Determination of herbicides residues in soil by small scale extraction

Herbicides such as trifluralin, simazine, atrazine, metribuzin and metolachlor are used in Brazilian agriculture. The efficiency of a small scale method for determination of these herbicides and two degradation products (deisopropylatrazine and deethylatrazine) in soil samples was evaluated. The compounds were extracted from soil samples (5 g) with 20 ml of ethyl acetate in a mechanical shaker for 50 min. Following the extraction, the supernatant was dried through anhydrous sodium sulphate, concentrated and analysed by high resolution gas chromatography (HRGC) with thermionic specific detection (TSD). Mean recoveries obtained from soil samples fortified at three different levels ranged from 81 to 115% with relative standard deviation (RSD) values varying from 1.2 to 12.7%. The method detection limits ranged from 0.01 to 0.06 mg kg-1. The methodology was applied using soil samples from farms located near the town of Araraquara, in the State of São Paulo, Brazil.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

Cysticercus antibodies and antigens in serum from blood donors from Pondicherry, India

The aim of the present study was to screen the serum of blood donors, which are apparently healthy and residing in Pondicherry or its neighboring districts of Tamil Nadu State, for specific detection of Cysticercus antigens and antibodies. A total of 216 blood samples were collected from blood donors at the Central Blood Bank, JIPMER Hospital, Pondicherry, India during January and February 2004. Enzyme-linked immunosorbent assay (ELISA) was used to demonstrate anti-Cysticercus antibodies and the Co-agglutination (CoA) was used to detect antigen in sera. 14 (6.48 %) males were positive for either anti-Cysticercus antibodies or antigens. Of these eight sera were positive for anti-Cysticercus antibodies and six were positive for antigens. Results of the present study show that serum Cysticercus antigen detection may be a useful adjunct to antibody testing for seroprevalence studies of cysticercosis in the community. The present study is the first kind of study, carried out to determine both cysticercal antibodies as well as antigens in the serum samples collected from the healthy blood donors.

First report on the occurrence of Trypanosoma rangeli Tejera, 1920 in the state of Ceará, Brazil, in naturally infected triatomine Rhodnius nasutus Stål, 1859 (Hemiptera, Reduviidae, Triatominae)

The aim of this work was to identify and report the occurrence of Trypanosoma rangeli and Trypanosoma cruzi in naturally infected Rhodnius nasutus (Hemiptera, Reduviidae, Triatominae) in the state of Ceará, Brazil. Triatomines feces, salivary glands, and hemolymph were collected for fresh examination, and specific detection of T. rangeli and T. cruzi DNA by polymerase chain reaction was carried out. The specific characterization of these two parasites showed the simultaneous presence of both parasites in two (7.7%) of the 26 positive insects. Our results provide further knowledge on the geographical distribution of T. rangeli in Brazil.

Microscopia de força atômica aplicada em imunoensaios

Affinity reactions have been used for specific detection of their complementary partners and an enormous variety of enzyme-linked immunosorbent assay (ELISA) formats are used in research and in routine serological tests. With the advent of the atomic force microscopy (AFM) technique, the immune reactions have been monitored by these devices. In the present article we focus on applications of AFM to immunoassays. After introducing the basic concepts of AFM, a brief discussion on the monitoring of the interactions between antigens and antibodies through both topographic image and biosensor systems is presented.

An isolate of Apple stem grooving virus associated with Cleopatra mandarin fruit intumescence

A citrus tatter leaf isolate (CTLV-Cl) of Apple stem grooving virus (ASGV) has been found to be associated with a fruit rind intumescence in Cleopatra mandarin (Citrus reshni) in Limeira (SP). The CTLV-Cl was mechanically transmitted to the main experimental herbaceous hosts of CTLV. Chenopodium quinoa and C. amaranticolor reacted with local lesions and systemic symptoms while other test plants reacted somewhat differently than what is reported for CTLV. A pair of primers designed for specific detection of ASGV and CTLV amplified the expected 801 bp fragment from the CTLV-Cl-infected plants. Typical capillovirus-like particles were observed by the electron microscope in experimentally infected C. quinoa and C. amaranticolor leaves.

Partial characterization of nif genes from the bacterium Azospirillum amazonense

Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense.

Human cryptosporidiosis: detection of specific antibodies in the serum by an indirect immunofluorescence

Cryptosporidium sp., a coccidian parasite usually found in the faeces of cattle, has been recently implicated as an agent of human intestinal disease, mainly in immunocompromised patients. In the study realized, by an indirect immunofluorescence technique, specific immunoglobulins (IgG and IgM) have been demonstrated in human serum against Cryptosporidium oocysts. Purified oocysts were used as antigens in the indirect immunofluorecence assay. After analyzing this test in sera from selected groups of patients, the frequency of both specific IgG and IgM of immunocompetent children who were excreting oocysts in their faeces was 62% and in children with negative excretion of oocysts was 20% and 40%, respectively. In adults infected with the human immunodeficiency virus (HIV) and who were excreting Cryptosporidium in their stools, the frequency was 57% for IgG but only 2% for IgM. Twenty three percent of immunocompromised adults with not determined excretion of oocysts in their stools had anti-Cryptosporidium IgG in their sera. Children infected with human immunodeficiency virus had no IgM and only 14% had IgG detectable in their sera. The indirect immunoflorescence assay, when used with other parasitological techniques appears to be useful for retrospective population studies and for diagnosis of acute infection. The humoral immune response of HIV positive patients to this protozoan agent needs clarification.

Antigens of worms and eggs showed a differentiated detection of specific IgG according to the time of Schistosoma mansoni infection in mice

INTRODUCTION: The correlation between the immunological assay and the antibody titer can offer a tool for the experimental analysis of different phases of the disease. METHODS: Two simple immunological assays for Schistosoma mansoni in mice sera samples based on specific IgG detection for worms soluble antigens and eggs soluble antigens were standardized and evaluated in our laboratory. Fifty mice were used in negative and positive groups and the results obtained by enzyme-linked immunosorbent assays (ELISA) assays were compared with the number of worms counted and the IgG titers at different times of infection. RESULTS: Data showed that ELISA using adult worm antigens (ELISA-SWAP) presented a satisfactory correlation between the absorbance value of IgG titers and the individual number of worms counted after perfusion technique (R²=0.62). In addition, ELISA-SWAP differentially detected positive samples with 30 and 60 days post infection (p=0.011 and 0.003, respectively), whereas ELISA using egg antigens (ELISA-SEA) detected samples after 140 days (p=0.03). CONCLUSIONS: These data show that the use of different antigens in immunological methods can be used as potential tools for the analysis of the chronological evolution of S. mansoni infection in murine schistosomiasis. Correlations with human schistosomiasis are discussed.

Rapid in vitro detection of HIV-1-specific antibody secretion by cells-culture with virus antigens

The present report describes an alternative method for in vitro detection of HIV-1 -specific antibody secretion in 24h of culture employing as stimulant of peripheral blood mononuclear cells the disrupted inactivated whole virus adsorbed onto microwells in a commercial ELISA kit plates. The results obtained from this technique have showed high sensitivity and specificity since it was capable of detecting HIV-1 infection early after birth. There were neither false-positivity nor false-negativity when blood samples obtained from HIV-1 seronegative asymptomatic individuals, and HIV-1 seropositive adult patients were analized. This rapid, low cost, simple, highly sensitive and specific assay can be extremely useful for early diagnosis of pediatric HIV infection.

Detection of Toxoplasma gondii-Specific Antibodies in Dogs. A Comparative Study of Immunoenzymatic, Immunofluorescent and Haemagglutination Titers

We evaluated the titers of anti-T. gondii antibodies by various serological tests in 40 serum samples from dogs exhibiting clinical signs of infectious diseases. Indirect immunofluorescence (IgG-IFI), indirect haemagglutination (IHA and M-Toxo) and immunoenzymatic (ELISA and PA-ELISA) tests were carried out. Titers ³ 64 were considered as positive. Anti-Toxoplasma antibodies were found in 9 (22.5%), 14 (35%), 14 (35%) and 12 (30%) samples, respectively for IHA, IgG-IFI, ELISA and PA-ELISA. The results showed that 57% were negative in all tests and 43% of the dogs presented antibodies to T. gondii; from these, 20% were positive in all three tests with high titers of antibodies and 23% were positive in only one or two tests with low titers of antibodies and mainly related to the IFI and ELISA tests. We observed 5 (12.5%) and 1 (2.5%) reactive samples, respectively, by M-Toxo and IHA with or without 2-mercapthoethanol, in the attempt to detect specific IgM. We can conclude that serodiagnosis of toxoplasmosis in dog have to be based on the combination of serological tests (IFI and ELISA) and with emphasis at the determination of the titers and the classes of the specific antibodies

Detection of the acute phase of abdominal angiostrongyliasis with a parasite-specific IgG enzyme linked immunosorbent assay

Angiostrongylus costaricensis may cause intestinal lesions of varied severity when it accidentally infects man in Central and South America. First-stage larvae have never been detected in stools. Therefore, a parasite-specific IgG ELISA was evaluated for the determination of the acute phase of infection. The specificity and the sensitivity of the immunoassay was shown to be 76.2% and 91.1%, respectively. Eight serum samples taken from patients with histopathological diagnosis, at different time points (3 to 15 months) after surgical treatment, showed a sharp and early decline in antibody reactivity. The titration of anti-A. costaricensis antibodies has proved to be a useful method for the diagnosis of acute abdominal angiostrongyliasis.