Marcos Heinemann | Universidade de São Paulo (original) (raw)

Papers by Marcos Heinemann

Journal of Medical Microbiology

Group A rotaviruses are the main cause of acute gastroenteritis in children throughout the world.... more Group A rotaviruses are the main cause of acute gastroenteritis in children throughout the world. The two outer capsid proteins, VP4 and VP7, define the P and G genotypes, respectively. Rotaviruses with P[8]G1, P[4]G2, P[8]G3 and P[8]G4 genotypes are predominant in infecting humans and the G9 genotype is emerging in most continents as the fifth most common G type worldwide. The inner capsid protein VP6 is responsible for subgroup (SG) specificities, allowing classification of rotaviruses into SG I, SG II, SG I+II and SG non-I-non-II. The non-structural protein 4 (NSP4) encoded by segment 10 has a role in viral morphogenesis and five genetic groups have been described, NSP4 genotypes A-E. The aim of this investigation was to characterize the NSP4 and VP6 genes of rotavirus strains recovered from hospitalized children. Thirty rotavirus strains were submitted to RT-PCR followed by sequencing and phylogenetic analysis. Among the different G and P genotype combinations, two distinct gene...

Journal of General Virology

The 3'-terminal 853 nt (and the putative 283 aa) sequence of the VP2-encoding gene from 29 fi... more The 3'-terminal 853 nt (and the putative 283 aa) sequence of the VP2-encoding gene from 29 field strains of porcine parvovirus (PPV) were determined and compared both to each other and with other published sequences. Sequences were examined using maximum-parsimony and statistical analyses for nucleotide diversity and sequence variability. Among the nucleotide sequences of the PPV field strains, 26 polymorphic sites were encountered; 22 polymorphic sites were detected in the putative amino acid sequence. Mapping polymorphic sites of protein data onto the three-dimensional (3D) structure of PPV VP2 revealed that almost all substitutions were located on the external surface of the viral capsid. Mapping amino acid substitutions to the alignment between PPV VP2 sequences and the 3D structure of canine parvovirus (CPV) capsid, many PPV substitutions were observed to map to regions of recognized antigenicity and/or to contain phenotypically important residues for CPV and other parvovir...

RESUMO Um método de ELISA policlonal tipo "duplo-sanduíche" foi desenvolvido para a det... more RESUMO Um método de ELISA policlonal tipo "duplo-sanduíche" foi desenvolvido para a detecção de rotavírus a partir de material fecal. A amostra NCDV de rotavírus do sorogrupo A foi concentrada por ultracentrifugação utilizando-se um colchão de sacarose a 45% (v/v), e inoculada em coelhos e carneiros. Em seguida, a fração IgG oriunda dos soros dos animais foi purificada por cromatografia de troca iônica e absorvida, utilizando-se polímero de glutaraldeído, de modo a eliminar reações inespecíficas. A presença dos rotavírus foi detectada pela IgG de carneiro e revelada pela IgG de coelho, usando-se IgG de cabra anti-IgG de coelho conjugada à peroxidase. Aplicado a um painel constituído de 86 amostras fecais diarréicas de leitões obteve-se 100% de sensibilidade; 98,79% de especificidade, com uma concordância de 98,83%, tendo como prova padrão a eletroforese em gel de poliacrilamida (PAGE). As variâncias entre 86 repetições da mesma amostra foram 0,001 (para a amostra positiva)...

Soroprevalência da anemia infecciosa eqüina, da arterite viral dos eqüinos e do aborto viral eqüi... more Soroprevalência da anemia infecciosa eqüina, da arterite viral dos eqüinos e do aborto viral eqüino no município de Uruará, PA, Brasil Seroprevalence of equine infection anemia, equine viral arteritis and equine viral abortion in Uruará municipality, Pará state, Brazil Recebido para publicação: 19/09/2001 Aprovado para publicação: 31/01/2002 HEINEMANN, M.B.; CORTEZ, A; SOUZA, M.C.C.; GOTTI, T.B. FERREIRA, F.; HOMEM, V.S.F.; FERREIRA NETO; J.S.; SOARES, R.M.; SAKAMOTO, S.M.; CUNHA, E.M.S.; RICHTZENHAIN, L.J. Soroprevalência da anemia infecciosa eqüina, da arterite viral dos eqüinos e do aborto viral eqüino no município de Uruará, PA, Brasil Braz. J. vet. Res. anim. Sci., São Paulo, v.39, n.1, p. 50-53, 2002.

Aspects of biological behavior of Brazilian rabies virus isolated from canine, bovine, equine, va... more Aspects of biological behavior of Brazilian rabies virus isolated from canine, bovine, equine, vampire and insectivorous bats were studied in mice. The oral infection occurred in mice fed with infected brain of insectivours bat (8.82%), canine (8.57%) and equine (3.03%). The mean period of incubation to all the isolates was 6 days after mice intracerebral inoculation, however, symptoms were variable, since hyperexcitability (canine sample), progressive paralysis of lower limbs and prolonged clinical course until death (equine sample), and mice without clinical signs before death (insectivorous bat). By immunohistochemistry IFN was detected in brains of mice inoculated with bovine and insectivorous bat samples, TNF and iNOS were detected in brains of those inoculated with insectivorous bat, bovine and canine samples, and positive GFAP astrocytes were found in all five samples. Two commercial inactivated rabies vaccines, one imported (vaccine 1) and another manufactured in Brazil (vac...

Genome Announcements, 2015

b A.M.S.G. and C.K.Z. contributed equally to this work. We report a draft genome sequence of Myco... more b A.M.S.G. and C.K.Z. contributed equally to this work. We report a draft genome sequence of Mycobacterium bovis strain SP38, isolated from the lungs of a cow in Brazil. The assembly of reads resulted in 36 contigs in a total of approximately 4.37 Mb. Comparison of M. bovis strains sequenced to date will aid in understanding bovine tuberculosis in Brazil. Citation Guimaraes AMS, Zimpel CK, Ikuta CY, do Nascimento NC, dos Santos AP, Messick JB, Heinemann MB, Ferreira Neto JS, Brandão PE. 2015. Draft genome sequence of Mycobacterium bovis strain SP38, a pathogenic bacterium isolated from a bovine in Brazil. Genome Announc 3(3):e00511-15.

Brazilian Journal of Microbiology, 2014

Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained pro... more Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 -1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% -100%) and 100% (CI = 93.98% -100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

Genetics and Molecular Research, 2014

This study aimed to develop and validate real-time PCR for the diagnosis of Mycobacterium bovis i... more This study aimed to develop and validate real-time PCR for the diagnosis of Mycobacterium bovis isolates. Two hundred and seventy-four M. bovis isolates and 156 M. tuberculosis isolates were tested. Both qPCRs amplified all of the 274 M. bovis samples, but none of the 156 M. tuberculosis samples. The qPCR for PE-PGRS 20 had 91% efficiency and a detection limit of 0.32 ng (sensitivity and specificity for qPCR "Mbovis.100" were 99.64 and 100%, respectively). The qPCR for RD4 had 100% efficiency, and a detection limit of 4 pg (diagnostic sensitivity and specificity were 100 and 100%. The qPCR tests were performed using 4 extraction sets, 3 qPCR kits, and with a range of equipment; yet, all combinations produced similar results in a diagnostic 4608 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (2): 4607-4616 (2014) M.L. Sales et al.

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, which is a se... more Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, which is a serious, economically important problem for sheep production. We examined the seroprevalence of infection by C. pseudotuberculosis and possible risk factors associated with caseous lymphadenitis in sheep herds of the state of Minas Gerais, Brazil. Samples were collected from 642 sheep from 97 farms. Sera of all of the sheep were tested with ELISA for antibodies against C. pseudotuberculosis. A questionnaire was applied to gather data on the farm, the sheep herd, the farmer, and individual animal data (breed, sex and age). This is the first sero-epidemiological survey for caseous lymphadenitis in sheep herds in Minas Gerais. We found a high real prevalence, much higher than that suggested from information obtained with the questionnaire, which points to the scarcity of vaccination against caseous lymphadenitis in the sample evaluated. Only a small proportion of the farmers declared that cases of this disease were present in their flocks. The frequency of seropositive sheep varied significantly with breed ( 2 test, P = 0.021). Age group also significantly affected the percentage of seropositivity ( 2 test, P = 0.049), the highest frequency being found in adult animals (more than 12 months old), when compared to the 5-12 months old group ( 2 test, P = 0.021). The prevalence of infection with C. pseudotuberculosis in sheep in the state of Minas Gerais was estimated to be 70.9% (95% confidence interval (CI): 64.7-77.0%) and the prevalence of infected flocks being 95.9% (95% CI: 89.8-98.9%). We concluded that C. pseudotuberculosis infection is widely disseminated in sheep flocks in Minas Gerais and that caseous lymphadenitis control and eradication programs are necessary in this state.

Brazilian Journal of Veterinary Research and Animal Science, 1999

... 13= grippotyphosa; 14= javanica; 15= panama; 16= pomona; 17= pyrogenes; 18= hardjo; 19= wolff... more ... 13= grippotyphosa; 14= javanica; 15= panama; 16= pomona; 17= pyrogenes; 18= hardjo; 19= wolffi; 20= shermani; 21= tarassovi; 22= andamana; 23= buenos aires; 24= garcia; 25= rufino and ... Part 1. Brucella abortus, Leptospira, Campylobacter fetus and Tritrichomonas foetus. ...

PLoS ONE, 2014

The aim of this study was to evaluate the Enterobacterial Repetitive Intergenic Consensus (ERIC-P... more The aim of this study was to evaluate the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis isolates from eight different hosts in twelve countries. Ninety-nine C. pseudotuberculosis field strains, one type strain (ATCC 19410 T ) and one vaccine strain (1002) were fingerprinted using the ERIC-1R and ERIC-2 primers, and the ERIC-1R+ERIC-2 primer pair. Twenty-nine different genotypes were generated by ERIC 1-PCR, 28 by ERIC 2-PCR and 35 by ERIC 1+2-PCR. The discriminatory index calculated for ERIC 1, ERIC 2, and ERIC 1+2-PCR was 0.89, 0.86, and 0.92, respectively. Epidemiological concordance was established for all ERIC-PCR assays. ERIC 1+2-PCR was defined as the best method based on suitability of the amplification patterns and discriminatory index. Minimal spanning tree for ERIC 1+2-PCR revealed three major clonal complexes and clustering around nitrate-positive (biovar Equi) and nitrate-negative (biovar Ovis) strains. Therefore, ERIC 1+2-PCR proved to be the best technique evaluated in this study for genotyping C. pseudotuberculosis strains, due to its usefulness for molecular epidemiology investigations. Citation: Dorneles EMS, Santana JA, Ribeiro D, Dorella FA, Guimarães AS, et al. (2014) Evaluation of ERIC-PCR as Genotyping Method for Corynebacterium pseudotuberculosis Isolates. PLoS ONE 9(6): e98758.

BMC Microbiology, 2014

The aims of the present study were (i) to assess the in vitro genetic stability of S19 and RB51 B... more The aims of the present study were (i) to assess the in vitro genetic stability of S19 and RB51 Brucella abortus vaccines strains and (ii) to evaluate the ability of multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) as a tool to be used in the quality control of live vaccines against brucellosis. Sixty-three batches of commercial S19 (n = 53) and RB51 (n = 10) vaccines, produced between 2006 and 2009, were used in this study. S19 and RB51 vaccines were obtained from, respectively, seven and two different manufacturers. Ten in vitro serial passages were performed on reference strains and on selected batches of commercial vaccines. All batches, reference strains and strains of serial passages were typed by the MLVA16. The results demonstrated that B. abortus S19 and RB51 vaccine strains are genetically stable and very homogeneous in their respective groups. Anyway, batches of S19 from one manufacturer and batches of RB51 from another presented genotypes distincts from the reference vaccine strains. In both cases, differences were found on locus Bruce07, which had addition of one repeat unit in the case of S19 batches and the deletion of one repeat unit in the case of RB51 batches. In summary, MLVA16 proved to be a molecular tool capable of discriminating small genomic variations and should be included in in vitro official tests.

Revista do Instituto de Medicina Tropical de São Paulo, 1997

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, t... more Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenically related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis and A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed.

Brazilian Journal of Veterinary Research and Animal Science, 2001

Ciência Rural, 2009

Arcanobacterium pyogenes, Bovine Herpesvirus 1, Brucella abortus, Campylobacter fetus subsp. vene... more Arcanobacterium pyogenes, Bovine Herpesvirus 1, Brucella abortus, Campylobacter fetus subsp. venerealis, Chlamydophila abortus, Leptospira sp., Listeria monocytogenes, Salmonella sp., Mycoplasma bovis, Mycoplasma bovigenitalium, Neospora caninum, and Tritrichomonas foetus. Tissue homogenates from 42 fetuses and paraffin-embedded tissues from 28 fetuses and 14 placentas/endometrium were included in this study. Brucella abortus was detected in 14.2% (12/84) of the samples. Salmonella sp. DNA was amplified from 2 fetuses, and there was one positive for Neospora caninum, and another for Listeria monocytogenes. This PCR-based approach resulted in identification of the etiology in 19% of samples, or 20% if considered fetal tissues only. RESUMO Aborto infeccioso é uma causa significativa de falhas reprodutivas e perdas econômicas na bovinocultura. O objetivo deste estudo foi detectar ácidos nucleicos de vários agentes infecciosos reconhecidos como causadores de aborto, incluindo-se Arcanobacterium pyogenes, Herpesvirus bovino tipo 1, Brucella abortus, Campylobacter fetus subsp. venerealis, Chlamydophila abortus, Leptospira sp., Listeria monocytogenes, Salmonella sp., Mycoplasma bovis, Mycoplasma bovigenitalium, Neospora caninum e Tritrichomonas foetus. Homogenados de tecidos de 42 fetos e tecidos incluídos em parafina de 28 fetos e 14 placentas/ endométrio foram incluídos neste estudo. Brucella abortus foi detectada em 14,2% (12/84) das amostras. DNA de Salmonella sp. foi amplificado de dois fetos e houve um feto positivo para Neospora caninum e outro para Listeria monocytogenes. Essa metodologia baseada em PCR resultou na identificação da etiologia em 19% das amostras ou 20% se considerados somente os tecidos fetais.

Virus Research, 2012

Bats are main reservoirs for Lyssavirus worldwide, which is an important public health issue beca... more Bats are main reservoirs for Lyssavirus worldwide, which is an important public health issue because it constitutes one of the big challenges in rabies control. Yet, little is known about how the virus is maintained among bats, and the epidemiological relationships remain poorly understood. The aim of the present study was to investigate the distribution of the rabies virus (RABV) in bat tissues and organs and to genetically characterize virus isolates from naturally infected non-hematophagous bats. The heminested reverse transcriptase polymerase chain reaction (hnRT-PCR) and sequencing using primers to the nucleoprotein coding gene were performed. The results showed a dissemination of the RABV in different tissues and organs, particularly in the salivary glands, tongue, lungs, kidneys, bladder, intestine and feces, suggesting other possible forms of RABV elimination and the possibility of transmission among these animals. The phylogenetic analysis confirmed that different variants of RABV are maintained by non-hematophagous bats in nature and have similar tissue distribution irrespective of bat species and phylogenetic characterization.

Veterinary Microbiology, 2002

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Bo... more Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, (L.J. Richtzenhain).

Veterinary Microbiology, 2000

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the ... more In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. bi¯exa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were ampli®ed and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp. at bovine arti®cial insemination centers. #

Research in Veterinary Science, 2008

Porcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, eight ... more Porcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, eight pig tissue samples from two Brazilian states were analyzed using six PCR primer pairs amplifying a 1705-bp fragment of the PCV-2 genome. The NJ distance-based method was used for the phylogenetic analysis with the eight field strains herein, 15 GenBank sequences and using PCV-1 as an out-group. This yielded two major clusters (A and B) for this viral species, with the Brazilian strains segregating with European and Asian sequences. Nucleotide identity was 99.7 to 100% among the sequences. This information can be used in further studies of pathogenesis related to PCV-2 in Brazil.