Prevalence of Bartonella henselae and Bartonella clarridgeiae in cats in the south of Brazil: a molecular study

Bartonella spp are the causative agent of cat scratch disease in humans. Cats are the natural reservoir of these bacteria and may infect humans through scratches, bites or fleas. Blood samples from 47 cats aged up to 12 months were collected for this study. All animals were lodged in municipal animal shelters in the Vale do Sinos region, Rio Grande do Sul, Brazil. Bartonella spp were detected by genus-specific polymerase chain reaction (PCR) and when the PCR was positive, the species were determined by DNA sequencing. A Giemsa-stained blood smear was also examined for the presence of intraerythrocytic elements suggestive of Bartonella spp infection. Phylogenetic analysis was also performed for all positive samples. Using molecular detection methods, Bartonella spp were detected in 17.02% (8/47) of the samples. In seven out of eight samples confirmed to be positive for Bartonella spp, blood smear examination revealed the presence of intraerythrocytic elements suggestive of Bartonella spp. Phylogenetic analysis characterized positive samples as Bartonella henselae (5) or Bartonella clarridgeiae (3). To the best of our knowledge, this is the first molecular study demonstrating the presence of Bartonella spp in cats from the Southern Region of Brazil.

Molecular identification of Rickettsia parkeri infecting Amblyomma triste ticks in an area of Argentina where cases of rickettsiosis were diagnosed

Specimens of the hard tick Amblyomma triste were found infected with Rickettsia parkeri in an area of Argentina (General Lavalle, Buenos Aires Province) where cases of human illness attributed to this microorganism have been reported. Molecular detection of R. parkeri was based on polymerase chain reactions that amplify a ca. 400-bp fragment of the 23S-5S intergenic spacer and a ca. 500-bp fragment of the gene encoding a 190-kDa outer membrane protein. Three (6.97%) of 43 A. triste ticks were determined to be positive for R. parkeri. These results provide strong evidence that A. triste is the vector of R. parkeri in the study area. The findings of this work have epidemiological relevance because human parasitism by A. triste ticks has been frequently recorded in some riparian areas of Argentina and Uruguay and new cases of R. parkeri rickettsiosis might arise in the South American localities where humans are exposed to the bites of this tick species.

Detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection

Viruses are the major contributors to the morbidity and mortality of upper and lower acute respiratory infections (ARIs) for all age groups. The aim of this study was to determine the frequencies for a large range of respiratory viruses using a sensitive molecular detection technique in specimens from outpatients of all ages with ARIs. Nasopharyngeal aspirates were obtained from 162 individuals between August 2007-August 2009. Twenty-three pathogenic respiratory agents, 18 respiratory viruses and five bacteria were investigated using multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay (IIF). Through IIF, 33 (20.4%) specimens with respiratory virus were recognised, with influenza virus representing over half of the positive samples. Through a multiplex real-time RT-PCR assay, 88 (54.3%) positive samples were detected; the most prevalent respiratory viral pathogens were influenza, human rhinovirus and respiratory syncytial virus (RSV). Six cases of viral co-detection were observed, mainly involving RSV. The use of multiplex real-time RT-PCR increased the viral detection by 33.9% and revealed a larger number of respiratory viruses implicated in ARI cases, including the most recently described respiratory viruses [human bocavirus, human metapneumovirus, influenza A (H1N1) pdm09 virus, human coronavirus (HCoV) NL63 and HCoV HKU1].

Orange rust in sugarcane: molecular identification in Rio de Janeiro State

Sugarcane (Saccharum spp.) cultivation is one of the major agricultural activities in the Brazilian states. This study aimed to molecularly identify the pathogen associated with rust in sugarcane cultivars in the state of Rio de Janeiro and to suggest a control strategy. Among the 14 PCR-tested cultivars, Puccinia kuehnii infection was identified for RB947520, RB92606, RB835486, RB72454, SP89-11I5, SP83-2847, both from infected leaf sample and from urediniospores. Puccinia kuehnii was not detected by PCR for the cultivars RB955971, RB951541, RB92579, RB867515, RB855536, SP91-1049, SP80-3280, SP80-1816. This is the first molecular detection of this fungus in the state of Rio de Janeiro for six of the 14 analyzed cultivars.

Molecular survey of porcine teschovirus, porcine sapelovirus, and enterovirus G in captive wild boars (Sus scrofa scrofa) of Paraná state, Brazil

AbstractPorcine teschovirus (PTV), porcine sapelovirus (PSV), and enterovirus G (EV-G) are infectious agents specific to pig host species that are endemically spread worldwide. This study aimed to investigate the natural infection by these porcine enteric picornaviruses in wild boars (Sus scrofa scrofa) of Paraná state, Brazil, and to evaluate peccaries (Pecari tajacu and Tayassu pecari) as alternative host species for these viruses. Fecal samples (n=36) from asymptomatic wild boars (n=22) with ages ranging from 2 to 7 months old (young, n=14) and 2 to 4 years old (adult, n=8) and from peccaries (6 to 8 months old, n=14) were collected from a farm and a zoo, respectively, both located in Paraná state. Reverse transcription-polymerase chain reaction (RT-PCR) and nested-PCR (n-PCR) assays targeting the 5'non-translated region of the virus genome were used for screening the viruses. Porcine enteric picornaviruses were detected in 12 out of the 22 wild boar fecal samples. According to each of the viruses, EV-G was most frequently (11/22, 50%) detected, followed by PTV (10/22, 45.5%) and PSV (4/22, 18.2%). Regarding the age groups, young wild boars were more frequently (9/14, 64.3%) infected with PTV, PSV, and EV-G than adult animals (3/8, 37.4%). One n-PCR amplified product for each of the viruses was submitted to sequencing analysis and the nucleotide sequences were compared with the related viruses, which showed similarities varying from 97.7% to 100% for PTV, 92.4% to 96.2% for PSV, and 87.1% to 100% for EV-G. Peccaries tested negative for the viruses and in this study they did not represent infection reservoirs. This study is the first to report the molecular detection of PTV, PSV, and EV-G from captive wild boars in a South American country and the first to screen peccaries as alternative host species for porcine enteric picornavirus.

Use of the polymerase chain reaction for detection of Fusarium graminearum in bulgur wheat

The detection of mycotoxigenic fungi in foodstuff is important because their presence may indicate the possible associated mycotoxin contamination. Fusarium graminearum is a wheat pathogen and a producer of micotoxins. The polymerase chain reaction (PCR) has been employed for the specific identification of F. graminearum. However, this methodology has not been commonly used for detection of F. graminearum in food. Thus, the objective of the present study was to develop a molecular methodology to detect F. graminearum in commercial samples of bulgur wheat. Two methods were tested. In the first method, a sample of this cereal was contaminated with F. graminearum mycelia. The genomic DNA was extracted from this mixture and used in a F. graminearum specific PCR reaction. The F. graminearum species was detected only in samples that were heavily contaminated. In the second method, samples of bulgur wheat were inoculated on a solid medium, and isolates having F. graminearum culture characteristics were obtained. The DNA extracted from these isolates was tested in F. graminearum specific PCR reactions. An isolate obtained had its trichothecene genotype identified by PCR. The established methodology could be used in surveys of food contamination with F. graminearum.

Sensibility of the PCR technique in the detection of Stenocarpella sp. associated with maize seeds

Maize seeds, infected by Stenocarpella species, are important sources of inoculum for the introduction and dissemination of stalk and ear rot and macrospore leaf spot diseases. The use of healthy seeds is an important strategy for the preventive control of these diseases. However, one of the difficulties in the health quality control programs for maize seeds is the availability of a reliable and quick method for detecting these fungi during routine seed analyses. Therefore, the objective of the present study was to investigate the possibility of using the PCR technique as an alternative method for accurately detecting these pathogens in maize seed samples. Maize seeds were kept in contact with S. maydis colonie developed in PDA media containing mannitol at -1.4 MPa for 72 h. The seed samples used in this study were prepared with infected seeds at incidences of 100, 20, 10, 2, 1 and zero %.The primers used were able to detect S. maydis fungi in association with seeds with a maximum of 2% , however those primers were not able to differentiate between the two species.

Detecting uterine cervical cancer cells using molecular biomarkers

Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie. Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire.

Biochemical and molecular investigations on Salmonella serovars from seafood

The present investigation was envisaged to determine the prevalence and identify the different Salmonella serovar in seafood from Cochin area. Though, the distribution of Salmonella serovars in different seafood samples of Cochin has been well documented, the present attempt was made to identify the different Salmonella serovars and determine its prevalence in various seafoods. First pan of this investigation involved the isolation and identification of Salmonella strains with the help of different conventional culture methods. The identified isolates were used for the further investigation i.e. serotyping, this provides the information about the prevalent serovars in seafood. The prevalent Salmonella strains have been further characterized based on the utilization of different sugars and amino acids, to identify the different biovar of a serovar.A major research gap was observed in molecular characterization of Salmonella in seafood. Though, previous investigations reported the large number of Salmonella serovars from food sources in India, yet, very few work has been reported regarding genetic characterization of Salmonella serovars associated with food. Second part of this thesis deals with different molecular fingerprint profiles of the Salmonella serovars from seafood. Various molecular typing methods such as plasmid profiling, characterization of virulence genes, PFGE, PCR- ribotyping, and ERIC—PCR have been used for the genetic characterization of Salmonella serovars.The conventional culture methods are mainly used for the identification of Salmonella in seafood and most of the investigations from India and abroad showed the usage of culture method for detection of Salmonella in seafood. Hence, development of indigenous, rapid molecular method is most desirable for screening of Salmonella in large number of seafood samples at a shorter time period. Final part of this study attempted to develop alternative, rapid molecular detection method for the detection of Salmonella in seafood. Rapid eight—hour PCR assay has been developed for detection of Salmonella in seafood. The performance of three different methods viz., culture, ELISA and PCR assays were evaluated for detection of Salmonella in seafood and the results were statistically analyzed. Presence of Salmonella cells in food and enviromnental has been reported low in number, hence, more sensitive method for enumeration of Salmonella in food sample need to be developed. A quantitative realtime PCR has been developed for detection of Salmonella in seafood. This method would be useful for quantitative detection of Salmonella in seafood.

Análisis de muestras de orina para la detección molecular de enfermedades infecciosas. Aplicación en la identificación de citomegalovirus humano

La implementación de metodologías de biología molecular como la reacción en cadena de la polimerasa (PCR), ha permitido la realización de diagnósticos sensibles y específicos para múltiples enfermedades, dentro de las cuales son de gran interés las infecciosas. Hasta hoy, los métodos de identificación se basan principalmente en cultivos y serología por su sensibilidad y especificidad, pero consumen tiempo y dinero. Las muestras de orina se han constituido en una alternativa no invasiva de obtención de ADN para la realización de análisis de biología molecular. Metodología: Implementación de una estrategia para la obtención de ADN a partir de muestras de orina. Las muestras fueron tomadas de niños de guardería, para documentar la presencia o no de inhibidores de PCR a través de la amplificación de genes de Citomegalovirus humano (CMVH). Resultados: En el 27,1% de las muestras analizadas se evidenció amplificación específica para CMVH, no se encontraron diferencias significativas en la presencia del virus en los tres estratos, pero sí en la intensidad de las bandas. Conclusión: Se verificó la ausencia de inhibidores de PCR mediante la amplificación del gen de la B-globina. Se estandarizó una metodología molecular para la identificación de CMVH, la cual puede ser aplicada

Molecular approaches for eco-epidemiological studies of Paracoccidioides brasiliensis

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

Molecular characterization of Hepatozoon canis in dogs from Colombia

Hepatozoonosis is a tick-borne disease whose transmission to dogs occurs by ingestion of oocysts infected ticks or feeding on preys infested by infected ticks. Until now, there is no previous report of molecular characterization of Hepatozoon sp. in dogs from Colombia. EDTA blood samples were collected from 91 dogs from central-western region of Colombia (Bogota, Bucaramanga, and Villavicencio cities) and submitted to 18S rRNA Hepatozoon sp. PCR and blood smears confection. Phylogenetic analysis was used to access the identity of Hepatozoon species found in sampled dogs. From 91 sampled dogs, 29 (31.8%) were positive to Hepatozoon sp. (25 dogs were only positive in PCR, 1 was positive only in blood smears, and 3 were positive in both blood smears and PCR). After sequencing, the found Hepatozoon sp. DNA showed 100% of identity with Hepatozoon canis DNA isolates. The phylogenetic tree supported the identity of the found Hepatozoon sp. DNA, showing that the isolates from Colombia were placed in the same clade than other H. canis isolates from Venezuela, Spain, and Taiwan. This is the first molecular detection of H. canis in dogs from Colombia.

Detection of Paracoccidioides spp. in environmental aerosol samples

Taking into account that paracoccidioidomycosis infection occurs by inhalation of the asexual conidia produced by Paracoccidioides spp. in its saprobic phase, this work presents the collection of aerosol samples as an option for environmental detection of this pathogen, by positioning a cyclonic air sampler at the entrance of armadillo burrows. Methods included direct culture, extinction technique culture and Nested PCR of the rRNA coding sequence, comprising the ITS1-5.8S-ITS2 region. In addition, we evaluated one armadillo (Dasypus novemcinctus) as a positive control for the studied area. Although the pathogen could not be isolated by the culturing strategies, the aerosol sampling associated with molecular detection through Nested PCR proved the best method for discovering Paracoccidioides spp. in the environment. Most of the ITS sequences obtained in this investigation proved to be highly similar with the homologous sequences of Paracoccidioides lutzii from the GenBank database, suggesting that this Paracoccidioides species may not be exclusive to mid-western Brazil as proposed so far. © 2013 ISHAM.

Detecção parasitológica, molecular e sorológica de Erlichia canis e babesia canis em cães da região Centro-Oriental da Colômbia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Detecção molecular e sorológica de Ehrlichia canis e Babesia canis em felídeos selvagens brasileiros mantidos em cativeiro

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