Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%)and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.

Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP)

Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP) assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.

A 28-fold increase in secretory protein synthesis is associated with DNA puff activity in the salivary gland of Bradysia hygida (Diptera, Sciaridae)

When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that these polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7, C5, C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time (Spradling AC and Mahowald AP (1980) Proceedings of the National Academy of Sciences, USA, 77: 1096-1100).

Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction

We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 µg = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25oC) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

5-Bromo-2’-deoxyuridine induces visible morphological alteration in the DNA puffs of the anterior salivary gland region of Bradysia hygida (Diptera, Sciaridae)

5-Bromo-2’-deoxyuridine (BrdUrd) has long been known to interfere with cell differentiation. We found that treatment ofBradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages.

Use of real-time PCR to evaluate two DNA extraction methods from food

The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R²) demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct) values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.

Identification of 'Ubá' mango tree zygotic and nucellar seedlings using ISSR markers

Polyembryonic seeds are characterized by the development of over one embryo in the same seed, which can be zygotic and nucellar. The objective of this work was to identify the genetic origin, whether zygotic or nucellar, of seedlings of polyembryonic seeds of 'Ubá' mango tree using ISSR markers, and relating them with the vigor of the seedlings. Thus, mangos were harvested in Visconde do Rio Branco (accession 102) and Ubá (accessions 112, 138, 152 and 159), whose seeds were germinated in plastic trays filled with washed sand. Fifty days after sowing, seedlings from five seeds of each one of the accessions 102, 112, 138, 159 and from 10 seeds of the accession 152, were analyzed. These sseedlings were characterized and evaluated for plant height, stem circumference and mass of fresh aerial part and the most vigorous seedling was the one displaying at least two of these traits higher than the other seedlings from seed. Leaves were collected for genomic DNA extraction, which was amplified using seven ISSR primers previously selected based on the amplification profile and considering the number and resolution of fragments. Zygotic seedlings were found in 18 seeds, which were the most vigorous in six seeds. The results evidenced the existence of genetic variability in orchards using seedlings grown from seeds, because the farmer usually uses the most vigorous ones, assuming that this is of nucellar origin. These results also indicate that the most vigorous seedling are not always nucellar, inasmuch as of 20% of the total seeds evaluated, the zygotic seedling was the most vigorous.

Detecção de DNA de Leishmania braziliensis em pacientes de leishmaniose tegumentar americana

Foi realizado diagnóstico para leishmaniose tegumentar americana a partir de sangue de pacientes residentes em dois municípios endêmicos do estado de Pernambuco. O DNA de 119 amostras de sangue foi extraído e submetido a reação em cadeia da polimerase. Utilizaram-se primers do minicírculo do DNA do cinetoplasto (kDNA) de Leishmania braziliensis, circulante em Pernambuco, cuja seqüência-alvo gera um fragmento de 750 pares de bases. No total 58 (48,7%) indivíduos apresentaram amplificação positiva e 61 (51,3%) negativa. Das amostras positivas para a PCR, 37 (≅ 64%) pertenciam a indivíduos tratados e sem lesão. Conclui-se que a técnica de PCR é eficaz para identificar o DNA de leishmânia em material de biópsias e em sangue venoso.

Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/Anti-HBe system, viral dna polymerase and HBV-DNA

The detection of HBV-DNA in serum by molecular hybridization is the most sensitive and specific marker of replication and infectivity of hepatitis B virus and currently is proposed as a routine diagnostic technique in the follow-up of HBV - related diseases. Comparing different techniques already described, we found that direct spotting of serum samples on nitrocellulose membranes under vacuum filtration, followed by denaturing and neutralizing washes is more practical, simple, sensible and reproducible. DNA polymerase assay using phosphonoformic acid as specific viral inhibitor has shown 86.8% of concordance with HBV-DNA detection, and so, it is an useful alternative in the follow-up of hepatitis B chronic patients. We found 19.2% HBeAg positive samples with no other markers of viral replication and no anti-HBe positive sample had detectable HBV-DNA. Discordance between the 2 systems have been extensively described, and we confirm this for the first time in our country. Molecular biological techniques are essential to determine the replication status of chronic hepatitis B patients.

Potential infectivity of blood from HBsAG asymptomatic carriers due to the presence of HBV-DNA and comparison with other markers of hbv infection

Serum samples from 356 HBsAg positive asymptomatic carriers, which were titrated by reverse passive hemagglutination, were analysed for the presence of HBV-DNA, HBsAg and IgM anti-HBc. The samples were divided in three classes, according to the titers of HBsAg and IgM anti-HBc and the distribution of HBV-DNA and HBsAg among these classes was studied. In the high titer class of HBsAg, 65% of samples have one or both markers against only 19% in the low titer class. From the total of 356 samples, 121 gave positive results for IgM anti-HBc (33.9%). From these, 38.9% of HBV-DNA and 47.9% of HBeAg were observed, whereas in samples with absence of IgM anti-HBc, 18.3% and 16.6% were respectively found. A higher frequency of agreement between all these markers was found in the class of high titers of HBsAg; however, HBV-DNA was detected in the low titer class of HBsAg and little or no IgM anti-HBc, showing potential blood infectivity even in HBsAg positive borderline samples.

Evolution of subpatent parasitaemia in Trypanosoma cruzi chronically infected mice with the help of a cyclophosphamide amplification transfer assay

We have evaluated the sensitivity of the classical blood subinoculation method, modified through cyclophosphamide treatment of transferred mice, for the detection of occult parasitaemias in Trypanosoma cruzi chronically infected mice. Besides its simplicity, the method was shown to be highly sensitive for both the "chronic" phase parasites (99% of chronic cases were shown to harbour occult parasitaemias) and for the acute phase parasites (T. cruzi could be detected in 53.8% of animals transferred with one Y strain parasite and in 20% of animals transferred with one CL strain parasite). Using acute phase bloodforms, the assay proved to be more sensitive than conventional subinoculation when dealing with the CL, but not the Y strain of the parasite. With the help of this parasite detection tool, we have studied during a one year period, the evolution of subpatent parasitaemias in a group of mice which survived through chemotherapy from lethal acute phase of T. cruzi infection. Cyclophosphamide transfer assay revealed occult parasitaemias in 100% of the chronic animals, nevertheless, continuous and discontinuous patterns of positivity were observed.

Human papillomavirus detection in cervical dysplasias or neoplasias and in condylomata acuminaata by in situ hybridization with biotinylated DNA probes

Specimens from cervical dysplasias or carcinomas and genital condylomata acuminata were retrospectively analysed by in situ hybridization (ISH) with bioti-nylated DNA probes for human papillomavirus (HPV) types 6, 11, 16 and 18. In the control group no case was positive for HPV DNA. In mild/moderate dysplasias, 4 cases (14%) were positive for HPV 6 or 11 and 2 cases (7%), for HPV 16. In the severe dysplasia/in situ carcinoma group, 9 cases (31%) showed presence of DNA of HPV types 16 or 18. Six invasive carcinomas (20%) were positive for HPV type 16 or 18. Among condylomata acuminata, 22 cases (73%) were positive for HPV types 6 or 11. In all ISH-positive cases only one viral type was detected. No correlation between HPV DNA positivity and histological findings of HPV infection was observed. Although less sensitive than some other molecular biology techniques, in situ hybridization with biotinylated DNA probes proved to be simple and useful for detecting and typing HPV in samples routinely received for histopathological analysis.

Immunization aganist hepatitis B in children from endemic zone: evaluation of the antibody response against the DNA recombinant vaccine (Engerix B-20 mcg)

A previous seroepidemiological study in the rural zone of Vargem Alta (ES) SouthEast of Brazil, showed a prevalence of up to 9% of hepatitis B surface antigen (HBsAg) in some areas. One hundred susceptible children aging 1 to 5 years old were selected and immunized with a recombinant DNA hepatitis B vaccine (Smith-Kline 20 mcg) using the 0-1-6 months vaccination schedule. Blood samples were collected at the time of the first vaccine dose (month 0) in order to confirm susceptible individuals and 1,3,6 and 8 months after the first dose , to evaluate the antibody response. Our results showed that two and five months after the second dose, 79% and 88% of children seroconverted respectively, reaching 97% after the third dose. The levels of anti-HBs were calculated in milli International Units/ml (mIU/ml) and demonstrated the markedly increase of protective levels of antibodies after the third dose. These data showed a good immunogenicity of the DNA recombinant hepatitis B vaccine when administered in children of endemic areas.

Detection of hepatitis B virus DNA by the polymerase chain reaction in anti-HBE positive chronic hepatitis B patients

Detection of HBV-DNA by PCR was compared with other serological markers (HBsAg, HBeAg and anti-HBe) in a series of49 Chronic Hepatitis B patients, including 12 with a spontaneous clearance of HBsAg. None of these HBsAg negative cases were PCR positive, but 33/37 (89.2%) HBsAg positive cases were PCR positive (p < 0.0001). Among HBsAg positive samples, nine cases were HBeAg positive and anti-HBe negative, all of them PCR positive. Other 3 patients were HBeAg and anti-HBe positive and these cases were also found PCR positive. A third group included 21 patients anti-HBe positive and HBeAg negative: 19 of them were PCR positive and 2 were PCR negative. The last 4 cases were HBeAg and anti-HBe negative, two of them were PCR positive. The detection of anti-HBe viremic cases in the present series suggest that preC variants could occur in our country. In conclusion, the integrated phase o f chronic hepatitis B seems to be less frequent than it was assumed, when only HBeAg or dot blot hybridization techniques were used. The new term "low replication phase" might favorably replace the former "integrated phase".