Foot-and-mouth disease virus: DNA aptamer selection for the 3ABC protein.

Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV), a member of the Picornaviridae family. The 3ABC is a non-structural protein of FMDV, produced during viral replication and absent from inactivated FMD vaccines. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain DNA aptamers specific for 3ABC protein with a view of their application in the FMD diagnosis. Aptamers are usually obtained through SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. In this study, an aptamer (termed FMDV1) was selected by a variation of this technique called Capillary Electrophoresis SELEX (CE-SELEX). The FMDV1 aptamer showed high binding affinity to the 3ABC protein with Kd value in the nano molar range: 22.69 ± 1.79 nM. The FMDV1 aptamer binding to 3ABC was significantly higher when compared with the BSA protein, used as control, demonstrating its specificity.

DNA aptamers selection for Staphylococcus aureus cells by SELEX and Cell-SELEX.

BACKGROUND: Staphylococcus aureus is the most common bacteria found in skin, soft tissues, bone, and bone prostheses infections. The aim of this study was to select DNA aptamers for S. aureus to be applied in the diagnosis of bacteria. METHODS AND RESULTS: We used SELEX (Systematic Evolution of Ligands by EXponencial Enrichment) for peptidoglycan followed by cell-SELEX with S. aureus cells as target. Four sequences showed significantly higher binding to S. aureus distinguishing it from the control cells of other significant microbial species: Escherichia coli, Candida albicans, Streptococcus pyogenes and Streptococcus pneumoniae. In particular, ApSA1 (Kd = 62.7 ± 5.6 nM) and ApSA3 (Kd = 43.3 ± 3.0 nM) sequences combined high affinity and specificity for S. aureus, considering all microorganisms tested. CONCLUSIONS: Our results demonstrated that these aptamers were able to identify peptidoglycan in the S. aureus surface and have great potential for use in the development of radiopharmaceuticals capable to identify S. aureus infectious foci, as well as in other aptamer-based methodologies for bacteria diagnosis.

Detection of mixed Leishmania infections in dogs from an endemic area in southeastern Brazil.

In Brazil, the visceral leishmaniasis (VL) is caused by Leishmania infantum, while the tegumentary leishmaniasis (TL) etiological agents are mainly Leishmania braziliensis and Leishmania amazonensis. The canine visceral leishmaniasis (CVL) diagnosis is an important step of the VL control program in Brazil, which involves the elimination of infected dogs, the main urban VL reservoirs. The current serology-based diagnostic tests have shown cross-reactivity between these three species, whereas molecular diagnosis allows high sensitivity and specie identification. In the present study, 349 dogs of the metropolitan region of Belo Horizonte (Minas Gerais state) were screened by conjunctival swab and the samples analyzed by ITS-1 nested PCR. Thirty dogs (8.5%) tested positive. The RFLP of amplicons using HaeIII demonstrated that 17/30 samples presented a banding pattern compatible with L. infantum, 4/30 matched with L. amazonenis, 1/30 with L. braziliensis and 8/30 showed a mixed infection pattern. The samples that were distinct of L. infantum or presented a mixed pattern were submitted to RFPL with HaeIII and RsaI enzymes that confirmed the mixed pattern. Such patterns were also confirmed by Sanger Sequencing. The results pointed eight dogs with mixed infections and the establishment of TL causing species in the Belo Horizonte dog population. These findings highlight the need for more comprehensive epidemiological studies, since the TL transmission profile might be changing. This study also shows the potential of the ITS1-nPCR associated with RFLP for the proper Leishmania diagnosis and typing in the dog population.

Mouse Immunization with Radioattenuated Yeast Cells of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America, and currently there is no effective vaccine. The present chapter describes the methodology to obtain radioattenuated yeast cells of P. brasiliensis and a protocol to evaluate protective response elicited by this immunogen in experimental paracoccidioidomycosis. The radioattenuated yeast provides a valuable tool for immunological studies in experimental paracoccidioidomycosis and vaccine research.

Evaluation of conjunctival swab as a mass-screening tool for molecular diagnosis of canine visceral leishmaniasis.

The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil once the dog is the main reservoir host of the disease. The aim of this study was to evaluate the conjunctival swab (CS) as a mass-screening tool for CVL molecular diagnosis in an endemic area classified as priority for the Brazilian Ministry of Healthy for surveillance action. A total of 1350 domiciled dogs were screened. The animals were evaluated by serological tests (enzyme-linked immunosorbent assay (ELISA) as screening and immunofluorescence antibody test (IFAT) for confirmation) and by CS associated to real-time PCR, using primers addressed to kinetoplast DNA (kDNA) minicircles and SYBR Green. Canine ß-globin gene amplification was used to evaluate the sample DNA integrity. A subgroup of 484 animals was also submitted to clinical evaluation. Among the 1350 dogs screened, 369 (27.3%) were positive by CS real-time PCR and 126 (9.3%) tested positive by ELISA. Thirty-one percent (39/126) of the ELISA-positive dogs were confirmed by IFAT. CS real-time PCR was able to detect infection in dogs independently of the symptomatology degree (p > 0.05), while ELISA was more sensitive in the group of dogs that present three or more clinical signs related to CVL. The results demonstrated that CS real-time PCR was able to detect a higher number of infected dogs than ELISA and that the prevalence of canine infections has been underestimated by the serological assays. The use of sensitive molecular diagnostic methods like CS real-time PCR, mainly in endemic areas, could greatly contribute to disease control.

Detection of Leishmania infantum in 4 different dog samples by real-time PCR and ITS-1 nested PCR.

The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil, which involves the elimination of infected dogs, the main animal reservoir host of the disease. The aim of the present study was to evaluate a sensitive real-time PCR method for Leishmania infantum detection in 4 different clinical samples of dogs, including the noninvasive conjunctival swab (CS) sample. The results of real-time PCR were compared with those obtained using internal transcribed spacer 1 nested PCR. Animals were divided into 2 groups based on the absence or presence of CVL clinical sings. The CS associated with real-time PCR, using primers addressed to kinetoplast DNA minicircles, was able to detect L. infantum infection in 96.7% of dogs without clinical signs and in 100% of the symptomatic animals, demonstrating the importance of these procedures for diagnosing CVL.

Nasal, oral and ear swabs for canine visceral leishmaniasis diagnosis: new practical approaches for detection of Leishmania infantum DNA.

BACKGROUND: The aim of this study was to evaluate the potential use of nasal, oral, and ear swabs for molecular diagnosis of canine visceral leishmaniasis (CVL) in an endemic urban area in Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Sixty-two naturally infected and ten healthy dogs were enrolled in this study. Bone marrow aspirates, peripheral blood, skin biopsy, and conjunctival, nasal, oral, and ear swabs were collected. All samples, except blood, were submitted to conventional PCR (cPCR) and quantitative real time PCR (qPCR) to detect and quantify Leishmania infantum DNA, respectively. All dogs were submitted to thorough clinical analysis and were included based on a combination of serological (ELISA immunoassay and immunofluorescent antibody test) and parasitological methods. The cPCR positivity obtained from nasal swab samples was 87% (54/62), equivalent to those from other samples (P>0.05). Positive results were obtained for 79% (22/28) in oral swabs and 43% (12/28) in ear swab samples. A significant difference was observed between these data (P=0.013), and the frequency of positive results from oral swab was equivalent to those from other samples (P>0.05). The use of ear swab samples for cPCR assays is promising because its result was equivalent to skin biopsy data (P>0.05). The qPCR data revealed that parasite loads in mucosal tissues were similar (P>0.05), but significantly lower than the parasite burden observed in bone marrow and skin samples (P<0.05). CONCLUSIONS: Nasal and oral swab samples showed a high potential for the qualitative molecular diagnosis of CVL because their results were equivalent to those observed in samples collected invasively. Considering that mucosae swab collections are painless, noninvasive, fast and practical, the combination of these samples would be useful in massive screening of dogs. This work highlights the potential of practical approaches for molecular diagnosis of CVL and human leishmaniasis infections.

Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantum DNA in an endemic urban area in Brazil.

BACKGROUND: We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively. METHODOLOGY/PRINCIPAL FINDINGS: Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups. CONCLUSIONS: The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.

Gamma radiation effects on Sporothrix schenckii yeast cells.

Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii. Zoonotic transmission to man can occur after scratches or bites of animals, mainly cats. In this study, the gamma radiation effects on yeast of S. schenckii were analyzed with a view of developing a radioattenuated vaccine for veterinary use. The cultures were irradiated at doses ranging from 1.0 to 9.0 kGy. The reproductive capacity was measured by the ability of cells to form colonies. No colonies could be recovered above 8.0 kGy, using inocula up to 10(7) cells. Nevertheless, yeast cells irradiated with 7.0 kGy already were unable to produce infection in immunosuppressed mice. Evaluation by the FungaLight™ Kit (Invitrogen) indicated that yeast cells remained viable up to 9.0 kGy. At 7.0 kGy, protein synthesis, estimated by the incorporation of [L-(35)S] methionine, continues at levels slightly lower than the controls, but a significant decrease was observed at 9.0 kGy. The DNA of 7.0 kGy irradiated cells, analyzed by electrophoresis in agarose gel, was degraded. Cytoplasmic vacuolation was the main change verified in these cells by transmission electron microscopy. The dose of 7.0 kGy was considered satisfactory for yeast attenuation since irradiated cells were unable to produce infection but retained viability, metabolic activity, and morphology.

PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples.

The efficacy of conjunctival swab (CS) as a sampling method for visceral leishmaniasis (VL) diagnosis by PCR of asymptomatic dogs was evaluated. The CS was compared to blood samples (B) and skin biopsies (SB), two less invasive samples potentially useful for massive screening of dogs. Thirty asymptomatic dogs, with serological and parasitological positive tests, were used. The samples were analyzed by two PCR methods: kDNA PCR-hybridization and ITS-1 nPCR. The DNA sample volume used was of 1.0 microL and 10.0 microL respectively. Using CS samples the kDNA PCR-hybridization was able to detected parasite DNA in 24/30 dogs (80%) using the right conjunctiva (RC) and 23/30 dogs (76.6%) with the left conjunctiva (LC), 17/30 dogs (56.7%) by means of SB and 4/30 dogs (13.3%) with B. The CS positivity obtained combining RC and LC results was of 90% (27/30 dogs). The assay of CS samples by ITS-1 nPCR revealed that 25/30 dogs (83.3%) were positive when using RC and 20/30 dogs (66.6%) were positive when using LC. Via the same method 15/30 dogs (50.0%) were positive by SB and 17/30 dogs (56.7%) with B. The CS positivity obtained by ITS-1 nPCR combining RC and LC was of 83.3%. The CS positivities for RC and LC were significantly higher (p<0.05) than SB and B for kDNA PCR-hybridization method. Statistical difference in relation to SB and B was verified by ITS-1 nPCR only for RC. The kDNA PCR-hybridization and ITS-1 nPCR methods showed similar sensitivities for CS and SB samples. On the other hand, for blood samples, the positivity of ITS-1 nPCR was significantly higher than the one obtained by the kDNA PCR-hybridization, indicating that sensitivity of PCR methods can vary according to the biological sample examined. Our results showed that CS was suitable to detect Leishmania DNA in asymptomatic animals when comparing to other low-invasive samples. The CS sensitivities obtained in this study were similar to the ones observed in other studies for VL diagnosis in symptomatic dogs. We concluded that the use of CS for regular screenings of dogs by PCR should be considered.

Mice immunization with radioattenuated yeast cells of Paracoccidiodes brasiliensis: influence of the number of immunizations.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. Up to the moment no vaccine has been reported. The aim of this study was to evaluate the influence of the number of immunizations on the protection elicited by radioattenuated yeast cells of P. brasiliensis. BALB/c mice were divided into two groups that were immunized once (Group 1) or twice (Group 2), respectively. In each group, mice were divided into sub-groups that were challenged 30, 45, or 60 days after the second immunization. Organ colony-forming units (CFUs) was determined 90 days post-challenge. A significant reduction in CFUs recovery was verified in both groups, but it was higher in Group 2. Histologic alterations were observed only in Group 1. The cytokines IL-4, IL-10, and IFN-gamma were produced in mice of Group 1. In Group 2, only IFN-gamma was significantly detected. IgG2a predominance relative to IgG1 was also observed in Group 2. Altogether, our results indicated that mice immunized once developed a mixed Th1/Th2 response, which was less efficient in the infection control, while a trend to a Th1 pattern was obtained with two immunizations, promoting optimal elimination of P. brasiliensis yeast cells from mice tissues.

Comparison of PCR methods for diagnosis of canine visceral leishmaniasis in conjunctival swab samples.

Four PCR assays for detection of Leishmania DNA in conjunctival swab samples were compared. All methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or seminested). Two methods (kDNA PCR-hybridization and kDNA snPCR) used primers targeted to the minicircles of kinetoplast DNA (kDNA) and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of ribosomal rRNA genes. kDNA PCR-hybridization was positive for 22/23 dogs (95.6%) and for 40/46 samples (86.9%), considering the right and the left conjunctivas. kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The ITS-1 nPCR and LnPCR were both able to detect the parasites in 17/23 dogs (73.9%) and 29/46 (63%) and 30/46 (65.2%) samples, respectively. The positivities of the kDNA based methods were significantly higher; however the choice of the best method will depend on the kind of information required with the diagnosis.

Evaluation of the conjunctival swab for canine visceral leishmaniasis diagnosis by PCR-hybridization in Minas Gerais State, Brazil.

The visceral leishmaniasis (VL) in Brazil is caused by Leishmania chagasi (L. infantum) and dogs are considered to be the main domestic reservoir. The epidemiological control involves the elimination of infected dogs. Therefore, the correct diagnosis is very important in order to avoid the disease transmission or unnecessary culling of dogs. Recently, an antileishmanial vaccine for dogs was licensed and commercialized in Brazil. Vaccinated dogs test positive in the conventional serological tests, rendering these assays useless for control programs involving vaccinated animals. The Polymerase Chain Reaction (PCR) is an attractive alternative to the diagnosis in this context; but non-invasive samplings have great importance because they are simpler, painless and less resisted by dog-owners. This study aimed at evaluating the conjunctival swab (CS) for canine VL diagnosis by the PCR-hybridization procedure. Two groups of 23 seropositive dogs were used. CS samples were obtained from both eyes of each animal. The DNA extraction from CS was performed by the phenol chloroform method in group 1 and by boiling in group 2. In addition, blood was collected from each animal so that 30 microl was spotted onto filter paper (FP) and 1.0 ml was treated to obtain the buffy coat (BC). The DNA extraction from the BC and FP was accomplished by identical procedures in both groups using commercial kits. The PCR positivities for both groups 1 and 2 were, respectively: 73.9% and 52.2% (CS), 13% and 30.4% (BC), 8.7% and 17.4% (FP). The hybridization step increased the positivities for: 91.3% and 65.2% (CS), 21.7% and 34.8% (BC), 30.4% and 43.5% (FP), respectively. The highest frequency of positivity was obtained by the association between CS and DNA extraction by phenol chloroform. This approach can be very useful for diagnosis of canine leishmaniasis and could be applied to the follow-up and regular screening of vaccinated dogs.

Immunization with radioattenuated yeast cells of Paracoccidioides brasiliensis induces a long lasting protection in BALB/c mice.

Paracoccidioides brasiliensis is the fungus agent of paracoccidioidomycosis, a chronic systemic disease prevalent in Latin America. The aim of the present work was to evaluate the protection elicited by the immunization of BALB/c mice with radioattenuated yeast cells of P. brasiliensis. The immunization promoted a long lasting protection against highly infective yeast forms of P. brasiliensis. A 99.5% decrease in CFUs recovery was verified 90 days post challenge. At the same time the levels of IgG2a and IFN-gamma were high while a very low production of IL-10 and IL-5 was verified, suggesting that a Th1 pattern was dominant. This work shows the potential of radioattenuated yeast cells for the development of vaccines against fungi infections.

Ultrastructural changes in Paracoccidioides brasiliensis yeast cells attenuated by gamma irradiation.

Paracoccidioides brasiliensis is a thermally dimorphic fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans with high prevalence in Latin America. Until now no vaccine has been reported. Ionizing radiation can be used to attenuate pathogens for vaccine development and we have successfully attenuated yeast cells of P. brasiliensis by gamma irradiation. The aim of the present study was to examine at ultrastructural level the effects of gamma irradiation attenuation on the morphology of P. brasiliensis yeast cells. Paracoccidioides brasiliensis (strain Pb-18) cultures were irradiated with a dose of 6.5 kGy. The irradiated cells were examined by scanning and also transmission electron microscopy. When examined 2 h after the irradiation by scanning electron microscopy, the 6.5 kGy irradiated cells presented deep folds or were collapsed. These lesions were reversible since when examined 48 h after irradiation the yeast had recovered the usual morphology. The transmission electron microscopy showed that the irradiated cells plasma membrane and cell wall were intact and preserved. Remarkable changes were found in the nucleus that was frequently in a very electrondense form. An extensive DNA fragmentation was produced by the gamma irradiation treatment.

Leishmania (Viannia) braziliensis is the prevalent species infecting patients with tegumentary leishmaniasis from Mato Grosso State, Brazil.

The frequency of Leishmania (Viannia) braziliensis infection among patients of Mato Grosso, Brazil was estimated by polymerase chain reaction-PCR, DNA hybridization and by isoenzyme electrophoresis. Analysis of DNA polymorphism was carried out using random amplified polymorphic DNA-PCR (RAPDPCR) with five different primers. The patients were attended from May 1997 to February 1998 at the Reference Ambulatory for American Tegumentary Leishmaniasis at Júlio Müller University Hospital of the Federal University of Mato Grosso, Brazil. In a first screening by PCR and DNA hybridization 94.1% of 68 patients, from whom parasites were isolated in culture medium, were found to be infected with species of the Le. braziliensis complex. Only four patients (5.9%) were infected with species of Le. mexicana complex. Thirty-three samples of Le. braziliensis complex and three of Le. mexicana complex were typed by isoenzyme analysis as Le. (V.) braziliensis sensu stricto and Le. (Leishmania) amazonensis, respectively. The predominant species was Le. (V.) braziliensis, although most of the patients of this study came from the northern area of Mato Grosso, which is part of the Amazonian region of Brazil, where other known species of both subgenus Viannia (Le. braziliensis complex) and Leishmania (Le. mexicana complex) are present. The results of RAPD showed higher genetic variability among the Le. (V.) braziliensis samples from Mato Grosso. The importance of these results concerning the taxonomic status of New World Leishmania, and their implications for both clinical and epidemiological data is discussed.

Paracoccidioides brasiliensis: attenuation of yeast cells by gamma irradiation.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America, and currently there is no effective vaccine. The aim of this study was to attenuate the yeast form of P. brasiliensis by gamma irradiation for further studies on vaccine research. Paracoccidioides brasiliensis (strain Pb 18) cultures were irradiated at doses between 0.5 and 8.0 kGy. After each dose the viability, reproductive ability and protein metabolism were evaluated. The comparison between the antigenic profile of irradiated and control yeast was made by Western blot and the virulence evaluated by the inoculation in C(57)Bl/J6 mice. At 6.5 kGy the yeast lost its reproductive capacity. The viability and the incorporation of [L-(35)S]-methionine were the same in control and up to 6.5 kGy irradiated cells, but 6.5 kGy-irradiated yeast secreted 40% less proteins. The Western blot profile was clearly similar in control and 6.5 kGy-irradiated yeast. No colony-forming unit (CFU) could be recovered from the tissues of the mice infected with the radioattenuated yeast. We concluded that for P. brasiliensis yeast it is possible to find a dose in which the pathogen loses its reproductive ability and virulence, while retaining its viability, metabolic activity and the antigenic profile.