Sixth Day Of Infection Research Articles

The Application of Impedance Spectroscopy for Pseudomonas Biofilm Monitoring during Phage Infection.

Bacterial biofilm prevention and eradication are common treatment problems, hence there is a need for advanced and precise experimental methods for its monitoring. Bacterial resistance to antibiotics has resulted in an interest in using a natural bacterial enemy—bacteriophages. In this study, we present the application of quartz tuning forks (QTF) as impedance sensors to determine in real-time the direct changes in Pseudomonas aeruginosa PAO1 biofilm growth dynamics during Pseudomonas phage LUZ 19 treatment at different multiplicities of infection (MOI). The impedance of the electric equivalent circuit (EEC) allowed us to measure the series resistance (Rs) corresponding to the growth-medium resistance (planktonic culture changes) and the conductance (G) corresponding to the level of QTF sensor surface coverage by bacterial cells and the extracellular polymer structure (EPS) matrix. It was shown that phage impacts on sessile cells (G dynamics) was very similar in the 10-day biofilm development regardless of applied MOI (0.1, 1 or 10). The application of phages at an early stage (at the sixth h) and on three-day biofilm caused a significant slowdown in biofilm dynamics, whereas the two-day biofilm turned out to be insensitive to phage infection. We observed an inhibitory effect of phage infection on the planktonic culture (Rs dynamics) regardless of the MOI applied and the time point of infection. Moreover, the Rs parameter made it possible to detect PAO1 population regrowth at the latest time points of incubation. The number of phage-insensitive forms reached the level of untreated culture at around the sixth day of infection. We conclude that the proposed impedance spectroscopy technique can be used to measure the physiological changes in the biofilm matrix composition, as well as the condition of planktonic cultures in order to evaluate the activity of anti-biofilm compounds.

Cytokine Profile in Early Infection by Leptospira interrogans in A/J Mice.

Leptospirosis is considered a neglected disease with an estimated more than one million cases every year. Since rodents are at the same time the main reservoir and generally asymptomatic to Leptospira infection, understanding why some animal species are resistant and others are susceptible to this infection would shed some light in how to control this important zoonosis. The innate immune response against Leptospira is mainly dependent on phagocytosis and activation of the Complement System. In this context, cytokines may drive the early control of infection and the adaptive response. Since the Complement System is important to eliminate leptospires in vivo, we investigated if Complement C5 in A/J mice would modulate the cytokine production during infection by Leptospira interrogans serovar Kennewicki type Pomona Fromm (LPF). Thus, our aim was to investigate the systemic levels of pro- and anti-inflammatory cytokines during Leptospira infection in the blood, liver, lung, and kidney on the third and sixth days of infection in A/J C5+/+ and A/J C5−/− mice. Blood levels of TNF-α, IL-6, IFN-γ, and MCP-1 reached a peak on the third day. Although both mouse strains developed splenomegaly, similar histopathological alterations in the liver and the lung, levels of pro- and anti-inflammatory cytokines were different. A/J C5+/+ mice had higher levels of liver IL-10, IL-1β, IL-12p40, and IL-12p70 and kidney IL-1β, IL-12p40, and IL-12p70 on the sixth day of infection when compared to A/J C5−/− mice. Our results showed that in A/J genetic background, the Complement component C5 modulates a cytokine profile in the liver and kidney of infected mice, which may play a role in the control of disease progression.

Role of Murine Complement Component C5 in Acute in Vivo Infection by Pathogenic Leptospira interrogans.

Leptospirosis is considered one of the most important zoonosis worldwide. The activation of the Complement System is important to control dissemination of several pathogens in the host. Only a few studies have employed murine models to investigate leptospiral infection and our aim in this work was to investigate the role of murine C5 during in vivo infection, comparing wild type C57BL/6 (B6 C5+/+) and congenic C57BL/6 (B6 C5−/−, C5 deficient) mice during the first days of infection. All animals from both groups survived for at least 8 days post-infection with pathogenic Leptospira interrogans serovar Kennewicki strain Fromm (LPF). At the third day of infection, we observed greater numbers of LPF in the liver of B6 C5−/− mice when compared to B6 C5+/+ mice. Later, on the sixth day of infection, the LPF population fell to undetectable levels in the livers of both groups of mice. On the third day, the inflammatory score was higher in the liver of B6 C5+/+ mice than in B6 C5−/− mice, and returned to normal on the sixth day of infection in both groups. No significant histopathological differences were observed in the lung, kidney and spleen from both infected B6 C5+/+ than B6 C5−/− mice. Likewise, the total number of circulating leukocytes was not affected by the absence of C5. The liver levels of IL-10 on the sixth day of infection was lower in the absence of C5 when compared to wild type mice. No significant differences were observed in the levels of several inflammatory cytokines when B6 C5+/+ and B6 C5−/− were compared. In conclusion, C5 may contribute to the direct killing of LPF in the first days of infection in C57BL/6 mice. On the other hand, other effector immune mechanisms probably compensate Complement impairment since the mice survival was not affected by the absence of C5 and its activated fragments, at least in the early stage of this infection.

Efecto inmunosupresor de <i>Trypanosoma lewisi</i> (Kinetoplastidae) sobre la multiplicación de <i>Toxoplasma gondii</i> (Sarcocystidae) en macrófagos alveolares y peritoneales de rata blanca

The immunosuppressant effect of T. lewisi infection on the multiplication of T. gondii was compared in peritoneal (MP) and alveolar macrophages (MA) of white rat. Two animal groups were infected with T. lewisi and sacrificed after four days and seven days post infection. A group without infection was maintained as a control. The number of intracellular parasites (tachyzoites) (IT) was counted by light microscopy, calculating the rate infection rate per 100 total cells (TC) and per infected cells (IC) for each group of phagocyte cells. The relation quotient IT, TC or IC multiplied percent, provided a statistical ratio (RE) of the relative number of parasites in both cellular types for each time interval. MA as well as MP obtained after 4 days showed a significant increase in the multiplication of T. gondii with respect to the control. Unlike the MP (which had an increase in the multiplication of T. gondii the fourth day of infection with T. lewisi diminishing towards the seventh day), the MA had an increase in the multiplication of the parasite from the fourth to the seventh day. This difference can be related to the route of infection used for the experiments, that affect the MP directly with a greater effect in comparison with the MA of the lungs. Lung compartment will be affected later, when the infection becomes systemic between the fourth and sixth day of infection. The immunity against T. gondii is similar between both phagocytes, but the time of infection and the compartment where the cells are located, makes the difference in the response time against T. gondii. Supernatants from macrophage cultures or T. lewisi by rat did not induced any immunosuppression.

Survival of Virulent Mycobacterium tuberculosis Involves Preventing Apoptosis Induced by Bcl‐2 Upregulation and Release Resulting from Necrosis in J774 Macrophages

Macrophage apoptosis plays a role in mycobacterial infection. To define the mechanism by which virulent Mycobacterium tuberculosis escapes apoptosis and killing in macrophages, J774 macrophages were infected with virulent M. tuberculosis H37Rv and attenuated H37Ra strains. H37Rv induced less apoptosis than H37Ra, and caspase 3 was activated in H37Ra- and H37Rv-infected macrophages. Intracellular H37Rv bacilli were released at a higher rate into the supernatant than were H37Ra by the sixth day of infection, and this was simultaneously accompanied by the increased necrosis of infected cells showing lactate dehydrogenase (LDH) release. Fas mRNA expression was downregulated and FasL was upregulated in H37Ra- and H37Rv-infected macrophages, while Bcl-2 was upregulated in H37Rv-infected macrophages but downregulated in H37Ra-infected macrophages as seen by real-time PCR. These results indicate that M. tuberculosis H37Ra and H37Rv proliferate in macrophages by preventing them from inducing apoptosis during the early phase of infection, and that M. tuberculosis H37Rv-infected macrophages are found to express Bcl-2 mRNA, which leads to anti-apoptotic activity, and that relatively distinct necrosis might occur during the later phase of infection.

In vivo development of Theileria annulata: major changes in efferent lymph following infection with sporozoites or allogeneic schizont-infected mononuclear cells.

The object of these experiments was to study the pathogenesis and kinetics of Theileria annulata infection in the efferent lymph of the draining lymph nodes of calves. Efferent lymphatics of calves were cannulated prior to infection with T. annulata sporozoite or an allogeneic schizont cell line. Potentially lethal sporozoite challenge induced cell shut-down from days 4-6 and then a massive increase in output of blasting cells (both infected and non-infected) in the efferent lymph. The rate of lymph flow and total cell output increased to 5 to 10-fold from day 6 onwards. Sporozoites were never isolated from the efferent lymph. However, large numbers of parasite-infected cells were seen in efferent lymph from the sixth day of infection. The animals inoculated with an allogeneic T. annulata-infected cell line exhibited only a small increase in flow rate and cell output. Parasite-infected cells of recipient origin were seen in efferent lymph from day 11 onwards. However, cells of donor origin were never isolated either from efferent lymph or peripheral blood. Thus the parasite transferred from the inoculated donor cell line to the cells of the recipient before schizonts appeared in efferent lymph.

Newcastle disease virus-induced apoptosis in the peripheral blood mononuclear cells of chickens

Specific pathogen-free chickens were inoculated with the GB strain of Newcastle disease virus by the ocular route, and were killed at daily intervals after infection. Peripheral blood mononuclear cells (PBMCs) were harvested for DNA extraction, electron microscopy, and flow cytometry studies to detect the presence of cellular apoptosis and necrosis. Extracted DNA showed fragmentation at 4 and 6 days after infection. Electron microscopy showed that the chromatin of the lymphocytes became condensed and occasionally crescent-shaped; apoptotic bodies could also be seen. Some heterophils became darkly stained and their nuclei degenerated; others seemed to have lost their cytoplasmic membrane, but had intact granules and condensed chromatin. Flow cytometry studies showed that the number of normal PBMCs gradually decreased from 99.04% at the beginning of the infection to 45.24% on the sixth day of infection, with a concomitant increase of apoptotic cells. The results show that PBMCs of chickens exhibit typical signs of apoptosis after infection by the Newcastle disease virus.

Complement Reversal of Immunosuppression Induced with Plasma of Rats Infected with Trypanosoma brucei rhodesiense

Suppression of antibody production by splenic lymphocytes from rats immunized with sheep red blood cells (SRBC) after incubation with plasma from rats infected with Trypanosoma brucei rhodesiense was confirmed. Suppressive activity became evident in plasma after the sixth day of infection and was manifested by reduction in the number of hemolytic Jerne plaques produced by the treated cells. The activity was temporally associated with increased amounts of soluble immune complex (SIC) reduced titers of lytic complement, elevated titers of immunoconglutinin (IK) and anemia. Treatment of suppressive plasma with hemolysin sensitized SRBC alexinated with horse complement to reduce IK did not reduce suppressive activity, and the activity appeared to have been enhanced when the plasma was heated to inactivate the remaining complement (C'). When fresh rat C' was added to the treated cells, the suppression was largely, though not completely, reversed. Treatment of spleen cells with SIC prepared in vitro from bovine serum albumin (BSA) and rabbit antiBSA also suppressed the plaque forming capacity of the cells. Complexes of BSA-antiBSA-C' and complexes of BSA-antiBSA-C'-IK were equally suppressive. Again, addition of fresh C' to cells treated with these complexes largely, though not completely, reversed the suppressive effect on the cells. From the results it is suggested that immunosuppression associated with experimental T. b. rhodesiense infection may be in part a suppression of the capacity of induced lymphocytes to produce antibody. It is possible that the suppression was mediated by SIC present in the plasma of the infected rats and this effect was probably enhanced by reduced levels of complement in the suppressive plasma.

Production and action of interferon in mice infected with rabies virus

Summary This study describes the production and action of interferon in mice infected with rabies virus (challenge virus strain). The production of interferon was monitored by an in vitro assay of plasma and extract of spleen, lung and brain for interferon activity. The action of interferon in mice was assessed by measuring two interferon-mediated enzyme activities, 2–5A synthetase and poly(I)poly(C)-Sepharose-bound protein kinase. Mice (strain C3H/He) infected with rabies virus were sacrificed at different time intervals, and the level of both enzyme activities was measured in extracts of spleen, lung and brain. Inoculation of mice with a mouse brain homogenate containing rabies virus resulted in the production of interferon with two different peaks detectable in the plasma: an early peak 24–48 h after inoculation followed by another peak on the sixth day of infection. The first peak was found to be independent of rabies virus, since it was also detectable in control mice injected with a brain homogenate from a normal uninfected mouse. The second peak of interferon, however, was probably due to the presence of high levels of interferon in the brain in response to rabies virus replication. In accord with the production of interferon, the level of 2–5A synthetase and protein kinase was enhanced several-fold in the different organs of mice infected with rabies virus. The level of protein kinase activity in the brain was strikingly higher than that of the spleen or the lung of mice 4 days after infection, and this level remained elevated until the death of the animals. These results indicate that infection of mice with rabies virus results in the production of interferon which is active in the infected mice, since different organs respond to it by elevated levels of 2–5A synthetase and protein kinase. Interferon produced during rabies virus infection, however, seems to be ineffective in inhibiting replication of virus, since these mice do not survive.

Immunization of guineapigs against Ascaris suum.

It has been shown that guineapigs can be effectively protected against re-infection by the subcutaneous inoculation of embryonated Ascaris eggs (Soulsby, 1957). The purpose of this communication is to report a successful attempt to immunize guineapigs against a lethal oral challenge dose of eggs by the intravenous inoculation of third stage larvae of A. suum.Three groups, each containing nine guineapigs, were infected with Ascaris as follows: Group I was given 10,000 eggs by mouth. Group II Was inoculated intravenously with 2,000 third stage Ascaris larvae which had been obtained from the lungs of other guineapigs on the sixth day of infection. Both groups were challenged at the same time with 250,000 eggs by mouth, along with a control group (Group III) which had no previous infection. The guineapigs in Group I were re-infected after 18 days and those in Group II after 12 days.