Monoamine Changes in the Brain of BALB/c Mice Following Sub-Lethal Infection with Nocardia asteroides (GUH-2)

BALB/c mice injected intravenously with a single, sub-lethal dose of Nocardia asteroides GUH-2 develop several levodopa responsive movement disorders. These included head-shake, stooped posture, bradykinesia, and hesitation to forward movement (6). The changes in monoamine levels in the brain of these mice were determined. There was a significant loss of dopamine with greatly increased dopamine turnover in the neostriatum 7 to 29 days after infection. These effects were specific for dopaminergic neurons since minimal changes were found in neostriatal norepinephrine and serotonin even though serotonin turnover was increased. Changes in monoamine metabolism were not limited to the neostriatum. There were reduced levels of serotonin and norepinephrine with increased serotonin turnover in the cerebellum. One year after infection, dopamine metabolism had returned to near normal levels, but many of the movement disorders persisted. Specific changes in neurochemistry did not always appear to correspond with these impairments. Nevertheless, these data are similar to those reported in MPTP treated BALB/c mice.     

Direct PCR detection of phytoplasmas in experimentally infected insects

Phytoplasmas in leafhoppers have been detected by PCR using chrysanthemum yellows (CY)-infected chrysanthemum as source plants, and two cicadellid Deltocephalinae species, Macrosteles quadripunctulatus and Euscelis incisus, as vectors. Three different primer pairs were used; two of these are universal and have been designed on conserved sequences of the 16S rRNA gene of phytoplasmas, and one was designed on extrachromosomal DNA of a severe strain of western aster yellows phytoplasma. They were used to amplify CY DNA obtained by two different extraction procedures; one was extraction with cetyl-trimethyl-ammonium-bromide (CTAB), and the other was boiling in Tris-EDTA buffer. The chromosomal primers amplified phytoplasma-specific bands only from “CTAB” samples, while the plasmid primers were successful with both procedures. Amplification of phytoplasma DNA was possible from as little as 1/10000 of total DNA extracted from a single hopper. Failure to amplify phytoplasma DNA from insects stored at –20^oC for 2 yr suggested that some kind of inhibition develops during long term tissue storage. Direct PCR appeared a very specific, sensitive and rapid method to detect phytoplasmas in fresh leafhoppers, provided that a proper combination of extraction and amplification procedures was used.     

感染小鼠组织中Q热立克次体的分子病理学检测

腹腔注射Q热立克次体悬液感染BALB／c小鼠,发病后解剖,取主要组织脏器,应用免疫组化和原位杂交检测感染小鼠体内Q热立克次体的抗原分布和DNA在靶细胞中的表达,探讨Q热病变规律及致病机理。结果显示阳性信号多位于单核-巨噬细胞系统细胞胞浆内。结果提示免疫组化和原位杂交技术可以作为Q热特异性的诊断方法,并为致病机理的研究提供线索。     

Leishmania infantum: mixed T-helper-1/T-helper-2 immune response in experimentally infected BALB/c mice

The main goal of the present study was to characterise the course of infection and immunological responses developed by Leishmania infantum infected BALB/c mice. Parasite load was determined by Real-time TaqMan PCR while cytokine and Immunoglobulin G (IgG) production were assessed by ELISA. Leishmania DNA was detected in spleen and liver as soon as day 1 post-inoculation (pi) and the parasitism was sustained until the end of the experiment. The cytokine kinetics in spleen and liver was generally associated with the oscillations of parasite load. Overall, it was not observed a distinct Th1 or Th2 pattern of cytokine production during the time of experiment. The infected mice developed a mixed immune response, with concomitant production of IFN-gamma, IL-4 and IL-10, both in spleen and liver, and both IgG isotypes. However, our results suggest that, compared to liver, the spleen is more susceptible to L. infantum infection.     

Nested PCR assay for the rapid detection of Naegleria fowleri from swimming pools in Egypt

The free-living amoeboflagellate Naegleria fowleri is the only species infects humans world widely distributed. N. fowleri is the causative agent of very rare but severe brain infection called primary amoebic meningo-encephalitis (PAM), a rapidly fatal disease of the central nervous system mainly in immuno-compromised individuals. N. fowleri infects human through the entry of the nose, and it happens when human swimming or diving in warm freshwater, such as lakes, rivers and swimming pools. The disease is acute, and patients often die within 5–10 days and before the infectious agent can be diagnosed. Limited information is available about the existence of pathogenic N. fowleri, in Egypt, so the present of N. fowleri is an important public health. In the present study, we examined hundred water, dust and swap samples collected from 5 swimming pools in Cairo, Egypt. Based on morphological characteristics of trophozoite and cyst, flagellation test 56% of thermo-tolerant Naegleria like amoeba was detected. The incidence of thermo-tolerant free-living amoebae reached 84, 80and 70% from water, cotton swap and dust samples, respectively at cultivation temperature of 45 °C. The highest occurrence of thermo-tolerant amoebae were recorded in summer (100 & 87.5%) while the lowest one were recorded in winter (58 & 37.5%) in both water and dust samples, respectively. In swap samples, the highest occurrence of thermo-tolerant free-living amoeba was recorded in both summer and spring (100%), while the lowest one was recorded in winter (40%). N. fowleri was performed on 24 samples from a total of 56 (42.2%) samples which are positive by culture. Nested PCR using Mp2Cl5 gene primers that is unique to N. fowleri was carried out. The N. fowleri specific primer showed band at 166 bp against 24 of 56 (42.2%) samples. The majority of positive samples unique to N. fowleri was detected in water samples followed by swap samples and finally dust samples 14 of 24 (58%), 7 of 24 (29%), 3 of 24 (13%), respectively. In conclusion, swimming pools water may be the source of Naegleria invasion. The use of molecular methods to identify free-living amoebae N. fowleri could provide a more rapid means to diagnose infections caused by those amoebae.     

Therapeutic effect of a novel oxazolidinone, DA-7867, in BALB/c mice infected with Nocardia brasiliensis

Background

Mycetoma is a chronic infectious disease of tropical and subtropical countries. It is produced by true fungi and actinobacteria. In México, Nocardia brasiliensis is the main causative agent of mycetoma, producing about 86% of the cases; the gold standard for the therapy of mycetoma by N. brasiliensis is the use of sulfonamides which give a 70% cure rate. The addition of amikacin to this regime increases to 95% the cure rate; however, the patients have to be monitored for creatinine clearance and audiometry studies because of the potential development of side effects. Because of that it is important to search for new active compounds. In the present work, we evaluated the in vivo effect of DA-7867, an experimental oxazolidinone, on the development of experimental mycetomas by N. brasiliensis in BALB/c mice.

Methodology/Principal Findings

In order to determine the optimal dose utilized to apply to the animals, we first determined by HPLC the plasma levels using several concentrations of the compounds. Based on these results, we used 10 and 25 mg/kg subcutaneously every 24 hr; DA-7867 was also supplied in the drinking water at a calculated dose of 25 mg/kg. As a control we utilized linezolid at 25 mg/kg, a compound active in murine and human infections, three times a day. The mice were infected in the right footpad with a young culture of N. brasiliensis HUJEG-1, and one week later we started the application of the antimicrobials for six more weeks. After that we compared the development of lesions in the groups injected with saline solution or with the antimicrobials; the results were analyzed by the variance ANOVA test. DA-7867 was able to reduce the production of lesions at 25 mg/kg, when given either subcutaneously or in the drinking water.

Conclusions/Significance

The experimental oxazolidinone DA-7867 is active in vivo against N. brasiliensis, which opens the possibility of using this drug once it is accepted for human application. Since oxazolidinones seem to be active against a wide spectrum of actinobacteria, it is possible they could be used in human cases of mycetoma by other actinomycetales, such as Streptomyces somaliensis, highly prevalent in Sudan, or Actinomadura madurae and A. pelletieri, which are commonly observed in Africa and India.     

Non-sand fly transmission of a North American isolate of Leishmania infantum in experimentally infected BALB/c mice

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, is endemic in the foxhound population in the United States and Canada. Leishmaniasis is usually transmitted by blood-feeding sand flies; however, epidemiological data do not support a significant role for sand flies in the maintenance of foxhound infections in North America, and an alternate mode of transmission may exist. The present study was conducted to determine if transplacental or direct transmission occurs in pregnant BALB/c mice experimentally infected with L. infantum isolated from a naturally infected foxhound from Virginia as well as to determine if the parasite was directly transmitted to the males used to breed the mice. Female BALB/c mice were intravenously inoculated with 1 x 10(6) promastigotes of the LIVT-1 strain of L. infantum. Mice were bred to uninfected male BALB/c mice 2 mo postinoculation. Pregnant mice were killed between days 13 and 18 of gestation. Pups and placentas were collected at necropsy, divided, and used for parasite culture and polymerase chain reaction (PCR) analyses. Culture and PCR analyses were performed on spleens from the male mice to determine the possibility of sexual transmission. Leishmania sp. DNA was detected in 4 of 88 pups and 3 of 16 placentas from LIVT-1-inoculated mice. One male mouse used to breed infected females was PCR positive. This work provides evidence for a low level of nonvector transmission of North American L. infantum in a mouse model.     

Maximum growth rate of Mycobacterium avium in continuous culture or chronically infected BALB/c mice

Mycobacterium avium is a human pathogen which may cause either chronic or disseminated disease and the organism exhibits a slow rate of growth. This study provides information on the growth rate of the organism in chronically infected mice and its maximal growth rate in vitro. M. avium was grown in continuous culture, limited for nitrogen with 0.5 mM ammonium chloride and dilution rates that ranged from 0.054 to 0.153 h-1. The steady-state concentration of ammonia nitrogen and M. avium cells for each dilution rate were determined. The bacterial saturation constant for growth-limiting ammonia was 0.29 mM (4 micrograms nitrogen/ml) and, from this, the maximal growth rate for M. avium was estimated to be 0.206 h-1 or a doubling time of 3.4 h. BALB/c mice were infected intravenously with 3 x 10(6) colony-forming units and a chronic infection resulted, typical of virulent M. avium strains. During a period of 3 months, the number of mycobacteria remained constant in the lungs, but increased 30-fold and 8,900-fold, respectively, in the spleen and mesenteric lymph nodes. The latter increase appeared to be due to proliferation in situ. The generation time of M. avium in the mesenteric lymph nodes was estimated to be 7 days.     

猪圆环病毒3型感染BALB/c小鼠的实验观察

目的研究猪圆环病毒3型(porcine circovirus3,PCV3)对BALB/c小鼠的感染情况。方法将雌性BALB/c小鼠分为两组,实验组感染PCV3组织毒,对照组接种同样剂量的PBS。感染后每天观察小鼠状态,并在第0,3,7,11和14天采血进行荧光定量PCR检测和ELISA检测。实验结束后,对所有动物进行安乐死和剖检,对心脏、肝、脾、肺、肾、脑和淋巴结取样进行荧光定量PCR检测,并制片进行组织病理学检查。选择PCR检测阳性组织进行PCV3CAP基因测序分析。结果PCV3组织毒感染小鼠不引起明显的临床症状和病理变化。病毒可在感染早期的血清中检测到,病毒含量最高的器官是肝脏和脾脏,PCV3感染小鼠后核酸序列未发生变化。随着感染时间的增加血清中抗体水平逐渐升高。结论PCV3可以感染BALB/c小鼠,并在小鼠体内增殖。本研究结果为猪圆环病毒3型致病性的研究以及防控提供了参考。     

Trapped in keratin; a comparison of dermatophyte detection in nail, skin and hair samples directly from clinical samples using culture and real-time PCR

Traditionally, laboratory detection and identification of dermatophytes consists of culture and microscopy which yields results within approximately 2-6 weeks. In 2007 our medical microbiological diagnostic laboratory implemented a molecular method for the detection of dermatophytes. A real-time PCR assay was developed which simultaneously detects and identifies the most prevalent dermatophytes directly in nail, skin and hair samples and has a turnaround time of less than two days. For 1437 clinical samples, received by our diagnostic laboratory, we compared the results obtained from both culture and real-time PCR. This study showed that real-time PCR significantly increased the detection rate of dermatophytes compared to culture. Furthermore, excellent concordance between culture and real-time PCR identification was achieved.     

博尔纳病病毒对BALB/c小鼠感染性检测

目的分析博尔纳病病毒（Borna disease virus,BDV）H1766株对BALB/c小鼠的感染性。方法选择病毒滴度为2.0×107FFU/ml的BDV病毒液分别对新生和成年BALB/c小鼠进行脑内接种,并用相同病毒液对原代培养的新生BALB/c小鼠脑细胞进行接种。经过一定时间的病毒作用后分别提取总RNA,采用巢式RT-PCR方法检测BDV-p40基因,并通过免疫组化方法检测脑内接种脑组织中BDV-P40蛋白。结果脑内接种病毒的小鼠脑组织中可以检测到BDV-p40基因和BDV-P40蛋白,培养的小鼠脑细胞中可以检测到BDV-p40基因。结论BDVH1766株可以感染新生和成年的BALB/c小鼠。     

Quantitative detection for low levels of Helicobacter pylori infection in experimentally infected mice by real-time PCR

Accurate diagnosis of Helicobacter pylori infection is important in both clinical practice and clinical research. Molecular methods are highly specific and sensitive, and various PCR-based tests have been developed to detect H. pylori in gastric biopsy specimens. We optimized a sensitive and specific quantitative SYBR Green I real-time PCR assay for detection of H. pylori based on amplification of the fragment of a 26-kDa Helicobacter species-specific antigen gene that allows for detection of 5 bacterial cells per PCR sample. Under the assay conditions, SYBR Green I real-time PCR is highly reproducible with a precise log-linear relation in the range of six orders of magnitude of bacterial DNA concentrations. For accurate comparison of H. pylori infection in different tissue samples, the amount of total host DNA in each sample is normalized by TaqMan real-time PCR of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pseudogenes. The developed method was validated in prophilactically immunized and experimentally infected mice and revealed a level of H. pylori gastric colonisation that was below the limit of detection for a rapid urease test. This new method established for a quantitative analysis of H. pylori in the host's stomach may be useful in experimental studies evaluating new anti-H. pylori drugs and vaccines.     

IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice

Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti- N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis . IgG mAb lacked this protective effect. IgM mAb showed a dose–response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection.     

Follow-up of antibody avidity in BALB/c mice infected with Toxocara canis

In human Toxocara canis infection, an association has been shown between high IgG avidity in the chronic phase and low IgG avidity in recently acquired toxocarosis. The evolution of the antibody response in terms of avidity has been carried out through a T. canis infection in BALB/c mice. Infection with T. canis embryonated eggs (EE) was carried out with single doses (SD) of 6, 12, 50, 100, 200 or 1000 EE/mouse and with multiple doses (MD) of 200 and 1000 EE. Specific antibodies against T. canis (IgM+G, IgG, IgG1 and IgM) were detected by ELISA and Western Blot (WB) techniques in the presence and absence of urea. With the ELISA method, an increase in the avidity index (AI) of around 50% was detected from days 40-80 p.i. to the end of the study, with all the doses studied. The WB method showed the presence of high avidity antibodies bound to 100 kDa and 75 kDa T. canis proteins in all the cases when the IgM+G and the IgG1 antibodies were investigated. Antibodies of variable avidity were observed in those sera that recognized the group of low molecular weight proteins, between 37 kDa and 25 kDa.     

Immune responses mediating survival of naive BALB/c mice experimentally infected with lethal rodent malaria parasite, Plasmodium yoelii nigeriensis

The rodent malaria parasite, Plasmodium yoelii nigeriensis is known to cause fatal malaria infections in BALB/c mice. However, we found that nearly 5% of inbred BALB/c mice could overcome primary infections initiated with lethal inoculum of P. y. nigeriensis asexual blood-stages, without any experimental intervention. These 'survivor' mice developed peak parasitemia levels of about 5% and successfully resolved their infections in about two weeks time; infected blood collected during the descending phase of infection in these mice and subinoculated in naive recipients resulted in a normal lethal course of infection. Typically, the parasites in survivor mice looked 'sick' compared to those in the susceptible mice. In experiments to define temporal basis of this protection, we found that purified splenic B cells isolated from such a survivor mouse, plus T cells from an infected or naive mouse, could adoptively transfer this protection to an X-irradiated, naive mouse against a lethal parasite challenge. Purified T cells or B cells alone from the survivor mouse donor provided no protection to the X-irradiated, naive recipient. Passive transfer of sera collected from survivor mice animals a week after recovery from infection was also able to substantially alter the course of preestablished P. y. nigeriensis infection. These findings are discussed in the light of recent reports on the genetic control of blood parasitemia in mouse malaria models. In the generally lethal malaria infections such as those caused by P. y. nigeriensis in mice and by Plasmodium falciparum in naive children, it is not clear what constitutes a protective immune response in cases which survive primary infections without any experimental or therapeutic intervention. An understanding of these mechanisms and their regulation would help design better vaccination strategies.     

儿童激素耐药型肾病综合征合并星形诺卡菌脑脓肿1例

本文报道1例激素耐药型肾病综合征儿童合并星形诺卡菌(Nocardia asteroides,N.asteroides)脑脓肿。患儿,男性,8岁,临床诊断为原发性肾病综合征(激素耐药型),病理诊断为局灶节段性肾小球硬化症(经典型)。肾穿后第4天患儿出现持续高热、抽搐,时有头痛,抗感染、抗凝治疗效果不佳。复查颅脑磁共振成像(magnetic resonance imaging,MRI)提示多发脑脓肿。头颅脓肿液经穿刺后培养显示为星形诺卡菌感染。予以多种抗生素联合糖皮质激素等治疗2个月,患儿体温正常,头痛缓解,脑脓肿范围明显缩小。因此,肾病综合征患儿在应用激素及免疫抑制剂治疗过程中如出现化脓性炎症,常规抗生素疗效差,应积极寻找病原,高度警惕诺卡菌病及其他机会性感染的可能。     

Immunogenicity is unrelated to protective immunity when induced by soluble and particulate antigens from Nocardia brasiliensis in BALB/c mice

Cell-mediated immunity plays a major role in protection against intracellular microbes. Nocardia brasiliensis is a facultative intracellular pathogen that causes chronic actinomycetoma. In this work, we injected BALB/c mice with soluble P24 and particulate antigens from N. brasiliensis. A higher antibody titer and lymphocyte proliferation was induced by the particulate antigen than by the soluble antigen. However, five months after antigen injection, antibody concentration and lymphocyte proliferation were similar. An increase in CD45R and CD4 T cells was unrelated to protective immunity. Active immunization with soluble or particulate antigens induced complete protection during the primary immune response. This protective response was IgM mediated. The higher immunogenicity was not related to protective immunity since the particulate antigen induced protection similar to the soluble antigen. Using particulate antigens for vaccination guarantees a stronger immune response, local and systemic side effects, but not necessarily protection.