Molecular Detection and Characterization of Ehrlichia canis Isolates from Three Geographic Regions in Mexico: A Retrospective Study.

Canine monocytic ehrlichiosis (CME) is the most common tick-borne disease affecting domestic dogs and other wild canids. It has a worldwide distribution and is associated with the presence of the brown dog tick. Few studies have been conducted in Mexico to identify and characterize Ehrlichia canis genetic variability. In the present study, 111 dogs of different sex, breed, and age from three geographic regions in Mexico were included. All of them had a previous history of tick infestation and/or the presence of one or more clinical signs compatible with CME. All dogs were tested by a commercial ELISA and nested PCR assay for the detection of E. canis. In addition, we analyzed the E. canis genetic diversity from the 16S rRNA gene sequences obtained in this study, along with 15 additional sequences described for E. canis in Mexico and obtained from GeneBank. Serological detection by commercial ELISA results showed overall infection rates of 85.58% (95/111), including 73.1% (30/41) in samples from Guerrero state; 75% (15/20) in Morelos; and 100% (50/50) in Chihuahua. On the other hand, molecular detection (nPCR assay) showed 31.5% (35/111) overall infection rate, with 41.4% (17/41) in Guerrero state; 55% (11/20) in Morelos; and 14% (7/50) in Chihuahua. We observed a high 16S rRNA gene sequence conservancy in most of the E. canis isolates in the three geographical areas from Mexico, including those analyzed in this research, suggesting a common geographic origin among isolates.

Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts.

In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.

Primer design to assess bacterial degradation of glyphosate and other phosphonates.

Phosphonates are organic phosphorous (P) compounds frequently detected in the environment due to a very stable CP bond that render them relatively recalcitrant. Glyphosate [N-phosphonomethyl glycine] is the most widely used and best-known synthetic phosphonate, and one of the most concerning herbicides in the world today. Microbial degradation of glyphosate and organophosphonates in general, is the main dissipation mechanism operating in most environments. One microbial metabolic pathway in this process is the CP lyase pathway, entailing an enzymatic complex encoded by about 14 genes (the Phn operon). Our goal was to develop a quantitative polymerase chain reaction (qPCR) assay for a key enzyme, the CP lyase that breaks down the CP bond, via quantification of the codifying phnJ gene. The primers designed in this study fulfill the requirements for a successful qPCR assay, with high efficiency and sensitivity, as well as specific detection of the target sequence in a wide range of taxonomic groups. This is, to our knowledge, the first report of primers designed to target phnJ in both pure cultures and metagenomic DNA from different environmental sources. Direct quantification of phnJ may be a cost-effective proxy to determine glyphosate degradation potential in different matrixes.

High prevalence of Gammapapillomaviruses (Gamma-PVs) in pre-malignant cutaneous lesions of immunocompetent individuals using a new broad-spectrum primer system, and identification of HPV210, a novel Gamma-PV type.

Genus Gammapapillomavirus (Gamma-PV) is the most diverse and largest clade within the Papillomaviridae family. A novel set of degenerate primers targeting the E1 gene was designed and further used in combination with the well-known CUT PCR assay to assess HPV prevalence and genus distribution in a variety of cutaneous samples from 448 immunocompetent individuals. General HPV, Gamma-PV and mixed infections prevalence were significantly higher in actinic keratosis with respect to benign and malignant neoplasms, respectively (p = 0.0047, p = 0.0172, p = 0.00001). Gamma-PVs were significantly more common in actinic keratosis biopsies than Beta- and Alpha-PVs (p = 0.002). The full-length genome sequence of a novel putative Gamma-PV type was amplified by 'hanging droplet' long-range PCR and cloned. The novel virus, designated HPV210, clustered within species Gamma-12. This study provides an additional tool enabling detection of HPV infections in skin and adds new insights about possible early roles of Gamma-PVs in the development of cutaneous malignant lesions.

Benznidazole therapy for Chagas disease in asymptomatic Trypanosoma cruzi -seropositive former blood donors: evaluation of the efficacy of different treatment regimens

Abstract: INTRODUCTION: Chagas disease currently affects 5.7 million people in Latin America and is emerging in non-endemic countries. There is no consensus concerning the efficacy of trypanocidal therapy for patients with the chronic form of the disease. We evaluated cardiac function and sociodemographic, clinical, and serologic characteristics of a group of asymptomatic Trypanosoma cruzi-seropositive former blood donors, and compared the effects of benznidazole treatment applied for different lengths of time. METHODS: Blood donors who screened positive for T. cruzi between 1998 and 2002 were recruited 10 years later for follow-up (n = 244); 46 individuals had received treatment. Three subjects had terminated treatment prematurely. The remaining 43 individuals were divided into two groups: individuals who had received benznidazole therapy for 50-60 days (n = 28; BT ≤60 group) or more than 60 days (n = 15; BT >60). Serologic assays, biochemical tests, electrocardiographic, echocardiographic, and clinical examinations were performed on all participants. Parasite loads were determined by qualitative and quantitative polymerase chain reaction. RESULTS: Parasitemia was significantly reduced in the BT ≤60 and BT >60 groups compared with the untreated group. There were no differences in epidemiologic profiles or clinical, biochemical, electrocardiographic, or echocardiographic data between any of the groups. CONCLUSIONS: Despite elimination or significant reduction in parasitemia in patients with chronic Chagas disease who received benznidazole, there was no clinical difference between those who were treated for >60 days and those treated for a shorter duration. Furthermore, the adverse effects of benznidazole appear to be less severe than previous reports would suggest.

Detection of different South American hantaviruses.

Hantaviruses are the etiologic agents of Hemorrhagic Fever with Renal Syndrome (HFRS) in Old World, and Hantavirus Pulmonary Syndrome (HPS)/Hantavirus Cardiopulmonary Syndrome (HCPS), in the New World. Serological methods are the most common approach used for laboratory diagnosis of HCPS, however theses methods do not allow the characterization of viral genotypes. The polymerase chain reaction (PCR) has been extensively used for diagnosis of viral infections, including those caused by hantaviruses, enabling detection of few target sequence copies in the sample. However, most studies proposed methods of PCR with species-specific primers. This study developed a simple and reliable diagnostic system by RT-PCR for different hantavirus detection. Using new primers set, we evaluated human and rodent hantavirus positive samples of various regions from Brazil. Besides, we performed computational analyzes to evaluate the detection of other South American hantaviruses. The diagnostic system by PCR proved to be a sensible and simple assay, allowing amplification of Juquitiba virus, Araraquara virus, Laguna Negra virus, Rio Mamore virus and Jabora virus, beyond of the possibility of the detecting Andes, Anajatuba, Bermejo, Choclo, Cano Delgadito, Lechiguanas, Maciel, Oran, Pergamino and Rio Mearim viruses. The primers sets designed in this study can detect hantaviruses from almost all known genetics lineages in Brazil and from others South America countries and also increases the possibility to detect new hantaviruses. These primers could easily be used both in diagnosis of suspected hantavirus infections in humans and also in studies with animals reservoirs.

Antibiotic resistance in lactic acid bacteria isolated from some pharmaceutical and dairy products.

A total of 244 lactic acid bacteria (LAB) strains were isolated from 180 dairy and pharmaceutical products that were collected from different areas in Minia governorate, Egypt. LAB were identified phenotypically on basis of morphological, physiological and biochemical characteristics. Lactobacillus isolates were further confirmed using PCR-based assay. By combination of phenotypic with molecular identification Lactobacillus spp. were found to be the dominant genus (138, 76.7%) followed by Streptococcus spp. (65, 36.1%) and Lactococcus spp. (27, 15%). Some contaminant organisms such as (Staphylococcus spp., Escherichia coli, Salmonella spp., mould and yeast) were isolated from the collected dairy samples but pharmaceutical products were free of such contaminants. Susceptibility of LAB isolates to antibiotics representing all major classes was tested by agar dilution method. Generally, LAB were highly susceptible to Beta-lactams except penicillin. Lactobacilli were resistant to vancomycin, however lactococci and streptococci proved to be very susceptible. Most strains were susceptible to tetracycline and showed a wide range of streptomycin MICs. The MICs of erythromycin and clindamycin for most of the LAB were within the normal range of susceptibility. Sixteen Lactobacillus, 8 Lactococcus and 8 Streptococcus isolates including all tetracycline and/or erythromycin resistant strains were tested for the presence of tetracycline and/or erythromycin resistant genes [tet(M) and/or erm(B)]. PCR assays shows that some resistant strains harbor tet(M) and/or erm(B) resistance genes.

COMPARISON BETWEEN AUTOMATED SYSTEM AND PCR-BASED METHOD FOR IDENTIFICATION AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF CLINICAL Enterococcus spp / Comparação entre o sistema automatizado e PCR na identificação e susceptibilidade de isolados clínicos de Enterococcus spp

Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci.

Os enterococos são cada vez mais responsáveis por infecções hospitalares em todo o mundo. Este estudo foi realizado para comparar a identificação e perfil de suscetibilidade entre o sistema automatizado MicrosScan e a técnica molecular de PCR em espécies de Enterococcus spp. Foram avaliados 30 isolados clínicos de Enterococcus spp. Os isolados foram identificados pelo sistema MicrosScan® e pela técnica de PCR. A detecção de genes de resistência a antibióticos (vancomicina, gentamicina, tetraciclina e eritromicina) foi determinada por PCR. Suscetibilidades antimicrobianas à vancomicina (30 µg), gentamicina (120 µg), tetraciclina (30 µg) e eritromicina (15 µg), foram testados pelos métodos automatizados e pelo disco difusão, de acordo com as orientações do CLSI. No que diz respeito à identificação de Enterococcus em geral entre os dados obtidos pelo método de PCR e pelo sistema automático foi de 90,0% (27/30). Para todos os isolados de E. faecium e E. faecalis observamos concordância de 100%. Freqüências de resistência foi maior em E. faecium do que em E. faecalis. As taxas de resistência obtidas foi maior para eritromicina (86,7%), vancomicina (80,0%), tetraciclina (43,35%) e gentamicina (33,3%). A correlação entre a técnica de disco difusão e automação revelou-se de acordo para maioria dos antibióticos com taxas > 80%. O gene van(A) foi detectado em 100% dos Enterococcus resistentes á vancomicina. O ensaio baseado em PCR é de simples realização e de confiança para identificação de enterococos clinicamente relevantes. Os dados obtidos reforçam a necessidade de melhoria no sistema automatizado para identificar alguns enterococos.

Antibiotic resistance in lactic acid bacteria isolated from some pharmaceutical and dairy products

A total of 244 lactic acid bacteria (LAB) strains were isolated from 180 dairy and pharmaceutical products that were collected from different areas in Minia governorate, Egypt. LAB were identified phenotypically on basis of morphological, physiological and biochemical characteristics. Lactobacillus isolates were further confirmed using PCR-based assay. By combination of phenotypic with molecular identification Lactobacillus spp. were found to be the dominant genus (138, 76.7%) followed by Streptococcus spp. (65, 36.1%) and Lactococcus spp. (27, 15%). Some contaminant organisms such as (Staphylococcus spp., Escherichia coli, Salmonella spp., mould and yeast) were isolated from the collected dairy samples but pharmaceutical products were free of such contaminants. Susceptibility of LAB isolates to antibiotics representing all major classes was tested by agar dilution method. Generally, LAB were highly susceptible to Beta-lactams except penicillin. Lactobacilli were resistant to vancomycin, however lactococci and streptococci proved to be very susceptible. Most strains were susceptible to tetracycline and showed a wide range of streptomycin MICs. The MICs of erythromycin and clindamycin for most of the LAB were within the normal range of susceptibility. Sixteen Lactobacillus,8 Lactococcus and 8 Streptococcus isolates including all tetracycline and/or erythromycin resistant strains were tested for the presence of tetracycline and/or erythromycin resistant genes [tet(M) and/or erm(B)]. PCR assays shows that some resistant strains harbor tet(M) and/or erm(B) resistance genes.(AU)

Antibiotic resistance in lactic acid bacteria isolated from some pharmaceutical and dairy products

A total of 244 lactic acid bacteria (LAB) strains were isolated from 180 dairy and pharmaceutical products that were collected from different areas in Minia governorate, Egypt. LAB were identified phenotypically on basis of morphological, physiological and biochemical characteristics. Lactobacillus isolates were further confirmed using PCR-based assay. By combination of phenotypic with molecular identification Lactobacillus spp. were found to be the dominant genus (138, 76.7%) followed by Streptococcus spp. (65, 36.1%) and Lactococcus spp. (27, 15%). Some contaminant organisms such as (Staphylococcus spp., Escherichia coli, Salmonella spp., mould and yeast) were isolated from the collected dairy samples but pharmaceutical products were free of such contaminants. Susceptibility of LAB isolates to antibiotics representing all major classes was tested by agar dilution method. Generally, LAB were highly susceptible to Beta-lactams except penicillin. Lactobacilli were resistant to vancomycin, however lactococci and streptococci proved to be very susceptible. Most strains were susceptible to tetracycline and showed a wide range of streptomycin MICs. The MICs of erythromycin and clindamycin for most of the LAB were within the normal range of susceptibility. Sixteen Lactobacillus,8 Lactococcus and 8 Streptococcus isolates including all tetracycline and/or erythromycin resistant strains were tested for the presence of tetracycline and/or erythromycin resistant genes [tet(M) and/or erm(B)]. PCR assays shows that some resistant strains harbor tet(M) and/or erm(B) resistance genes.

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India.

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.(AU)

Incidence and multiplex PCR based detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly harvested Maize kernels in Southern India

Hundred Fusarium culmorum strains, isolated from freshly harvested maize grain samples from Southern parts of India, were incubated in czapek-dox medium and analyzed for trichothecene (DON/NIV) production. The mPCR assay was standardized targeting trichothecene metabolic pathway genes viz., Tri6, Tri7, Tri13 for detection of trichothecene (DON/NIV) chemotypes and rDNA gene for specific detection of F. culmorum species. Primers for targeted genes were designed and used to predict whether these isolates could produce deoxynivalenol/nivalenol, 94 isolates were able to produce DON/NIV by mPCR assay. Chemical analysis of DON/NIV was carried out for mPCR positive isolates by high performance-thin layer chromatography (HPTLC). To check the practical usefulness of developed mPCR assay, 150 field samples of maize were evaluated and results were compared with conventional HPTLC method. Out of 150 samples, 34% samples stayed as a positive for NIV contamination whereas 44% were found to have deoxynivalenol contamination. Moreover, mPCR results are equivocally matched with the HPTLC chemical analysis for field samples. Chemotyping of F. culmorum isolates were reported for the first time from India, and highlights the important potential of F. culmorum to contaminate maize with DON/NIV.

A RT-PCR assay for the detection of encephalomycarditis virus infections in pigs

Encephalomyocarditis virus (EMCV) infections can cause losses in pig farms all over the world. Rapid, sensitive and unequivocal detection of this virus is therefore essential for the diagnosis and control of the disease. An RT-PCR assay was developed, optimized and evaluated for encephalomyocarditis virus detection in organ based on a pair of primers that amplifies a 165 bp DNA fragment from a highly conserved nucleotide region of the viral 3D glycoprotein. PCR products of the expected size were obtained from Cuban EMCV 744/03 strain. Non-specific reactions were not observed when other porcine RNA genome viruses and uninfected cells were used. The analytical sensitivity of the test was estimated to be 2 TCID50/50 mL. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases.

Development of a polymerase chain reaction assay for the detection of pseudorabies virus in clinical samples / Desenvolvimento de um ensaio de reação de polimerase em cadeia para detecção do vírus da pseudo-raiva em amostras clínicas

Aujeszky's disease, also known as pseudorabies causes severe economic losses in swine industry and affects the pig husbandry all over the world. The conventional diagnostic procedure is time-consuming and false-negative results may occur in submissions from latently infected animals. The development, optimization and evaluation of a polymerase chain reaction (PCR) assay are presented for the diagnosis of pseudorabies infection. This assay was based on the amplification of a highly conserved viral gD gene fragment. PCR products of the expected size were obtained from PRV strains. Non-specific reactions were not observed when a related herpesvirus, other porcine DNA genome viruses and uninfected cells were used to assess PCR. The analytical sensitivity of the test was estimated to be 1.34 TCID50/50 uL. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases. A rapid, sensitive and specific PCR-based diagnostic assay to detect pseudorabies virus in clinical samples is provided.

A doença de Aujeszky, também conhecida como pseudo-raiva, causa perdas econômicas graves na indústria suína e afeta a criação de suínos em todo o mundo. O procedimento de diagnóstico convencional é demorado, podendo ocorrer resultados falso-negativos em animais infectados de forma latente. Este estudo apresenta o desenvolvimento, otimização e avaliação de um ensaio de Reação de Polimerase em Cadeia para o diagnóstico da pseudo-raiva. O ensaio baseou-se na amplificação do fragmento genético viral gD altamente conservado. Os produtos da PCR de tamanho esperado foram obtidos a partir de isolados de PRV. Não foram observadas reações inespecíficas quando foram testados herpes-vírus relacionados, outros vírus DNA de suínos e células não infectadas. A sensibilidade analítica estimada do teste foi 1,34 TCID50/50 uL. A análise de homogenatos feitos com tecidos de animais naturalmente infectados mostrou que o método é útil para o diagnóstico rápido da doença no campo, sendo um ensaio rápido, sensível e específico para detectar o vírus da pseudo-raiva em amostras clinicas.

Development of a polymerase chain reaction assay for the detection of pseudorabies virus in clinical samples

Aujeszky's disease, also known as pseudorabies causes severe economic losses in swine industry and affects the pig husbandry all over the world. The conventional diagnostic procedure is time-consuming and false-negative results may occur in submissions from latently infected animals. The development, optimization and evaluation of a polymerase chain reaction (PCR) assay are presented for the diagnosis of pseudorabies infection. This assay was based on the amplification of a highly conserved viral gD gene fragment. PCR products of the expected size were obtained from PRV strains. Non-specific reactions were not observed when a related herpesvirus, other porcine DNA genome viruses and uninfected cells were used to assess PCR. The analytical sensitivity of the test was estimated to be 1.34 TCID50/50 uL. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases. A rapid, sensitive and specific PCR-based diagnostic assay to detect pseudorabies virus in clinical samples is provided.

A doença de Aujeszky, também conhecida como pseudo-raiva, causa perdas econômicas graves na indústria suína e afeta a criação de suínos em todo o mundo. O procedimento de diagnóstico convencional é demorado, podendo ocorrer resultados falso-negativos em animais infectados de forma latente. Este estudo apresenta o desenvolvimento, otimização e avaliação de um ensaio de Reação de Polimerase em Cadeia para o diagnóstico da pseudo-raiva. O ensaio baseou-se na amplificação do fragmento genético viral gD altamente conservado. Os produtos da PCR de tamanho esperado foram obtidos a partir de isolados de PRV. Não foram observadas reações inespecíficas quando foram testados herpes-vírus relacionados, outros vírus DNA de suínos e células não infectadas. A sensibilidade analítica estimada do teste foi 1,34 TCID50/50 uL. A análise de homogenatos feitos com tecidos de animais naturalmente infectados mostrou que o método é útil para o diagnóstico rápido da doença no campo, sendo um ensaio rápido, sensível e específico para detectar o vírus da pseudo-raiva em amostras clinicas.

Development of a polymerase chain reaction assay for the detection of pseudorabies virus in clinical samples.

Aujeszky's disease, also known as pseudorabies causes severe economic losses in swine industry and affects the pig husbandry all over the world. The conventional diagnostic procedure is time-consuming and false-negative results may occur in submissions from latently infected animals. The development, optimization and evaluation of a polymerase chain reaction (PCR) assay are presented for the diagnosis of pseudorabies infection. This assay was based on the amplification of a highly conserved viral gD gene fragment. PCR products of the expected size were obtained from PRV strains. Non-specific reactions were not observed when a related herpesvirus, other porcine DNA genome viruses and uninfected cells were used to assess PCR. The analytical sensitivity of the test was estimated to be 1.34 TCID50/ 50 uL. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases. A rapid, sensitive and specific PCR-based diagnostic assay to detect pseudorabies virus in clinical samples is provided.

A RT-PCR assay for the detection of encephalomycarditis virus infections in pigs.

Encephalomyocarditis virus (EMCV) infections can cause losses in pig farms all over the world. Rapid, sensitive and unequivocal detection of this virus is therefore essential for the diagnosis and control of the disease. An RT-PCR assay was developed, optimized and evaluated for encephalomyocarditis virus detection in organ based on a pair of primers that amplifies a 165 bp DNA fragment from a highly conserved nucleotide region of the viral 3D glycoprotein. PCR products of the expected size were obtained from Cuban EMCV 744/03 strain. Non-specific reactions were not observed when other porcine RNA genome viruses and uninfected cells were used. The analytical sensitivity of the test was estimated to be 2 TCID50/50 µL. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases.

A RT-PCR assay for the detection of encephalomycarditis virus infections in pigs

Encephalomyocarditis virus (EMCV) infections can cause losses in pig farms all over the world. Rapid, sensitive and unequivocal detection of this virus is therefore essential for the diagnosis and control of the disease. An RT-PCR assay was developed, optimized and evaluated for encephalomyocarditis virus detection in organ based on a pair of primers that amplifies a 165 bp DNA fragment from a highly conserved nucleotide region of the viral 3D glycoprotein. PCR products of the expected size were obtained from Cuban EMCV 744/03 strain. Non-specific reactions were not observed when other porcine RNA genome viruses and uninfected cells were used. The analytical sensitivity of the test was estimated to be 2 TCID50/50 mL. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases.

Development of a polymerase chain reaction assay for the detection of pseudorabies virus in clinical samples

Aujeszky's disease, also known as pseudorabies causes severe economic losses in swine industry and affects the pig husbandry all over the world. The conventional diagnostic procedure is time-consuming and false-negative results may occur in submissions from latently infected animals. The development, optimization and evaluation of a polymerase chain reaction (PCR) assay are presented for the diagnosis of pseudorabies infection. This assay was based on the amplification of a highly conserved viral gD gene fragment. PCR products of the expected size were obtained from PRV strains. Non-specific reactions were not observed when a related herpesvirus, other porcine DNA genome viruses and uninfected cells were used to assess PCR. The analytical sensitivity of the test was estimated to be 1.34 TCID50/50 uL. The analysis of tissue homogenate samples from naturally infected animals proved the potential usefulness of the method for a rapid disease diagnosis from field cases. A rapid, sensitive and specific PCR-based diagnostic assay to detect pseudorabies virus in clinical samples is provided.

A doença de Aujeszky, também conhecida como pseudo-raiva, causa perdas econômicas graves na indústria suína e afeta a criação de suínos em todo o mundo. O procedimento de diagnóstico convencional é demorado, podendo ocorrer resultados falso-negativos em animais infectados de forma latente. Este estudo apresenta o desenvolvimento, otimização e avaliação de um ensaio de Reação de Polimerase em Cadeia para o diagnóstico da pseudo-raiva. O ensaio baseou-se na amplificação do fragmento genético viral gD altamente conservado. Os produtos da PCR de tamanho esperado foram obtidos a partir de isolados de PRV. Não foram observadas reações inespecíficas quando foram testados herpes-vírus relacionados, outros vírus DNA de suínos e células não infectadas. A sensibilidade analítica estimada do teste foi 1,34 TCID50/50 uL. A análise de homogenatos feitos com tecidos de animais naturalmente infectados mostrou que o método é útil para o diagnóstico rápido da doença no campo, sendo um ensaio rápido, sensível e específico para detectar o vírus da pseudo-raiva em amostras clinicas.