Novel MLST sequence types of pathogenic Leptospira spp.: Opening the black box of animal leptospirosis in Brazil.

In the current context of the emergence of certain infectious diseases and discussion of the One Health concept for many of these, the study of leptospirosis - both in domestic and wild hosts - cannot be neglected. The study of animal leptospirosis has evolved in recent years. It has been demonstrated that the human-animal-environment interface is more important than previously thought. In the present study, 35 strains of five pathogenic Leptospira species were isolated from different animal species in Brazil and characterized by rrs, secY, and Multilocus Sequence Typing (MLST) sequencing. Phylogenetic inferences were performed and the molecular diversity of the populations (intra- and inter-population levels) was evaluated. Among the five studied species, 18 different sequence types (STs) were found (22 new alleles and 11 new STs). eBURST analysis revealed two clonal complexes (CCs) and seven singletons. A high genetic diversity was demonstrated (H = 0.954 ± 0.017), mainly for the L. santarosai population (H = 0.942 ± 0.034, n = 20). The same strain was identified in different host species, as well as strains with zoonotic potential circulating in the country. Although the difficulty of culturing Leptospira strains is well known, the high variability of the strains found in Brazil highlights the importance of animals in maintaining the biological cycle of the bacterium in nature. Moreover, the selection of autochthonous strains for the development of vaccines becomes a challenge.

Detection of East/Central/South African genotype Chikungunya virus during an outbreak in a southeastern state of Brazil.

Chikungunya virus (CHIKV) is an arbovirus transmitted by Aedes mosquitoes that was first identified in Brazil in 2014. It causes a febrile illness characterised by severe arthralgia and rash. Our group investigated a suspected CHIKV outbreak in Governador Valadares, state of Minas Gerais, Brazil and from 25 acute-phase patients, 10 had qRT-PCR positive sera samples and had E1 partial sequence amplified and Sanger sequenced. Samples were identified as East/Central/South African (ECSA) genotype by phylogenetic analysis and clustered with CHIKV sequences isolated in the neighbour state of Bahia. Our findings confirm previous predictions that ECSA genotype would spread through northeast and southeast of Brazil.

High frequency of leptospiral vaginal carriers among slaughtered cows.

Bovine leptospirosis is one of the most important reproductive diseases that compromise the productivity of cattle farming. However, the presence of the agent on vaginal environment is still poorly understood in cattle. Considering this context, the present study aimed to detect the presence of pathogenic Leptospira sp. in vaginal fluid (VF) of cows. VF and urine were collected from 254 cows from a slaughterhouse for bacteriological culture and PCR (lipL32 gene). Overall, eleven pure culture (4.3%) of leptospiral isolates were obtained. Leptospiral DNA was detected in 128 (50.4%) of VF samples and 81 (31.0%) of urine samples, while on 75 (29.5%) it was exclusively in VF and 28 (11.3%) only in the urine. Detection of leptospiral DNA and the recovery of viable leptospires from VF of a high number of cows without apparent symptoms highlight the role of vaginal carriers and indicate that venereal transmission (female-to-male) could occur in that species. Moreover, VF should be encouraged as a valuable sample for diagnosis of bovine genital leptospirosis.

Genotyping of Leptospira directly in urine samples of cattle demonstrates a diversity of species and strains in Brazil.

The aim of this study was to identify Leptospira in urine samples of cattle by direct sequencing of the secY gene. The validity of this approach was assessed using ten Leptospira strains obtained from cattle in Brazil and 77 DNA samples previously extracted from cattle urine, that were positive by PCR for the genus-specific lipL32 gene of Leptospira. Direct sequencing identified 24 (31·1%) interpretable secY sequences and these were identical to those obtained from direct DNA sequencing of the urine samples from which they were recovered. Phylogenetic analyses identified four species: L. interrogans, L. borgpetersenii, L. noguchii, and L. santarosai with the most prevalent genotypes being associated with L. borgpetersenii. While direct sequencing cannot, as yet, replace culturing of leptospires, it is a valid additional tool for epidemiological studies. An unexpected finding from this study was the genetic diversity of Leptospira infecting Brazilian cattle.

Urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock.

The aim of the present study was to consider the wide usage of urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock. A total of 512 adult animals (300 cattle, 138 horses, 59 goats and 15 pigs), from herds/flocks with reproductive problems in Rio de Janeiro, Brazil was studied by serology and urinary PCR. From the 512 serum samples tested, 223 (43.5 %) were seroreactive (cattle: 45.6 %, horses: 41.3 %, goats: 34%and pigs: 60 %). PCR detected leptospiral DNA in 32.4 % (cattle: 21.6 %, horses: 36.2 %, goats: 77.4 % and pigs: 33.3 %. To our knowledge there is no another study including such a large number of samples (512) from different species, providing a comprehensive analysis of the usage of PCR for detecting leptospiral carriers in livestock. Serological and molecular results were discrepant, regardless the titre, what was an expected outcome. Nevertheless, it is impossible to establish agreement between these tests, since the two methodologies are conducted on different samples (MAT - serum; PCR - urine). Additionally, the MAT is an indirect method and PCR is a direct one. In conclusion, we have demonstrated that urinary PCR should be considered and encouraged as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock.

(+)α-Tocopheryl succinate inhibits the mitochondrial respiratory chain complex I and is as effective as arsenic trioxide or ATRA against acute promyelocytic leukemia in vivo.

The vitamin E derivative (+)α-tocopheryl succinate (α-TOS) exerts pro-apoptotic effects in a wide range of tumors and is well tolerated by normal tissues. Previous studies point to a mitochondrial involvement in the action mechanism; however, the early steps have not been fully elucidated. In a model of acute promyelocytic leukemia (APL) derived from hCG-PML-RARα transgenic mice, we demonstrated that α-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that α-TOS induces an early dissipation of the mitochondrial membrane potential in APL cells and studies with isolated mitochondria revealed that this action may result from the inhibition of mitochondrial respiratory chain complex I. Moreover, α-TOS promoted accumulation of reactive oxygen species hours before mitochondrial cytochrome c release and caspases activation. Therefore, an in vivo antileukemic action and a novel mitochondrial target were revealed for α-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy.

Absence of mutagenicity of acid pyrogallol-containing hair gels.

In the present work, three commercial acid (pH 3.5-4) pyrogallol-containing hair gels, SunSet Alizador Negro (two formulations) and Embelleze Henê Gel, were tested for mutagenicity using two well-established assays. In the Salmonella mutagenicity assay using 648-5000 microg/plate of cosmetic samples, none of the samples reached a 2-fold increase in revertants relative to the controls. Both in the absence and in the presence of S9, the dose-response relation in strains TA98, TA100, TA102, TA1535, and TA1537 was not significant (p>0.01). In the mouse bone marrow micronucleus assay, 10 Swiss male mice were orally administered 2000 mg/kg of sample per body weight/day. The ratio between polychromatic and normochromatic erythrocytes as well as the presence of micronuclei in bone marrow cells were determined. Equal numbers of micronucleated polychromatic erythrocytes were detected between the cells of each treated group and the negative control, using ANOVA and chi-square analyses. Thus, none of the products induced mutagenesis in either assay. Previous studies have shown pyrogallol is mutagenic in various test systems, including Salmonella. However studies have also shown that acidic conditions may repress the reactive-oxygen species (ROS) produced by pyrogallol, and ROS is considered the primary mechanism for the mutagenicity of pyrogallol. Consistent with this are our results, which show that acidic, commercially available pyrogallol-containing hair gels are neither mutagenic in Salmonella nor induce micronuclei in mouse bone marrow in vivo.

Immunohistochemical markers in the identification of metastatic breast cancer.

A panel of nine monoclonal and polyclonal antibodies were tested regarding specificity for metastatic breast cancer. A hundred metastatic tumors were stained, 50 of breast origin and 50 of other origins. Antibodies used were anti-alpha-lactalbumin, anti-lactoferrin, anti-casein, E29 (Dako-EMA), anti-secretory component, anti-gross cystic disease fluid protein (GCDFP15), BRST1, BRST2, and MC5. Analyses of the results were performed using chi-square and logistic regression. Positivity for MC5, BRST1, BRST2, lactoferrin, EMA, and GCDFP15 was significantly higher in tumors of breast origin than in others (p less than 0.05). Analyses of the whole panel indicated that GCDEP15 and MC5 were the best markers for identification of breast cancer metastases. When both were positive (58% of breast origin cases), the predicted probability of breast origin was 98%, compared to only 5% when both were negative. Comparison of anti-GCDFP15 with BRST2, a monoclonal antibody against the same protein, showed a slightly better sensitivity of the former, and a similar degree of specificity for breast tissue. In conclusion, a panel of antibodies can be used to securely differentiate metastatic breast cancer from other cancers in a large number of metastatic tumors of unknown origin.

Sequential determination of immunocytochemical estrogen receptor and nuclear DNA content in fine needle biopsies from breast carcinoma.

The application of fine needle biopsy as a tool for early detection of breast cancer is becoming extensive, therefore parameters reported to be associated with prognosis should be standardized in this material. We propose the sequential determination of estrogen receptor (ER) status and DNA ploidy on the same smear obtained from a fine needle biopsy of a breast carcinoma, since both parameters seem to reflect properties associated with tumor behaviour and biological aggressiveness. Fifty fine-needle biopsies were investigated for presence of ER by the monoclonal antibody D75 followed by DNA content quantification using Feulgen-DNA cytophotometry. Overall, 66% of the tumors showed immunoreactivity for ER and 66% were classified as aneuploid. Forty-one percent of the aneuploid tumors were negative for ER, while only 7% of the diploid tumors showed no immunoreaction (p less than 0.05). The significant association between absence of immunocytochemical ER and DNA aneuploidy on the same fine-needle smear is consistent with data obtained through other methods previously reported using much larger tissue samples.

Tissue carcinoembryonic antigen in the prognosis of early invasive breast cancer.

Twenty-five patients with stage II ductal breast carcinoma followed up for ten years were studied for the presence of tissue carcinoembryonic antigen (CEA). Overall expression of CEA was 60%. The ten year survival rate was significantly higher for patients with CEA-negative tumours (70%) than for patients with CEA-positive tumours (27%), while the difference between the survival rate of patients with (30%) or without (53%) lymph node involvement did not reach significance. Among the 10 patients with lymph node involvement, CEA-negative patients had a better outcome. These results suggest that there is a correlation between the presence of tissue CEA and the prognosis of the disease, and that CEA status might possibly be more important than lymph node involvement, at least within stage II breast carcinomas.

[High resolution light microscopy: adaptation of the method and its use in the study of experimental leptospirosis in guinea pigs]. / Microscopia óptica de alta resolução: adaptação do método e sua aplicação ao estudo da leptospirose experimental em cobaias.

Morphological lesions in parenchimal and mesenchimal structures of liver and kidney were studied in guinea-pigs experimentally infected with Leptospira interrogans serogroup icterohaemorrhagiae in comparison with a group of non-infected guinea-pigs. All specimens were submitted to conventional light microscopy as well as to high resolution light microscopy, in one micrometer sections of tissue embedded in glycolmethacrylate. High resolution light microscopy, applied for the first time in leptospirosis, was proved very useful, since it enabled us to visualize cellular structures in the same slide used for panoramic view. Cell cohesion, brush borders, pynocytotic vesicles and organellae distributions were parameters especially suitable for analysis at this low-cost, highly precise procedure in microscopy.

Glial fibrillary acidic protein and cytokeratin in choroid plexus tumors. An immunohistochemical study.

Ten cases of choroid plexus tumors (3 papillomas and 7 carcinomas) were tested for the presence of glial fibrillary acidic protein (GFAP) and cytokeratin. None of the papillomas and one of the carcinomas were positive with GFAP antisera. Cytokeratin-positive cells were present in 2 of 7 carcinomas and in all papillomas. There seems to be a positive correlation between the degree of the tumor differentiation and the expression of intermediate filaments.

Glicolipoproteína de Leptospira interrogans sorogrupo icterohaemorrhagiae: distribuiçäo em fígado e rim de cobaias experimentalmente infectadas / Glycoproteins from Leptospira interrogans serogroup icterohaemorrhagiae: distribution in the liver and kidney of experimentally infected guinea pigs

Acredtita-se que as lesöes teciduais na leptospirose possam decorrer da açäo direta das leptospiras, de toxinas sintetizadas ou liberadas durante sua lise. O presente estudo visou a extraçäo química da glicolipoproteína (GLP) da aleptospira, a produçäo de anti-soro anti-GLP e a avaliaçäo de sua distribuiçäo em cortes de fígado e rim de cobaias inoculadas e sacrificadas em estudo sequencial diário até o 6§ dia de infecçäo, correspondente ao pico da doença. Procurou-se também correlacionar a expressäo tecidual da GLP com o grau de lesöes locais, em busca de novos subsídios para a compreensäo da patogenia da leptospiros. A GLP foi detectada em fígado e rim de 2 dentre 6 cobaias no 5§ dia e em todas as 6 no 6§ dia de infecçäo, sob a forma de grânulos no citoplasma de macrófagos, livres no interstício ou acolados à membrana de células endoteliais e parenquimatosas, especialmente nas regiöes mais lesadas. A cronologia do aparecimento da GLP e sua distribuiçäo sugerem tratar-se de produto de lise de leptospiras fagocitadas por macrófagos e que esta substância, conquanto näo comprovada como iniciadora das lesöes, asocia-se a seu agravamento nas etapas mais avançadas da leptospirose

[Glycoproteins from Leptospira interrogans serogroup icterohaemorrhagiae: distribution in the liver and kidney of experimentally infected guinea pigs]. / Glicolipoproteína de Leptospira interrogans sorogrupo icterohaemorrhagiae: distribuição em fígado e rim de cobaias experimentalmente infectadas.

Tissue damage in leptospirosis has been ascribed to direct effect of the microorganisms and/or their virulence, including products synthetized by leptospires or released during their lysis. This study aimed at chemical extraction of the glycolipoprotein (GLP) from virulent leptospires, production of a rabbit anti-GLP and analysis of its distribution in liver and kidney of inoculated guinea-pigs, sacrificed sequentially from the 1st to 6th day of infection, covering the whole, spectrum of acute leptospirosis. The comparison of GLP expression to local injuries aimed at new pathogenetic data. GLP was detected in liver and kidney in 2 out of 6 guinea-pigs on the 5th day and in all 6 animals on the 6th day of infection. Granular forms were seen in the cytoplasm of macrophages, free in interstitium or adhered to endothelial and parenchymal cell membranes, especially in the most damaged sites. These findings lead us to the hypothesis of GLP as a toxic factor resulting from leptospiral lysis by macrophages. Although it was not proved as a promoter of initial lesions, it seems to be related to the enhancement of tissue damage late in the course of the disease.

[Use of immunohistochemistry in detecting the primary site in neoplasm metastasis]. / Utilização da imuno-histoquímica na detecção da sede primária em neoplasias metastáticas.

The aim of the present study is to demonstrate the sensitivity, specificity and applicability of several tissue markers in the determination of the primary sites of metastatic tumors. The immunoperoxidase technique was used in 19 metastatic tumors from breast (6), gastrointestinal tract (6), thyroid (3), prostate (1), ovary (1), pancreas (1) and melanoma (1). Polyclonal antisera against thyroglobulin and prostatic specific antigen were used. The following monoclonal antibodies were employed: BRST-1, BRST-2, CAR-3, BD-5 and HMB-45. BRST-1 and BRST-2 are considered to be breast cancer markers, while CAR-3 and BD-5 gastrointestinal markers. HMB-45 was described as a melanoma marker. Breast markers were positive for 3 out of 6 breast metastases. BRST-1 was also positive for metastases from melanoma and prostate. CAR-3 and BD-5 were positive for 5 out of 6 gastrointestinal metastases. CAR-3 also presented focal positivity for 4 out of 6 breast metastasis, 1 out of 3 thyroid metastasis and for metastasis from ovary, prostate, pancreas and melanoma. BD-5 was also positive for prostate metastasis. Thyroglobulin and prostatic specific antigen were only positive for thyroid and prostate metastasis, respectively. In conclusion, immunocytochemistry and monoclonal antibodies are useful tools in the detection of the primary sites of metastatic tumors of unknown origin. In some of the fields, the results are already satisfactory. Nevertheless, further studies should be carried out to improve this promising technique.