Ribotyping and virulence markers of Yersinia pseudotuberculosis strains isolated from animals in Brazil

Ribotyping and virulence markers has been used to investigate 68 Yersinia pseudotuberculosis strains of serogroups O:1a and O:3. The strains were isolated from clinical material obtained from healthy and sick animals in the Southern region of Brazil. Ribotypes were identified by double digestion of extracted DNA with the restriction endonucleases SmaI and PstI, separation by electrophoresis and hybridization with a digoxigenin-labeled cDNA probe. The presence of the chromosomal virulence marker genes inv, irp1, irp2, psn, ybtE, ybtP-ybtQ, and ybtX-ybtS, of the IS100 insertion sequence, and of the plasmid gene lcrF was detected by polymerase chain reaction. The strains were grouped into four distinct ribotypes, all of them comprising several strains. Ribotypes 1 and 4 presented distinct profiles, with 57.3% genetic similarity, ribotypes 2 and 3 presented 52.5% genetic similarity, and genetic similarity was 45% between these two groups (1/4 and 2/3). All strains possessed the inv, irp1, and irp2 genes. Additionally, strains of serogroup O:1a carried psn, ybtE, ybtP-ybtQ, ybtX-ybtS, and IS100. As expected lcrF was only detected in strains harboring the virulence plasmid. These data demonstrate the presence of Y. pseudotuberculosis strains harboring genotypic virulence markers in the livestock from Southern Brazil and that the dissemination of these bacteria may occur between herds.

The involvement of tetA and tetE tetracycline resistance genes in plasmid and chromosomal resistance of Aeromonas in Brazilian strains

This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5% of the samples were positive for tetA, and also 37.5% were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.

Isolation and molecular identification of Leishmania chagasi from a bat (Carollia perspicillata) in northeastern Venezuela

This report describes the isolation of a Leishmania chagasi strain from a bat (Carollia perspicillata), and its identification using biological methods and molecular characterization. The parasites were isolated in an artificial culture medium from a blood sample extracted from a bat heart. The isolate was then inoculated into the footpads of Balb/c mice, which subsequently developed a typical nodular leishmanial lesion; the parasites were confirmed as Leishmania by smear and histopathology. Molecular characterization of the parasites was performed by polymerase chain reaction with species-specific primers, kDNA restriction pattern following Hae III endonuclease digestion and dot blot hybridization using a kDNA probe. This report demonstrates that bats can be hosts for L. chagasi species and suggests the need for studies to determine whether they may be involved in foci of visceral leishmaniasis.

Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.

Mucosal Healing with Anti-TNF Antibodies.

Nowadays, mucosal healing is regarded as a major end point in clinical trials and is increasingly used in clinical practice for the management of patients with inflammatory bowel disease. The definition of mucosal healing varies across studies and validated endoscopic scoring indices are still lacking. The advent of anti-tumor necrosis factor agents has changed the way of treating inflammatory bowel disease and high rates of induction and maintenance of mucosal healing can be achieved with this drug class. Mucosal healing is desirable as it may change the natural course of the disease by decreasing surgery and hospitalization rates in both ulcerative colitis and Crohn's disease.

Larval recovery of Toxocara cati in experimentally infected Rattus norvegicus and analysis of the rat as potential reservoir for this ascarid

Toxocara cati is a common feline parasite transmitted by the ingestion of embryonated eggs, by the transmammary route or by predation of paratenic hosts harbouring third-stage larvae in their bodies. In the present study, the larval distribution of T. cati in tissues and organs of Rattus norvegicus experimentally infected with 300 embryonated eggs was analysed. Third-stage larvae were recovered from livers, lungs, kidneys, eyes, brains and carcasses of infected rats, following tissue digestion with HCl 0.5% for 24 h at 37°C. Some differences from the known larval distribution of Toxocara canisin the same rodent species were found.

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

Molecular mechanisms of Trypanosoma cruzi infection by oral route

Frequent reports on outbreaks of acute Chagas' disease by ingestion of food contaminated with parasites from triatomine insects illustrate the importance of this mode of transmission. Studies on oral Trypanosoma cruzi infection in mice have indicated that metacyclic trypomastigotes invade the gastric mucosal epithelium. A key molecule in this process is gp82, a stage-specific surface glycoprotein that binds to both gastric mucin and to target epithelial cells. By triggering Ca2+ signalling, gp82 promotes parasite internalisation. Gp82 is relatively resistant to peptic digestion at acidic pH, thus preserving the properties critical for oral infection. The infection process is also influenced by gp90, a metacyclic stage-specific molecule that negatively regulates the invasion process. T. cruzi strains expressing high gp90 levels invade cells poorly in vitro. However, their infectivity by oral route varies considerably due to varying susceptibilities of different gp90 isoforms to peptic digestion. Parasites expressing pepsin-susceptible gp90 become highly invasive against target cells upon contact with gastric juice. Such is the case of a T. cruzi isolate from an acute case of orally acquired Chagas' disease; the gp90 from this strain is extensively degraded upon short period of parasite permanence in the gastric milieu. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of Chagas' disease reported in outbreaks of oral infection.

Do Archaea and bacteria co-infection have a role in the pathogenesis of chronic chagasic cardiopathy?

Chronic cardiopathy (CC) in Chagas disease is a fibrotic myocarditis with C5b-9 complement deposition. Mycoplasma and Chlamydia may interfere with the complement response. Proteolytic enzymes and archaeal genes that have been described in Trypanosoma cruzi may increase its virulence. Here we tested the hypothesis that different ratios of Mycoplasma, Chlamydia and archaeal organisms, which are frequent symbionts, may be associated with chagasic clinical forms. MATERIALS AND METHODS: eight indeterminate form (IF) and 20 CC chagasic endomyocardial biopsies were submitted to in situ hybridization, electron and immunoelectron microscopy and PCR techniques for detection of Mycoplasma pneumoniae (MP), Chlamydia pneumoniae(CP), C5b-9 and archaeal-like bodies. RESULTS: MP and CP-DNA were always present at lower levels in CC than in IF (p < 0.001) and were correlated with each other only in CC. Electron microscopy revealed Mycoplasma, Chlamydia and two types of archaeal-like bodies. One had electron dense lipid content (EDL) and was mainly present in IF. The other had electron lucent content (ELC) and was mainly present in CC. In this group, ELC correlated negatively with the other microbes and EDL and positively with C5b-9. The CC group was positive for Archaea and T. cruzi DNA. In conclusion, different amounts of Mycoplasma, Chlamydia and archaeal organisms may be implicated in complement activation and may have a role in Chagas disease outcome.

Proline racemases: insights into Trypanosoma cruzi peptides containing D-proline

Trypanosoma cruzi proline racemases (TcPRAC) are homodimeric enzymes that interconvert the L and D-enantiomers of proline. At least two paralogous copies of proline racemase (PR) genes are present per parasite haploid genome and they are differentially expressed during T. cruzi development. Non-infective epimastigote forms that overexpress PR genes differentiate more readily into metacyclic infective forms that are more invasive to host cells, indicating that PR participates in mechanisms of virulence acquisition. Using a combination of biochemical and enzymatic methods, we show here that, in addition to free D-amino acids, non-infective epimastigote and infective metacyclic parasite extracts possess peptides composed notably of D-proline. The relative contribution of TcPRAC to D-proline availability and its further assembly into peptides was estimated through the use of wild-type parasites and parasites over-expressing TcPRAC genes. Our data suggest that D-proline-bearing peptides, similarly to the mucopeptide layer of bacterial cell walls, may be of benefit to T. cruzi by providing resistance against host proteolytic mechanisms.

Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

JNK3 is abundant in insulin-secreting cells and protects against cytokine-induced apoptosis.

AIMS/HYPOTHESIS: In insulin-secreting cells, activation of the c-Jun NH(2)-terminal kinase (JNK) pathway triggers apoptosis. Whereas JNK1 and JNK2 are ubiquitously produced, JNK3 has been described exclusively in neurons. This report aims to characterise the expression and role in apoptosis of the three JNK isoforms in insulin-secreting cells exposed to cytokines. METHODS: Sections of human and mouse pancreases were used for immunohistochemistry studies with isoform-specific anti-JNK antibodies. Human, pig, mouse and rat pancreatic islets were isolated by enzymatic digestion and RNA or protein extracts were prepared. RNA and protein levels were determined by quantitative RT-PCR and western blotting respectively, using JNK-isoform-specific primers and isoform-specific antibodies; activities of the three JNK isoforms were determined by kinase assays following quantitative immunoprecipitation/depletion of JNK3. JNK silencing was performed with small interfering RNAs and apoptotic rates were determined in INS-1E cells by scoring cells displaying pycnotic nuclei. RESULTS: JNK3 and JNK2 mRNAs are the predominant isoforms expressed in human pancreatic islets. JNK3 is nuclear while JNK2 is also cytoplasmic. In INS-1E cells, JNK3 knockdown increases c-Jun levels and caspase-3 cleavage and sensitises cells to cytokine-induced apoptosis; in contrast, JNK1 or JNK2 knockdown is protective. CONCLUSIONS/INTERPRETATION: In insulin-secreting cells, JNK3 plays an active role in preserving pancreatic beta cell mass from cytokine attacks. The specific localisation of JNK3 in the nucleus, its recruitment by cytokines, and its effects on key transcription factors such as c-Jun, indicate that JNK3 is certainly an important player in the transcriptional control of genes expressed in insulin-secreting cells.

Rotavirus A genotype G1P[8]: a novel method to distinguish wild-type strains from the Rotarix® vaccine strain

Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix®. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix® vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.

Schistosoma mansoni triggers Dectin-2, which activates the Nlrp3 inflammasome and alters adaptive immune responses.

The propensity of helminths, such as schistosomes, to immunomodulate the host's immune system is an essential aspect of their survival. Previous research has demonstrated how soluble schistosomal egg antigens (SEA) dampen TLR-signaling during innate immune responses. We show here that the suppressive effect by SEA on TLR signaling is simultaneously coupled to the activation of the Nlrp3 (NLR family, pyrin domain containing 3) inflammasome and thus IL-1β production. Therefore, the responsible protein component of SEA contains the second signal that is required to trigger proteolytic pro-IL-1β processing. Moreover, the SEA component binds to the Dectin-2/FcRγ (Fc receptor γ chain) complex and activates the Syk kinase signaling pathway to induce reactive oxygen species and potassium efflux. As IL-1β has been shown to be an essential orchestrator against several pathogens we studied the in vivo consequences of Schistosoma mansoni infection in mice deficient in the central inflammasome adapter ASC and Nlrp3 molecule. These mice failed to induce local IL-1β levels in the liver and showed decreased immunopathology. Interestingly, antigen-specific Th1, Th2, and Th17 responses were down-regulated. Overall, these data imply that component(s) within SEA induce IL-1β production and unravel a crucial role of Nlrp3 during S. mansoni infection.

Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.