Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1). B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis. (c) 2008 Elsevier Ltd. All rights reserved.

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of noninfected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen. (c) 2007 Elsevier B.V. All rights reserved.

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations. (C) 2007 Elsevier B.V. All rights reserved.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Avaliacão das técnicas de cultivo microbiológico e soroaglutinação rápida em cartão com e se 2-Mercaptoetanol da Brucelose canina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação de seis testes sorológicos no diagnóstico da brucelose bubalina

ABSTRACT: Four hundred and forty buffalo sera, selected from about 1,200 blood samples of another study, were examined. The samples were tested by six serological methods: two of agglutination, two of indirect ELISA and two of competitive ELISA. To determine the relative sensitivity and specificity of different tests, animals with a positive result to competitive ELISA of the FAO/IAEA were considered as infected. The relative sensitivity of competitive ELISA, indirect ELISA with conjugate anti-bovine light chain monoclonal antibody labelled with HRPO, indirect ELISA with anti-bovine IgG conjugate, rose Bengal test and rapid slide agglutination test was 100%, 98.57%, 97.14%, 91.42% and 79.28%, and the relative specificity 99.33%, 97.33%, 95.66%, 94.00% and 86.33%, respectively. The value of the different serological tests for the diagnosis of brucellosis is discussed.

Rapid detection of white spot syndrome virus (WSSV) of Penaeus monodon by latex agglutination test using monoclonal antibodies

Latex beads were sensitized with monoclonal antibodies (MAb) rose against VP28 of WSSV. The optimum concentration of MAb required to sensitize the latex beads was 125 µg/ml. The sensitized latex beads were used to detect WSSV from PCR-positive stomach tissue homogenates obtained from infected shrimp. Stomach tissue homogenates from WSSV-infected shrimp agglutinated the sensitized latex beads within 10 minutes, while uninfected samples did not produce any agglutination, although non-specific agglutinations were observed in some samples. The analytical sensitivity, analytical specificity, diagnostic sensitivity and diagnostic specificity of the (LAT) agglutination test were assessed. The analytical sensitivity of the test was 40 ng of purified WSSV (2 µg/ml). The sensitized latex beads did not agglutinate with normal shrimp tissue or MBV-infected tissue homogenate. The test has a diagnostic sensitivity of 70 and 45%, respectively, compared to single-step and nested PCR. The diagnostic specificity of the test was 82%. This test is a simple and rapid on-farm test which can be used to corroborate clinical signs for the detection of WSSV in grow-out ponds.

Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against Anaplasma marginale in cattle

The recombinant protein MSP5 has been established as an important antigen for serological diagnosis of Anaplasma marginale by enzyme-linked immunosorbent assay (ELISA). However, due to the high cost of specialized equipment, this technique is not accessible to all laboratories, especially in developing countries in areas where the disease is endemic. The present study describes the standardization of a latex agglutination test (LAT) to detect antibodies against A. marginale based on recombinant MSP5. Compared with indirect enzyme-linked immunosorbent assay (iELISA), the relative sensitivity and specificity of the LAT were 95.21% and 91.86% respectively, with an almost perfect agreement between tests (kappa index = 0.863). These results can be considered important for the serological diagnosis of A. marginale, as they indicate that the test represents a rapid and low cost alternative to ELISA.

Preliminary assessment of a rapid leaf nitrogen test in mango

Nitrogen (N) is an essential nutrient in mango, influencing both productivity and fruit quality. In Australia, tree N is traditionally assessed once a year in the dormant pre-flowering stage by laboratory analysis of leaf N. This single assessment is insufficient to determine tree N status at all stages of the annual phenological cycle. Development of a field-based rapid N test would allow more frequent monitoring of tree N status and improved fertiliser management. This experiment examined the accuracy and useability of several devices used in other horticultural crops to rapidly assess mango leaf N in the field; the Konica Minolta 'SPAD-502 chlorophyll meter', Horiba 'Cardy Meter' and the Merck 'RQflex 10'. Regression and correlation analyses were used to determine the relationship between total leaf N and the measurements from the rapid test devices. The relationship between the chlorophyll index measured by the SPAD-502 meter and leaf N is highly significant at late fruit set (R 2=0.72, n=40) and post-harvest (R2=0.81, n=40) stages in the mango cultivar 'Kensington Pride' and significant (R2=0.51, n=40) at the flowering stage, indicating the device can be used to rapidly assess mango leaf N in the field. Correlation analysis indicated the relationship between petiole sap measured with the Cardy or Merck devices and leaf N is non-significant. © 2013 ISHS.

Preparation and characterization of a novel pyrrole-benzophenone copolymerized silica nanocomposite as a reagent in a visual immunologic-agglutination test.

Biological sensing is explored through novel stable colloidal dispersions of pyrrole-benzophenone and pyrrole copolymerized silica (PPy-SiO(2)-PPyBPh) nanocomposites, which allow covalent linking of biological molecules through light mediation. The mechanism of nanocomposite attachment to a model protein is studied by gold labeled cholera toxin B (CTB) to enhance the contrast in electron microscopy imaging. The biological test itself is carried out without gold labeling, i.e., using CTB only. The protein is shown to be covalently bound through the benzophenone groups. When the reactive PPy-SiO(2)-PPyBPh-CTB nanocomposite is exposed to specific recognition anti-CTB immunoglobulins, a qualitative visual agglutination assay occurs spontaneously, producing as a positive test, PPy-SiO(2)-PPyBPh-CTB-anti-CTB, in less than 1 h, while the control solution of the PPy-SiO(2)-PPyBPh-CTB alone remained well-dispersed during the same period. These dispersions were characterized by cryogenic transmission microscopy (cryo-TEM), scanning electron microscopy (SEM), FTIR and X-ray photoelectron spectroscopy (XPS).

Rapid serum agglutination and agar gel immunodiffusion tests associated to clinical signs in rams experimentally infected with Brucella ovis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of serum sample inactivation on the performance of latex agglutination test for paracoccidioidomycosis serodiagnosis

Paracoccidioidomycosis is diagnosed from the direct observation of the causative agent, but serology can facilitate and decrease the time required for diagnosis. The objective of this study was to determine the influence of serum sample inactivation on the performance of the latex agglutination test (LAT) for detecting antibodies against Paracoccidioides brasiliensis. The sensitivity of LAT from inactivated or non-inactivated samples was 73% and 83%, respectively and the LAT selectivity was 79% and 90%, respectively. The LAT evaluated here was no more specific than the double-immunodiffusion assay. We suggest the investigation of other methods for improving the LAT, such as the use of deglycosylated antigen.