Development of instrumentation for amperometric and coulometric detection using ultramicroelectrodes

In this work it is presented the development of a simple, portable and inexpensive instrumentation for amperometric and coulometric detection in different analytical instrumentation systems utilizing ultramicroelectrodes. The software, developed in LabVIEW 7.1TM, is capable to carry out three main detection techniques (amperometric, pulsed amperometric and coulometric detection) and a voltammetric technique (cyclic voltammetry). The instrumentation was successfully evaluated using the following systems: cyclic voltammograms of metallic electrodes in alkaline solutions, flow electrochemical detection of glucose and glycine and direct determination of herbicide glyphosate (electrochemical detection coupled to HPLC).

Determination of picloram in waters by sequential injection chromatography with UV detection

This paper describes a sequential injection chromatography procedure for determination of picloram in waters exploring the low backpressure of a 2.5 cm long monolithic C18 column. Separation of the analyte from the matrix was achieved in less than 60 s using a mobile phase composed by 20:80 (v v-1) acetonitrile:5.0 mmol L-1 H3PO4 and flow rate of 30 μL s-1. Detection was made at 223 nm with a 40 mm optical path length cell. The limits of detection and quantification were 33 and 137 μg L-1, respectively. The proposed method is sensitive enough to monitor the maximum concentration level for picloram in drinking water (500 μg L-1). The sampling frequency is 60 analyses per hour, consuming only 300 μL of acetonitrile per analysis. The proposed methodology was applied to spiked river water samples and no statistically significant differences were observed in comparison to a conventional HPLC-UV method.

Evaluation of a recombinant p24 antigen for the detection of Feline Immunodeficiency Virus-specific antibodies

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

Detection of anti-Toxoplasma gondii antibodies in experimentally and naturally infected non-human primates by Indirect Fluorescence Assay (IFA) and indirect ELISA

The Indirect Fluorescence Assay (IFA) and the indirect ELISA were comparatively used to detect IgG and IgM antibodies for Toxoplasma gondii in experimentally and naturally infected primates. In the experimentally infected group, antibodies of diagnostic value were detected at day 9 post-infection (PI) with the IFA (IgG and IgM) and with IgG-ELISA. IgM-ELISA detected antibodies for T. gondii starting at day 3 PI until the end of the experiment (102 days PI). Of the 209 naturally infected sera tested, from many zoos of State of Sao Paulo, 64.59 and 67.94% were positive in the IgG-IFA test and IgG-ELISA respectively. IgM-ELISA test detected seropositivity in 52.63% of the sera although IgM-IFA test detected it in only in 0.96% of the samples. The differential toxoplasmosis diagnosis was accomplished with Neospora caninum by IFA, observing 61 (29.2%) seropositive animals for this parasite and 149 (70.8%) negative. Sixty animals were positive for both T. gondii and N. caninum. Pneumonia, splenomegaly, and intestinal ulcers were macroscopically observed. Unremarkable interstitial pneumonia, enteritis, colitis, splenitis, and glomerulitis were microscopically observed. The immunohistochemical stain could not detect the presence of T. gondii in the tissues of the animals infected experimentally.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

Performance of transport and selective media for swine Bordetella bronchiseptica recovery and it comparison to polymerase chain reaction detection

Three comparative assays were performed seeking to improve the sensitivity of the diagnosis of Bordetella bronchiseptica infection analyzing swine nasal swabs. An initial assay compared the recovery of B. bronchiseptica from swabs simultaneously inoculated with B. bronchiseptica and some interfering bacteria, immersed into three transport formulations (Amies with charcoal, trypticase soy broth and phosphate buffer according to Soerensen supplemented with 5% of bovine fetal serum) and submitted to different temperatures (10ºC and 27ºC) and periods of incubation (24, 72 and 120 hours). A subsequent assay compared three selective media (MacConkey agar, modified selective medium G20G and a ceftiofur medium) for their recovery capabilities from clinical specimens. One last assay compared the polymerase chain reaction to the three selective media. In the first assay, the recovery of B. bronchiseptica from transport systems was better at 27ºC and the three formulations had good performances at this temperature, but the collection of qualitative and quantitative analysis indicated the advantage of Amies medium for nasal swabs transportation. The second assay indicated that MacConkey agar and modified G20G had similar results and were superior to the ceftiofur medium. In the final assay, polymerase chain reaction presented superior capability of B. bronchiseptica detection to culture procedures.

Molecular detection of Hepatozoon canis and Babesia canis vogeli in domestic dogs from Cuiabá, Brazil

The objective of this study was to report for the first time infection by Hepatozoon spp. and Babesia spp. in 10 dogs from the city of Cuiabá, State of Mato Grosso, central-western Brazil. A pair of primers that amplifies a 574 bp fragment of the 18S rRNA of Hepatozoon spp., and a pair of primers that amplifies a 551 bp fragment of the gene 18S rRNA for Babesia spp. were used. Six dogs were positive for Babesia spp., and 9 were positive for Hepatozoon spp. Co-infection of Babesia spp. and Hepatozoon spp. was seen in 5 dogs. Sequenced samples revealed 100% identity with B. canis vogeli, and H. canis. This is the first molecular detection of H. canis in domestic dogs from Cuiabá. Additionally, it is described for the first time the presence of B. canis vogeli circulating among dogs in Cuiabá.

Detection of Mycobacterium avium in pet birds

The present study is a report on the presence of Mycobacterium avium in four birds of the psittaciform order kept as pets. Anatomopathological diagnosis showed lesions suggestive of the agent and presence of alcohol-acid resistant bacilli (AARB) shown by the Ziehl-Neelsen staining. The identification of Mycobacterium avium was performed by means of PRA (PCR Restriction Analysis). DNA was directly extracted from tissue of the lesions and blocked in paraffin. The role of this agent in pet bird infection is discussed, as well as its zoonotic potential.

Detection of a new mumps virus genotype during parotitis epidemic of 2006-2007 in the State of São Paulo, Brazil

Nucleotide sequence analyses of the SH gene of 18 mumps virus isolates collected in the 2006-2007 parotitis epidemic in the state of São Paulo identified a new genotype, designated genotype M. This new designation fulfills all the parameters required to define a new mumps virus genotype. The parameters were established by an expert panel in collaboration with the World Health Organization (WHO) in 2005. This information will enhance the mumps virus surveillance program both at the national and global levels

Aeromonas detection and their toxins from drinking water form reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in São Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A.allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%).The results revealed that 70% of A. caviae, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern

Detection of Giardia and Cryptosporidium cysts/oocysts in watersheds and drinking water sources in Brazil urban areas

The protozoan parasites Giardia and Cryptosporidium have been described as important waterbone disease pathogens, and are associated with severe gastrointestinal illnesses. The objective of this paper was to investigate the presence of Giardia cysts and Cryptosporidium oocysts in sample from wtershed catchments and treated water sources. A total of 25 water samples were collected and examined according to the EPA - Method 1623, 2005, consisting of 12 from drinking water and 13 from raw water. Positive samples from raw water for Giardia cysts and Cryptosporidium oocysts were 46.1 and 7.6%, respectively. In finished water, positive samples were 41.7 per centfor Giardia cysts and 25 per cent for Cryptosporidium oocysts. Concentrations of Giardia cysts found in raw water samples ranged from "not detected" to 0.1oocysts/L, whereas concentrations of Cryptosporidium oocystsranged from "not detected" to 0.1 oocysts/L. In finished water, Giardia concentrations ranged from "not detected" to 0.06 cysts/L, and Cryptosporidium oocysts were not high in the samples analyzed. Nevertheless, the results of this study highlight the need to monitor these organisms in both raw and drinking water.

Detection of metallo-'beta'-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei

Aims: To determine the prevalence and expression of metallo-beta-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil. Methods and Results: Eighty-seven Aeromonas isolates belonging to Aeromonas hydrophila (n = 41) and Aer. jandaei (n = 46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97.6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla(IMP), bla(VIM) and bla(SPM-1) were not detected. On the other hand, production of MBL activity was detectable in 87.8% and 10.9% of the cphA-positive Aer. hydrophila and Aer. jandaei isolates respectively. Conclusions: Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity. Significance and Impact of the Study: These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of water-borne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view

Development of a Multicommutated Flow System with Chemiluminometric Detection for Quantification of Gentamicin in Pharmaceuticals

A new flow procedure based on multicommutation with chemiluminometric detection was developed to quantify gentamicin sulphate in pharmaceutical formulations. This approach is based on gentamicin's ability to inhibit the chemiluminometric reaction between luminol and hypochlorite in alkaline medium, causing a decrease in the analytical signal. The inhibition of the analytical signal is proportional to the concentration of gentamicin sulphate, within a linear range of 1 to 4 mu g mL(-1) with a coefficient variation <3%. A sample throughput of 55 samples h(-1) was obtained. The developed method is sensitive, simple, with low reagent consumption, reproducible, and inexpensive, and when applied to the analysis of pharmaceutical formulations (eye drops and injections) it gave results with RSD between 1.10 and 4.40%.

Border figure detection using a phase oscillator network with dynamical coupling

Oscillator networks have been developed in order to perform specific tasks related to image processing. Here we analytically investigate the existence of synchronism in a pair of phase oscillators that are short-range dynamically coupled. Then, we use these analytical results to design a network able of detecting border of black-and-white figures. Each unit composing this network is a pair of such phase oscillators and is assigned to a pixel in the image. The couplings among the units forming the network are also dynamical. Border detection emerges from the network activity.

Molecular basis for porcine parvovirus detection in dead fetuses

Reproductive failures are still common grounds for complaint by commercial swine producers. Porcine parvovirus (PPV) is associated with different clinical reproductive signs. The aim of the present study was to investigate PPV fetal infection at swine farms having ongoing reproductive performance problems. The presence of virus in fetal tissues was determined by nested-polymerase chain reaction assay directed to the conserved NS1 gene of PPV in aborted fetuses, mummies and stillborns. Fetuses show a high frequency of PPV infection (96.4%; N = 28). In 60.7% of the fetuses, PPV were detected in all tissue samples (lung, heart, thymus, kidney, and spleen). Viral infection differed among fetal tissues, with a higher frequency in the lung and heart (P < 0.05). Fetuses with up to 99 days of gestational age and from younger sows showed a higher frequency of PPV (P < 0.05). No significant difference in the presence of PPV was detected among the three clinical presentations. The results suggest that PPV remains an important pathogenic agent associated with porcine fetal death.