ABSTRACT
The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.
Subject(s)
Baculoviridae/chemistry , Baculoviridae/metabolism , Hepatitis A virus/chemistry , Viral Proteins/biosynthesis , Baculoviridae/genetics , Gene Expression Regulation, Viral , Genetic Vectors , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Solubility , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
The purpose of this study was to report the first recovery and characterization of Leptospira interrogans (serogroup Australis) from urine of swine in Brazil. The isolate was studied by serogrouping, MLVA, PGFE, and partial sequencing of rrs and secY. It was serogrouped as serogroup Australis, probably serovar Bratislava (titre 1,600), and sequenced as Leptospira interrogans. The MLVA and PGFE profiles also suggested the isolate as serovar Bratislava, since they were indistinguishable from reference strains Balico and Jez Bratislava. This is the first Leptospira interrogans serogroup Australis isolate, probably serovar Bratislava, obtained in Brazil...
O objetivo deste estudo foi relatar o primeiro isolamento e caracterização de Leptospira interrogans (sorogrupo Australis) a partir da urina de suínos no Brasil. O isolado foi caracterizado por sorogrupagem, MLVA, PGFE, e sequenciamento parcial de rrs e secY. Este foi identificado como pertencente ao sorogurpo Australis, provavelmente sorovar Bratislava (título 1600) e sequenciado como Leptospira interrogans. Os perfis de MLVA e PGFE também sugeriram o isolado como sorovar Bratislava, uma vez que estes foram indistinguíveis das cepas de referência Balico e Jez Bratislva. Este é o primeiro isolado de Leptospira interrogans sorogrupo Australis, provavelmente sorovar Bratislava, obtido no Brasil...
Subject(s)
Animals , Male , Female , Leptospira interrogans serovar australis/isolation & purification , Swine/microbiology , Urinalysis/veterinary , Leptospirosis/epidemiologyABSTRACT
The purpose of this study was to analyze the usefulness of PCR for the detection of leptospiral carriers in sheep under tropical field conditions. Two flocks, previously reported as seroreactive (A) and seronegative (B), were selected for this study. From those, the totality of animals of each flock, urine and vaginal fluid (VF)/semen were collected for bacteriological culture and PCR, as well as serum samples for serology. Serology confirmed the previous status of the two flocks. Culture was negative for all the samples. In PCR, animals of Flock A presented 26.7% (VF), 33.3% (semen) and 38.9% (urine) of positivity. Flock B presented 40.0% (VF), 33.3% (semen) and 5.6% (urine) of positivity by PCR. In conclusion, PCR was important to identify carriers of leptospires, including animals from a seronegative flock, what reinforces the advantages of the usage of this tool for the detection of carriers in sheep as part of control programs of leptospirosis under tropical field conditions.
O objetivo do presente estudo foi analisar a aplicabilidade da PCR na detecção de ovinos carreadores de Leptospira em ambiente tropical. Dois rebanhos ovinos, previamente reportados como sororeativo (A) e soronegativo (B) foram selecionados para este estudo. Da totalidade de animais dos rebanhos, amostras de urina e fluido vaginal (FV)/sêmen foram colhidas para cultura bacteriológica e PCR. Além disso, amostras de soro foram colhidas e utilizadas na sorologia (teste da soroaglutinação microscópica). Essa técnica confirmou o estado prévio dos dois rebanhos. Nenhuma amostra pura de leptospiras foi obtida no cultivo. Já na PCR, animais do Rebanho A apresentaram 26,7% (FV), 33,3% (sêmen) e 38,9% (urina) de amostras positivas. O Rebanho B apresentou 40,0% (FV), 33,3% (sêmen) e 5,6% (urina) de positividade pela PCR. Em conclusão, a PCR foi uma importante ferramenta na identificação de carreadores de leptospiras, incluindo animais do rebanho soronegativo, o que reforça as vantagens do uso desta técnica para a detecção de ovinos portadores como parte dos programas de controle da leptospirose em ambiente tropical.
Subject(s)
Animals , Leptospira , Sheep/classification , Polymerase Chain ReactionABSTRACT
In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during metacyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.