Galvanic current dosage and bacterial concentration are determinants of the bactericidal effect of percutaneous needle electrolysis: an in vitro study.

Percutaneous needle electrolysis (PNE) is a physiotherapy technique that has been shown to be effective in different pathologies such as tendinopathies or mammary fistula. For many years, theoretical bactericidal and germicidal effects have been attributed to this type of galvanic currents, partly explained by the changes in pH that it generates. However, these effects have not yet been demonstrated. The aim of this study was to evaluate the bactericidal effect and the changes in pH caused by PNE. S. aureus were prepared in two different solutions (TSB and saline solution) and in different concentrations (from 9 to 6 Log10 CFU/mL). Bacteria were treated with three experimental PNE doses to assess bacterial death levels and the changes caused to the pH of the medium. The viable cell count showed that all experimental PNE doses had a bactericidal effect against a high concentration (9 Log10 CFU/mL) of S. aureus in saline solution (p < 0.001). Furthermore, we found that when the concentration of bacteria decreased, a lower dose of galvanic current generated the same effect as a higher dose. Changes in pH were registered only in experiments performed with saline solution. PNE had a bactericidal effect against S. aureus and the level of this effect was mainly modulated by the solution, the bacterial concentration and the dose. Changes affecting pH were modulated by the type of solution and there was no relationship between this and bacterial death.

Immune transcriptome analysis in predatory beetles reveals two cecropin genes overexpressed in mandibles.

The great complexity and variety of the innate immune system and the production of antimicrobial peptides in insects is correlated with their evolutionary success and adaptation to different environments. Tiger beetles are an example of non-pest species with a cosmopolitan distribution, but the immune system is barely known and its study could provide useful information about the humoral immunity of predatory insects. Suppression subtractive hybridization (SSH) was performed in Calomera littoralis beetles to obtain a screening of those genes that were overexpressed after an injection with Escherichia coli lipopolysaccharide (LPS). Several genes were identified to be related to immune defense. Among those genes, two members of the cecropin antimicrobial peptides were characterized and identified as CliCec-A and CliCec-B2. Both protein sequences showed cecropin characteristics including 37 and 38 residue mature peptides, composed by two α-helices structures with amphipathic and hydrophobic nature, as shown in their predicted three-dimensional structure. Chemically synthesized CliCec-B2 confirmed cecropin antimicrobial activity against some Gram (+) and Gram (-) bacteria, but not against yeast. Expression of both cecropin genes was assessed by qPCR and showed increases after a LPS injection and highlighted their overexpression in adult beetle mandibles, which could be related to their alimentary habits.

Isolation of Chlamydia abortus from a laboratory worker diagnosed with atypical pneumonia.

BACKGROUND: Identifying the aetiological agent of atypical pneumonia in human can sometimes be a tedious process, especially in cases where Mycoplasma pneumoniae, Legionella species and Chlamydia pneumoniae are ruled out. In such cases, a correct anamnesis of the patient is basic to clarify which pathogens might have produced the infection. For this reason, health professionals including veterinarians and laboratory personnel working with zoonotic pathogens should keep their doctors informed. CASE PRESENTATION: A human case of atypical pneumonia linked to Chlamydia abortus is reported. A 47-year-old male, a veterinarian researcher into chlamydiae, developed respiratory symptoms, breathing problems and high fever. Serological analyses ruled out the involvement of several respiratory pathogens, such as M. pneumoniae, Legionella pneumophila, Rickettsia conorii and C. pneumoniae, and Chlamydia abortus was identified as the possible aetiological agent of the infection. The isolation of C. abortus from the patient's sputum and subsequent molecular analysis confirmed the presence of this microorganism. CONCLUSION: As far as we know, although C. abortus has not been previously described as capable of causing pneumonia in humans, this is the first reported case of atypical pneumonia in which C. abortus is thought to have played an aetiological role.

B cells are essential for moderating the inflammatory response and controlling bacterial multiplication in a mouse model of vaccination against Chlamydophila abortus infection.

The use of inactivated vaccines associated with suitable adjuvants has been demonstrated to confer a good level of protection against Chlamydophila abortus. However, the basis of the immune protective response induced by these vaccines has been poorly studied. B cells act as an immune regulatory population during primary infection by C. abortus. Thus, it was considered of interest to study the role of B cells in an infection after immunization with a killed vaccine. For this, C57BL/6 and B-cell-deficient mice were immunized with a killed vaccine against C. abortus using QS-21 as the adjuvant. After challenge, the course of infection was established by analysis of morbidity, C. abortus burden in the liver, and histopathological changes. The immune response induced was studied by real-time PCR techniques. Experiments involving transfer of immune serum from vaccinated or previously infected mice were also carried out. The lack of B cells reduced the protection conferred by the QS-21 adjuvant vaccine. Vaccinated B-cell-deficient mice showed a 1,000-fold-greater bacterial burden in the liver than their wild-type counterparts. Obvious differences existed in the liver, where a severe neutrophilic reaction and extended areas of necrosis were observed with vaccinated B-cell-deficient mice. An analysis of the immune response pointed to a significant increase in inflammatory cytokines and chemokines and the deficient production of transforming growth factor beta. The transfer of antibodies restored the level of protection. This study demonstrates that B cells play a crucial role in controlling C. abortus multiplication and prevent an exacerbated inflammatory response.

Evaluation of Chlamydophila abortus DNA extraction protocols for polymerase chain reaction diagnosis in paraffin-embedded tissues.

Polymerase chain reaction (PCR) has gained increasing importance as a tool for directly demonstrating the presence of Chlamydophila in the placentas of aborted sheep and goats. However, because of the zoonotic potential of the disease, it is advisable to use fixed materials. To evaluate 4 different DNA extraction protocols in paraffin-embedded sections for PCR, previously immunohistochemically diagnosed placental samples from outbreaks of abortions in goats and sheep were used. The samples were also used to evaluate the effect of the duration of fixation in formalin on PCR. A protocol that uses Tris-HCl pH 8.5 with EDTA and subsequent digestion with proteinase K was found to be an easy protocol for obtaining excellent PCR products for Chlamydophila abortus diagnosis from formalin-fixed and paraffin-embedded specimens. It was also found that if samples are fixed in formalin for more than 2 weeks, the PCR technique is affected more adversely than immunohistochemical methods.

Relationship between the immune response and protection conferred by new designed inactivated vaccines against ovine enzootic abortion in a mouse model.

Chlamydophila abortus is a gram-negative obligate intracellular bacterium and the etiological agent of ovine enzootic abortion (OEA), an economically important disease in many countries. Inactivated vaccines have been reported to induce immunity in ewes and they have been used for many years. However, some outbreaks have been reported in correctly vaccinated flocks, so it is clear that new vaccines are necessary to address adequate protection and to avoid the shedding of the microorganism. This idea lead us to design inactivated vaccines, in a previously established mouse model, evaluating different inactivation procedures and new adjuvants. To assess the protection conferred, the results were analyzed on the basis of clinical signs and the isolation of C. abortus from spleen. These findings were correlated with the immune response induced by the vaccines, as determined by the production of C. abortus-specific IFN-gamma and IL-4 from splenocyte cultures and the detection of IgG isotypes in serum. BEI was found to be the best C. abortus-inactivation procedure. The inactivated vaccines adjuvated with QS-21 (QS) or Montanide 773 (M7) induced the best protection both against homologous and heterologous challenge, with an adequate (Th1-like) immune response. Finally, these selected vaccines were evaluated in a pregnant mouse model, in which they were seen to confer good protection and to avoid the C. abortus persistence in uterus after delivery. With these results, this mouse model could be considered as an adequate tool for selecting and optimizing effective vaccines against OEA.

B-cell-deficient mice show an exacerbated inflammatory response in a model of Chlamydophila abortus infection.

The resolution of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection is dependent on gamma interferon and CD8(+) T cells, and classically, B cells have been considered to play a minimal role in host defense. The role of B cells in the immune response was studied by using a model of infection in mice with genetically modified immunoglobulin M transmembrane domains ( micro MT). In the absence of B cells, infection with C. abortus leads to an acute severe fatal disease that involves a disseminated intravascular coagulation syndrome. micro MT mice displayed an increased level of proinflammatory cytokines in serum, and an increased number of neutrophils was observed in the lesions. The possible deleterious role of neutrophils in the pathogenesis of disease in micro MT mice was determined by depletion of the neutrophils with the monoclonal antibody RB6-8C5. This led to an enhancement of the bacterial burden and early mortality in both micro MT and wild-type mice, while necrotic lesions remained. Analysis of the presence of immunoregulatory cytokines showed significantly lower levels of transforming growth factor beta in the sera of micro MT mice. However, mice lacking mature B cells were able to establish a specific immune response that protected them from a secondary challenge. Taken together, these data suggest an immunomodulatory role for B cells in the early events of C. abortus primary infection that can protect mice against an exaggerated inflammatory response.