38 research outputs found
Seroprevalence of Toxoplasma gondii and Toxocara canis in a human rural population of Southern Rio Grande do Sul
Due to the growing population of pets, especially homeless dogs and cats, zoonoses still represent a significant public health problem. Toxoplasma gondii and Toxocara spp. are epidemiologically important zoonotic agents as they are etiological factors of human toxoplasmosis and toxocariasis, respectively. These parasites remain neglected even though they are substantially prevalent in rural areas. The aim of this study was to investigate T. gondii and T. canis seroprevalence and risk factors of seropositivity in a rural population in Pelotas municipality, Brazil. The study participants (n=344) were patients of a Basic Healthcare Unit (BHU) located in Cerrito Alegre. Blood samples were collected and tested for T. gondii antibodies by indirect immunofluorescence and T. canis antibodies by an indirect ELISA that targets an excreted-secreted antigen (TES). T. gondii seropositivity was 53.2%, with higher titers (1:256 - 1:1,024) in individuals who habitually eat pork, beef, or chicken, while T. canis seropositivity was 71.8% and concomitant T. gondii and T. canis seropositivity was 38.3%. Among the seropositivity risk factors assessed, only habitual undercooked meat consumption was significant (p = 0.046; OR = 3.7) for T. gondii and none of them were associated with T. canis seropositivity. Both parasites have a high prevalence in rural areas, which reinforces the need to invest in rural community education and health
Construction of auxotrophic marker in Mycobacterium bovis BCG, knockout strain for the DPPD and proteomic study of tuberculin
Mycobacterium bovis BCG has the potential to be an effective live vector for
multivalent vaccines. However, there are two problems regarding the utilization
of recombinant BCG as vaccine. The first one is that most mycobacterial
cloning vectors rely on antibiotic resistance gene as selectable marker, which is
used for genetic transformation. The second one is the limited use of BCG in
animals because it interferes in the tuberculosis diagnosis by tuberculin skin
test, which elicits delayed type hypersensitivity to the purified protein derivative
(PPD). In this work we developed and evaluated the use of auxotrophic
complementation as a new selectable marker, characterized the proteins that
are present in the bovine and avium PPD and developed a knockout BCG strain
by homologous recombination. To test the auxotrophic complementation as
selectable marker, an auxotrophic BCG strain for the amino acid leucine was
constructed by knocking out the leuD gene by homologous recombination.
Expression of leuD on a plasmid acted as a selectable marker in the
auxotrophic M. bovis BCG leuD and M. smegmatis mc2144. The auxotrophic
complementation selection was similar to selection by antibiotic resistance, but
with the advantage of promoting stability of the plasmid. The new system was
highly stable even during in vivo BCG growth. The identification of proteins from
PPD was archived by LC-MS/MS (Liquid Chromatography/Mass
Spectrometry/Mass Spectrometry). A total of 147 proteins among five PPD
samples (2 bovine PPD and 3 avium PPD) were identified. The bovine PPD had
a considerable higher number of proteins comparing to the avium PPD. We
identifying a group of 28 proteins present only in bovine PPD and a group of five
proteins deleted in M. bovis BCG vaccinal strain. These two groups are of
special interest as they can be used in tests with improved specificity, and
potentially able to differentiate vaccinated and infected individuals. A mutant
BCG strain with the DPPD antigen deleted was constructed. The Mb0092
coding sequence was knocked out by homologous recombination. The
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sequences flanking the target gene were cloned into a suicide vector. Double
crossovers were selected using sacB. The knockout genotype was determined
by PCR and by Southern blot. This mutant BCG strain can be useful in animal
vaccination as it will not interfere in the tuberculosis diagnostic test, when
performed using recombinant DPPD. The results show alternatives for the
problems related to the use of M. bovis BCG as a recombinant vaccine. The
auxotrophic complementation system was highly stable, efficient and it is
suitable for expressing heterologous antigens in BCG. The identification of
proteins present in PPD preparations and the mutant BCG obtained provide the
possibility for the development of differential diagnostic test, thus allowing the
use of BCG as vaccine also in animals.Mycobacterium bovis BCG tem o potencial para ser um vetor efetivo para
vacinas recombinantes multivalentes. No entanto, existem dois problemas
quanto a sua utilização como vetor vacinal. O primeiro é a presença de genes
que conferem resistência a antibióticos nos vetores utilizados para
transformação genética. O segundo é a limitação de uso de BCG em animais,
principalmente por comprometer o teste de tuberculina, utilizado como
diagnóstico de tuberculose, o qual se baseia em reação de hipersensibilidade
ao PPD (Derivado Protéico Purificado). Neste trabalho desenvolvemos e
avaliamos a complementação auxotrófica como novo marcador de seleção,
fizemos a caracterização das proteínas componentes de amostras de PPD
aviário e bovino e desenvolvemos um mutante de BCG por recombinação
homóloga. Para o uso de complementação auxotrófica como marcador de
seleção, uma cepa de BCG auxotrófica para o aminoácido leucina foi
construída por knockout do gene leuD por recombinação homóloga. A
expressão do gene leuD em um plasmídio atuou como marcador de seleção
nas cepas auxotróficas de M. bovis BCG leuD e M. smegmatis mc2144. A
seleção por complementação de BCG auxotrófica se mostrou equivalente à
seleção por resistência a antibiótico, com a vantagem adicional de proporcionar
maior estabilidade do vetor plasmidial, já que a pressão seletiva é mantida
mesmo durante multiplicação da bactéria in vivo. A identificação das proteínas
que compõem o PPD foi feita por espectrometria de massa utilizando-se LCMS/
MS (cromatografia líquida associada à espectrometria de massa em
tandem). Foram identificadas 147 proteínas entre 5 amostras de PPD (2 PPD
bovino e 3 PPD aviário). O PPD bovino teve um número maior de proteínas
comparado ao PPD aviário. Foi identificado um grupo de 28 proteínas
presentes em PPD bovino, mas ausentes em PPD aviário. Além disso, 5
proteínas encontradas no PPD estão ausentes em M. bovis BCG. Estes são de
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especial interesse, pois poderão vir a contribuir para o desenvolvimento de um
teste de diagnóstico mais específico, e possivelmente capaz de diferenciar
indivíduo vacinado com BCG e infectado com o bacilo da tuberculose. Um
mutante de M. bovis BCG Pasteur foi construído. O gene Mb0092 (dppd) foi
alvo de inativação gênica por recombinação homóloga. Seqüências que
flanqueiam o gene alvo foram clonadas em um vetor suicida. Duplo crossover
foi selecionado utilizando sacB. O genótipo mutante foi determinado por PCR e
por Southern blot. Esta cepa poderá ser utilizada como vacina em animais,
quando o diagnóstico for feito com DPPD recombinante. Os resultados obtidos
apresentam alternativas para os problemas envolvidos quanto à utilização de
M. bovis BCG como vacina recombinante. O sistema de seleção por
complementação auxotrófica foi estável, e pode ser empregado na expressão
de antígenos heterólogos em BCG. A identificação dos principais componentes
protéicos do PPD e o desenvolvimento da cepa mutante de BCG possibilitam o
desenvolvimento de testes diagnósticos diferencias, permitindo a utilização de
BCG como vacina também em animas
Auxotrophic <i>Mycobacterium bovis</i> BCG: Updates and Perspectives
Mycobacterium bovis BCG has been used for a century as the only licensed vaccine against tuberculosis. Owing to its strong adjuvant properties, BCG has also been employed as an oncological immunotherapeutic as well as a live vaccine vector against other pathogens. However, BCG vaccination has limited efficacy in protecting against adult forms of tuberculosis (TB), raises concerns about its safety in immunocompromised populations, compromises the diagnosis of TB through the tuberculin test and lacks predictability for successful antigen expression and immune responses to heterologous antigens. Together, these factors propelled the construction and evaluation of auxotrophic BCG strains. Auxotrophs of BCG have been developed from mutations in the genes required for their growth using different approaches and have shown the potential to provide a model to study M. tuberculosis, a more stable, safe, and effective alternative to BCG and a vector for the development of recombinant live vaccines, especially against HIV infection. In this review, we provide an overview of the strategies for developing and using the auxotrophic BCG strains in different scenarios
Rational design of diagnostic and vaccination strategies for tuberculosis
The development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals is crucial for the wider utilization of bacillus Calmette-Guérin (BCG) as vaccine in humans and animals. BCG_0092 is an antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both animals and humans infected with the tubercle bacilli. We carried out bioinformatics analyses of the BCG_0092 and designed a diagnostic test by using the predicted MHC class I epitopes. In addition, we performed a knockout of this gene by homologous recombination in the BCG vaccine strain to allow differentiation of vaccinated from infected individuals. For that, the flanking sequences of the target gene (BCG_0092)were cloned into a suicide vector. Spontaneous double crossovers, which result in wild type revertants or knockouts were selected using SacB. BCG_0092 is present only in members of the Mycobacterium tuberculosis complex. Eight predicted MHC class I epitopes with potential for immunological diagnosis were defined, allowing the design of a specific diagnostic test. The strategy used to delete the (BCG_0092) gene from BCG was successful. The knockout genotype was confirmed by PCR and by Southern blot. The mutant BCG strain has the potential of inducing protection against tuberculosis without interfering with the diagnostic test based on the use of selected epitopes from BCG_0092
Atividade in vitro de 3,7-dimethyl-2,6-octadienal contra trofozoítos de Trichomonas vaginalis
A tricomoníase é a infecção sexualmente transmissível não viral mais comum no mundo. Destacando a importância desse patógeno como um problema de saúde pública, o Trichomonas vaginalis (TV) pode aumentar o risco de adquirir e transmitir o vírus da imunodeficiência humana (HIV), além dos riscos de complicações na gravidez (SERWIN et al., 2013). Uma revisão atualizada de vários estudos que avaliam a eficácia de plantas e seus derivados contra TV, mostrou atividade potencial e resultados promissores, sendo produtos naturais uma alternativa para o tratamento da tricomoníase. Neste contexto, tendo em vista a busca de novos agentes a partir dos recursos naturais, a atividade antiparasitária do óleo essencial de capim-limão e a resistência desenvolvida por algumas linhagens de TV, o presente estudo teve como objetivo avaliar a atividade anti- T. vaginalis do citral, o principal composto de Cymbopogon sp. Este teste foi realizado para a atividade in vitro contra TV isolado ATCC 30236 (American Type Culture Collection, ATCC) e também foi avaliado em comparação com o metronidazol