.

New method for standardization of electropherograms obtained in capillary zone electrophoresis.

A new method for standardization of electropherograms obtained by capillary zone electrophoresis was proposed, where the migration time axis was replaced by the effective mobility axis. The mobility increase due to temperature increase by Joule heating and the relaxation effect of the potential gradient were eliminated successfully by introducing a temperature coefficient for mobility expression and a delay time, respectively. The precision of the mobility evaluated by the proposed conversion methods was evaluated for a model sample. By using the conversion method, almost the same electropherograms could be obtained even from the electropherograms originally obtained by using different hardware conditions.

Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA.

The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.