Treatment and prevention of Pneumocystis carinii pneumonia and further elucidation of the P. carinii life cycle with 1,3-beta-glucan synthesis inhibitor L-671,329.

Two different classes of 1,3-beta-glucan synthesis inhibitors, the echinocandins and papulacandins, have anti-Pneumocystis activity in an immunosuppressed rat model for acute P. carinii pneumonia (PCP). This activity combined with potent anti-Candida activity makes the echinocandins attractive agents for treating both Pneumocystis and candidiasis in the immunocompromised patient. Natural product echinocandin L-671,329 rapidly eliminates greater than 99% of the P. carinii cysts after 4 days of treatment at a dose of 1 mg/kg twice daily while 2-3 weeks of therapy with trimethoprimsulfamethoxazole (TMP-SMZ) or pentamidine was required to achieve the same degree of cyst clearance. Effects of L-671,329, TMP-SMZ and pentamidine on the trophozoite stage of P. carinii were also explored using a P. carinii-specific DNA probe to quantitate organism load. Although L-671,329 was not as effective as the known agents against the trophozoite stage, prophylactic use of L-671,329 at a daily dose of 1 mg/kg prevented the development of cysts and trophozoites in the rat model. The foamy exudate commonly seen in lungs of animals with PCP is also absent in rats receiving L-671,329 prophylaxis. In addition to demonstrating the potential of L-671,329 as a prophylactic agent these studies also help in elucidating the life cycle of P. carinii. The observation that L-671,329 prophylaxis prevents the appearance of trophozoites, while acute therapy does not directly affect trophozoites, provides the first evidence that the cyst stage is required for trophozoite proliferation. The rapid elimination of cysts by L-671,329 in animals with acute PCP also indicates that all cysts are turning over within 4 days since it is the development of new cysts which is prevented with this compound.     

CoMFA/CoMSIA 3D-QSAR of pyrimidine inhibitors of Pneumocystis carinii dihydrofolate reductase

Pneumocystis carinii is typically a non-pathogenic fungus found in the respiratory tract of healthy humans. However, it may cause P. carinii pneumonia (PCP) in people with immune deficiency, affecting mainly premature babies, cancer patients and transplant recipients, and people with acquired immunodeficiency syndrome (AIDS). In the latter group, PCP occurs in approximately 80% of patients, a major cause of death. Currently, there are many available therapies to treat PCP patients, including P. carinii dihydrofolate reductase (PcDHFR) inhibitors, such as trimetrexate (TMX), piritrexim (PTX), trimethoprim (TMP), and pyrimethamine (PMT). Nevertheless, the high percentage of adverse side effects and the limited therapeutic success of the current drug therapy justify the search for new drugs rationally planned against PCP. This work focuses on the study of pyrimidine inhibitors of PcDHFR, using both CoMFA and CoMSIA 3D-QSAR methods.     

An improved rat model of Pneumocystis carinii pneumonia: induced infections in Pneumocystis-free animals.

An immunosuppressed rat model of Pneumocystis carinii pneumonia (PCP) is described that results in a predictable course of disease development which includes moderate P. carinii (Pc) infections in 2 to 3 weeks, heavy infections in 4 to 5 wk, and a high percentage of mortality due to PCP in 6 wk. The model also provides uninfected, immunosuppressed contemporary controls, an experimental compartment that is needed to correctly interpret results obtained from many different studies. Non-invasive intratracheal inoculation of cryopreserved parasites into Pc- and virus-free rats immunosuppressed by weekly injections of methylprednisolone are key features of the model that result in the development of consistent heavy Pc infections and very few secondary infections by bacteria and fungi. This model is useful for (1) maintaining isolates or strains of Pc over time, (2) producing large numbers of parasites for laboratory studies, and (3) evaluating the anti-Pc activity of experimental compounds and approved drugs.     

A Bead-Based Multiplex Assay for the Detection of DNA Viruses Infecting Laboratory Rodents

The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals’ physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.     

Lurking in the shadows: emerging rodent infectious diseases

Rodent parvoviruses, Helicobacter spp., murine norovirus, and several other previously unknown infectious agents have emerged in laboratory rodents relatively recently. These agents have been discovered serendipitously or through active investigation of atypical serology results, cell culture contamination, unexpected histopathology, or previously unrecognized clinical disease syndromes. The potential research impact of these agents is not fully known. Infected rodents have demonstrated immunomodulation, tumor suppression, clinical disease (particularly in immunodeficient rodents), and histopathology. Perturbations of organismal and cellular physiology also likely occur. These agents posed unique challenges to laboratory animal resource programs once discovered; it was necessary to develop specific diagnostic assays and an understanding of their epidemiology and transmission routes before attempting eradication, and then evaluate eradication methods for efficacy. Even then management approaches varied significantly, from apathy to total exclusion, and such inconsistency has hindered the sharing and transfer of rodents among institutions, particularly for genetically modified rodent models that may not be readily available. As additional infectious agents are discovered in laboratory rodents in coming years, much of what researchers have learned from experiences with the recently identified pathogens will be applicable. This article provides an overview of the discovery, detection, and research impact of infectious agents recently identified in laboratory rodents. We also discuss emerging syndromes for which there is a suspected infectious etiology, and the unique challenges of managing newly emerging infectious agents.     

Killing of Flies in Electrocuting Insect Traps Releases Bacteria and Viruses

Electrocuting insect traps (EIT) are popular devices frequently used by homeowners and food handlers attempting to localize the control of flying insects, including the ubiquitous house fly (Musca domestica L.). The traps contain a visual attractant and a high-voltage metal grid. Upon contact with the grids, the insects are disintegrated by the high voltage. As part of a systematic evaluation of EITs and their role in infectious disease spread, we quantitated spread of bacteria and a bacterial virus during electrocution of house flies. We loaded flies with Serratia marcescens or with the Escherichia coli phage ΦX174 and placed sprayed or fed flies into a room containing an EIT. While flies were being electrocuted, liberated particles and bacteria were assayed via agar plates or via air filtration samplers. Sprayed flies released one of every 10,000 of the added bacteria or viruses, and fed flies released one of every 1,000,000 of the consumed bacteria or viruses. Results of our studies suggest EITs could play a role in the spread of infectious disease agents, but the potential is influenced by the insect's route of contamination. Received: 26 February 2000 / Accepted: 2 May 2000     

地塞米松抑制卡氏肺孢子菌肺炎β2-防御素和Dectin-1受体的表达

目的研究卡氏肺孢子菌肺炎（Pneumocystis carinii pneumonia,PCP）鼠肺Dectin-1和β2-防御素的表达变化,探讨地塞米松对Deetin-1和B2-防御素的影响与疾病发生的相互关系。方法实验分4组：正常对照组、Pc刺激组、PCP模型组以及PCP模型恢复组。免疫抑制方法建立PCP动物模型,改良四胺银（Groeoti’s methenamine—silver nitrate method,GMS）染色检测Pc包囊;肺组织切片HE染色观察肺组织病理变化;实时荧光定量和Westernblot检测Deetin-1和B2-防御素的mRNA以及蛋白的表达。结果Pc刺激组的Dectin．1和β2-防御素的mRNA以及蛋白的表达明显高于正常对照组（P〈0．05）;Pc刺激组和PCP恢复组的Dectin-1和β2-防御素的mRNA以及蛋白显著高于PCP组（P〈0．05）,而PCP恢复组与PCP组肺部炎症无明显差别。结论对免疫功能正常宿主,Dectin-1受体和p2-防御素可能在防Pc感染中起重要作用;地塞米松抑制了鼠肺Deetin-1和β2-防御素的表达,这可能与PCP疾病的发生和发展有关。     

Pneumocystis carinii pneumonia in the rat model.

Groups of barrier-raised but not certified virus-free Sprague-Dawley rats, obtained from the same source over the course of several years, were placed on an identical immunosuppressive regimen. This caused reactivation of latent Pneumocystis carinii infection, manifest as P. carinii pneumonia (PCP) of varying severity. Rats were euthanized after 9-12 wk of immunosuppression. An assessment of the severity of the induced PCP was made, based on the total number of organisms extracted from the lungs and their ability to proliferate in short-term cell culture. Serum samples obtained at sacrifice were tested by indirect immunofluorescence for antibodies to coronavirus, parvovirus, Sendai virus, pneumonia virus of mice (PVM) and Mycoplasma pulmonis. A total of 60 rats were examined. Thirty-four of these (57%) developed moderate or severe PCP. No antibodies were detected to either coronavirus or Mycoplasma pulmonis in any of the rats. Although antibodies were detected to parvovirus in 13/60 (22%), to PVM in 29/60 (48%), and to Sendai virus in 47/60 (78%), there was no apparent correlation between the presence or absence of antibodies to these agents and the severity of PCP. Sequential observations during the course of immunosuppression are needed to clarify the role of concomitant infections in the development of PCP.     

Epidemiology and disease control in everyday beef practice

It is important for food animal veterinarians to understand the interaction among animals, pathogens, and the environment, in order to implement herd-specific biosecurity plans. Animal factors such as the number of immunologically protected individuals influence the number of individuals that a potential pathogen is able to infect, as well as the speed of spread through a population. Pathogens differ in their virulence and contagiousness. In addition, pathogens have various methods of transmission that impact how they interact with a host population. A cattle population's environment includes its housing type, animal density, air quality, and exposure to mud or dust and other health antagonists such as parasites and stress; these environmental factors influence the innate immunity of a herd by their impact on immunosuppression. In addition, a herd's environment also dictates the "animal flow" or contact and mixing patterns of potentially infectious and susceptible animals. Biosecurity is the attempt to keep infectious agents away from a herd, state, or country, and to control the spread of infectious agents within a herd. Infectious agents (bacteria, viruses, or parasites) alone are seldom able to cause disease in cattle without contributing factors from other infectious agents and/or the cattle's environment. Therefore to develop biosecurity plans for infectious disease in cattle, veterinarians must consider the pathogen, as well as environmental and animal factors.     

Co-infection of Ticks: The Rule Rather Than the Exception

IntroductionTicks are the most common arthropod vectors of both human and animal diseases in Europe, and the Ixodes ricinus tick species is able to transmit a large number of bacteria, viruses and parasites. Ticks may also be co-infected with several pathogens, with a subsequent high likelihood of co-transmission to humans or animals. However few data exist regarding co-infection prevalences, and these studies only focus on certain well-known pathogens. In addition to pathogens, ticks also carry symbionts that may play important roles in tick biology, and could interfere with pathogen maintenance and transmission. In this study we evaluated the prevalence of 38 pathogens and four symbionts and their co-infection levels as well as possible interactions between pathogens, or between pathogens and symbionts.Conclusion/significanceOur study reveals high pathogen co-infection rates in ticks, raising questions about possible co-transmission of these agents to humans or animals, and their consequences to human and animal health. We also demonstrated high prevalence rates of symbionts co-existing with pathogens, opening new avenues of enquiry regarding their effects on pathogen transmission and vector competence.     

Microbial considerations in genetically engineered mouse research

Microbial infections have long been of concern to scientists using laboratory rodents because of their potential to confound and invalidate research. With the explosion of genetically engineered mice (GEM), new concerns over the impact of microbial agents have emerged because these rodents in many cases are more susceptible to disease than their inbred or outbred counterparts. Moreover, interaction between microbe and host and the resulting manifestation of disease conceivably differ between GEM and their inbred and outbred counterparts. As a result, infections may alter the GEM phenotype and confound interpretation of results and conclusions about mutated gene function. In addition, because GEM are expensive to produce and maintain, contamination by pathogens or opportunists has severe economic consequences. This review addresses how microbial infections may influence phenotype, how immunomodulation of the host as the result of induced mutations may modify host susceptibility to microbial infections, how novel host:microbe interactions have led to the development of new animal models for disease, how phenotype changes have led to the discovery of new pathogens, and new challenges associated with prevention and control of microbial infections in GEM. Although the focus is on naturally occurring infections, extensive literature on the use of GEM in studies of microbial pathogenesis also exists, and the reader is referred to this literature if microbial infection is a suspected culprit in phenotype alteration.     

Do viruses use vectors to penetrate mucus barriers?

I propose a mechanism by which viruses successfully infect new individuals, despite being immotile particles with no ability for directed movement. Within cells, viral particle movements are directed by motors and elements of the cytoskeleton, but how viruses cross extracellular barriers, like mucus, remains a mystery. I propose that viruses cross these barriers by hitch-hiking on bacteria or sperm cells which can transport themselves across mucosal layers designed to protect the underlying cells from pathogen attack. An important implication of this hypothesis is that agents that block interactions between viruses and bacteria or sperm may be new tools for disease prevention.     

实验用生物样品的啮齿类微生物污染的检测管理

实验用生物样品在生物医学研究中有着广泛的应用.生物样品的微生物污染可危及实验动物设施的生物安全以及对动物实验研究造成负面影响.因此在将生物样品接种到啮齿类动物之前,要对其进行检测以确保无啮齿类动物传染性病原体的污染.因为检测技术和经济上的限制和局限性,不可能对所有的啮齿类动物传染性病原都进行检测.本文依据科学文献、笔者的经验以及专家的意见,尝试着去阐述如何建立一个有效可行的生物样品微生物污染检测计划.笔者建议对一些重要的微生物进行检测,并简要综述了选取这些微生物的理由即它们对科研、实验动物生物安全以及职业健康安全的影响.     

P. carinii DNA Detected by ITSs Nested PCR in Serum and PBMC of AIDS Patients with PCP

SUMMARY Pneumocystis carinii pneumonia (PCP) is usually diagnosed by examination of BAL, a sample often unpleasant to be collected from immunocompromised host affected by acute respiratory disease. We studied by the Internal Transcribed Spacers (ITSs) nested PCR the presence of P. carinii DNA in serum and Peripheral Blood Mononuclear Cells (PBMC) during acute episodes of PCP to test blood as a possible noninvasive diagnostic tool.     

Molecular diagnostic assays for detection of viral respiratory pathogens in institutional outbreaks

Outbreaks of viral respiratory disease in institutions may be associated with high morbidity and mortality, depending upon the viral etiology and the age and immune status of the affected patients. Control of outbreaks may include isolation and/or cohorting, and prophylaxis or treatment with specific antiviral agents may be indicated, all dependent upon the specific cause of the outbreak. Conventional methods of viral diagnosis detect only a limited number of the viruses that are known to cause outbreaks. The availability of sensitive and specific molecular assays has facilitated rapid diagnosis of a wider range of viruses from respiratory outbreaks. Molecular methods have distinct advantages over conventional methods, including the ability to rapidly develop assays for emerging viruses and new variants of existing viruses. In addition, molecular testing allows rapid detection of resistance to antiviral agents or mutations leading to increased virulence. However, high-throughput molecular testing requires batch processes that may compromise the ability to respond quickly to urgent testing demands.     

Recent developments in new and old viral infections

Until recently, concerns regarding viral infections and hematologic malignancies were primarily focused on the transplantation of an allogeneic graft. In the last years, the use of immunomodulatory agents such as monoclonal antibodies (e.g. anti CD20, anti CD52) directed against lymphocyte antigens in the treatment of hematopoietic malignancies (e.g. lymphoma, chronic lymphocytic leukemia) has added a great potential to impact on the incidence, severity and timing of viral infections. Patients may acquire viral infections through several mechanisms including transfusion, community exposure or via the donor origin in the case of stem cell transplant. Endogenous reactivation of latent viruses is also commonly observed. Viral replication may lead directly to viral diseases or induce indirect effects such as increased incidence of opportunistic infections and decreased patient survival. Traditionally, herpesviruses have been and are still today the most important viruses in patients with hematologic malignancies. Nowadays, several emerging viral infections have been highlighted as being of concern in this patients' population.     

Phencyclidine-Induced Dysregulation of Dopamine Response to Amphetamine in Prefrontal Cortex and Striatum

Phencyclidine (PCP) administration in rodents has been used to model aspects of schizophrenia. One aspect of such treatment has been the enhancement of amphetamine-induced increase of dopamine in the prefrontal cortex and striatum. To further characterize this mechanism rats were treated for 2 weeks with continuous PCP (15 mg/kg per day via Alzet minipump). Rats were implanted with a microdialysis probe into the prefrontal cortex (PFC) or striatum. Amphetamine was administered locally via the dialysis probe during one collection period and changes in extracellular dopamine were monitored. The effect of local administration of the dopamine uptake blocker nomifensine was also measured. Amphetamine (10 M) and nomifensine (10 M) increased the level of dopamine in both the PFC and striatum. PCP administration did not alter the response to amphetamine or nomifensine in the PFC, but reduced this response about 2-fold in striatum. To examine effects of continuous PCP administration on dopamine autoreceptor function, release of [³H]dopamine in response to electrical stimulation and in the presence of a dopamine agonist or antagonist was tested in striatal and prefrontal cortical tissue. Autoreceptor responses were similar in control and PCP-treated tissues. We conclude that the brain region-specific enhancement of dopamine release by peripheral amphetamine administration in rats after PCP is not likely mediated by alterations in the dopamine autoreceptors or changes in the dopamine transporter. The selective local responses of amphetamine indicates heterogeneous regional effects of continuous PCP on NMDA receptor function; effects that influence both regional excitatory responses and the overall dynamics of tonic excitatory/inhibitory inputs to the PFC and striatum.     

Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem/progenitor cells

The potential of neural stem and progenitor cell (NSPC) transplantation in neurodegenerative disease raises a concern about immunosuppressive agents and opportunistic neurotropic pathogens that may interfere with engraftment. Cytomegalovirus (CMV) is an important opportunistic pathogen infecting the central nervous system, where it may remain latent for life, following transplacental transmission. Cyclosporine (Cs), an immunosuppressive drug used in organ transplantation, where its use is associated with CMV reactivation, suppressed murine CMV (MCMV) infection in cultured NSPCs but not in fibroblasts. This activity of Cs appears to be mediated via cyclophilin (CyP) rather than via calcineurin. First, the calcineurin-specific inhibitor FK506 failed to suppress replication. Second, the CyP-specific inhibitor NIM811 strongly suppressed replication in NSPC. NSPCs maintained in the presence of NIM811 retained viral genomes for several weeks without detectable viral gene expression or obvious deleterious effects. The withdrawal of NIM811 reactivated viral replication, suggesting that the inhibitory mechanism was reversible. Finally, inhibition of endogenous CyP A (CyPA) by small interfering RNA also inhibited replication in NSPCs. These results show that MCMV replication depends upon cellular CyPA pathways in NSPCs (in a specific cell type-dependent fashion), that CyPA plays an important role in viral infection in this cell type, and that inhibition of viral replication via CyP leads to persistence of the viral genome without cell damage. Further, the calcineurin-signaling pathway conferring immunosuppression in T cells does not influence viral replication in a detectable fashion.     

Use of endophytes as biocontrol agents

Plant diseases, caused by various microorganisms, including viruses, bacteria, fungi, protozoa and nematodes, affect agricultural practices and result in significant crop losses. Fungal pathogens are the major cause of plant diseases and infect most plants. Agrochemicals play a significant role in plant disease management to ensure a sustainable and productive agricultural system. However, the intensive use of chemicals has adverse effects on humans and ecosystem functioning and also reduces agricultural sustainability. A sustainable agriculture is achieved through reduction or elimination of fertilizers and agrochemicals, resulting in minimal impact to the environment. Recently, the use of antagonistic endophytes as biocontrol agents is drawing special attention as an attractive option for management of some plant diseases, resulting in minimal impact to the environment. Endophytes that resides asymptomatically within a plant, have the potential to provide a source of candidate strains for potential biocontrol applications. This review addresses biocontrol methods using endophytic fungi such as Colletotrichum, Cladosporium, Fusarium, Pestalotiopsis and Trichoderma species as an attractive option for management of some plant diseases. Potential endophytes are screened in vitro and in vivo to test their antagonistic actions by different mechanisms, including mycoparasitism, production of lytic enzymes and/or antibiotics and induction of plant defenses. Currently, efforts are being made to commercialize these biocontrol agents. A continued research pipeline consisting of screening, in vitro and in vivo testing, biomass production and commercialization of endophytes as biocontrol agents may contribute to sustainable agriculture.     

Generalized immune response to Pneumocystis carinii infection in the lung.

We studied inflammatory cells retrieved by bronchoalveolar lavage (BAL) from immunocompromised patients with or without Pneumocystis carinii pneumonia (PCP). Twenty-four patients with PCP, and 20 patients without PCP underwent lavages of both an uninvolved lobe and the lobe involved in pulmonary infection. Patients without P. carinii, had a significant increase (p less than 0.02) in the percentages of neutrophils (22 +/- 7.1%, mean +/- SEM) and lymphocytes (16 +/- 3.8%) in the involved lobe compared to those in the uninvolved area (neutrophils: 9 +/- 4.8%; lymphocytes: 10 +/- 2.4%). Patients with PCP, had no differences between the % neutrophils or % lymphocytes in the involved vs. uninvolved lobes. Patients with PCP had more (p less than 0.01) P. carinii in the upper lobe (23 +/- 4.6 P. carinii clusters/500 cells) than the middle lobe (11 +/- 3.6). In PCP, despite regional infections, there was a diffuse inflammatory response.