A new model for Hendra virus encephalitis in the mouse

Hendra virus (HeV) infection in humans is characterized by an influenza like illness, which may progress to pneumonia or encephalitis and lead to death. The pathogenesis of HeV infection is poorly understood, and the lack of a mouse model has limited the opportunities for pathogenetic research. In this project we reassessed the role of mice as an animal model for HeV infection and found that mice are susceptible to HeV infection after intranasal exposure, with aged mice reliably developing encephalitic disease. We propose an anterograde route of neuroinvasion to the brain, possibly along olfactory nerves. This is supported by evidence for the development of encephalitis in the absence of viremia and the sequential distribution of viral antigen along pathways of olfaction in the brain of intranasally challenged animals. In our studies mice developed transient lower respiratory tract infection without progressing to viremia and systemic vasculitis that is common to other animal models. These studies report a new animal model of HeV encephalitis that will allow more detailed studies of the neuropathogenesis of HeV infection, particularly the mode of viral spread and possible sequestration within the central nervous system; investigation of mechanisms that moderate the development of viremia and systemic disease; and inform the development of improved treatment options for human patients.     

Polyvalent 23 epitope polysaccharide pneumonia vaccine induced effective protection through strain-adapted effector mechanisms as demonstrated by the different cytokine responses in mice challenged with two different strains of Streptococcus pneumoniae

We used a Balb/c mouse model of pneumococcal pneumonia to investigate the protection mechanisms induced by immunization with a polyvalent 23 epitope polysaccharide pneumonia vaccine. Groups of mice were injected x 4 times s.c. within one month, with this vaccine preparation. Mice were subsequently challenged at day 45, with a lethal, intratracheal inoculum of two strains of Streptococcus pneumoniae - either a highly virulent and strongly immunogenic serotype 3 strain (P4241), or a less virulent and weakly immunogenic serotype 19F strain (P15986). The intratracheal S. pneumoniae challenge-induced lethality, antibody response, bacterial clearance, and cytokine secretions were monitored to analyze the strain-adapted effector mechanisms. Pulmonary levels of TNFalpha, IL-6, IL-1 beta, MIP-1 alpha, KC, MCP-1/JE and MIP-2 cytokines were determined up to 48 hours post-infection. Survival rates were 82% and 100% among vaccinated animals challenged at day 45 with P4241, and P1598 mice respectively, and 0% in non-vaccinated mice (p<0.001). Survival was associated with a rapid bacterial clearance from blood and lungs, which similar for the two strains. Immunization induced a serotype-specific antibody response. Kinetics of the cytokine profile in the lung following intratracheal inoculation with the 4241 strain was different in animals vaccinated 45 days previously, compared to na?ve, control mice. Generally speaking the bacterial-induced inflammatory cytokine response induced with the 4241 strain was much weaker in vaccinated animals than in control mice. The only cytokines showing a greater increase in vaccinated mice compare to control animals were IL-1 beta, KC and MCP-1. Production of TNFalpha and IL-6 was lower in vaccinated animals than in controls. At variance with the previous bacteria strain-induced cytokine profile, infection with the P15986 strain induced a strong inflammatory response, with a substantial increase in all the cytokine tested, which was similar in vaccinated and in na?ve, control animals, except for MIP-1 alpha, which was the only mediator significantly more produced by vaccinated animals than by na?ve, control mice following P15986 infection. The distinct cytokine profiles, which were observed in this study depending upon the two strains of S. pneumoniae used for challenge, demonstrated that protection against each strain was obtained through a different defence strategy.     

Does Helicobacter pylori infection per se cause gastric cancer or duodenal ulcer? Inadequate evidence in Mongolian gerbils and inbred mice

A role for Helicobacter pylori infection in the development of gastric cancer in humans is well established; however, evidence for its carcinogenicity in animals remains inadequate. Mongolian gerbils and mice are commonly used to investigate the carcinogenicity of H. pylori, yet it is unclear whether H. pylori infection per se causes gastric cancer or duodenal ulcers in these animal models. Gastric adenocarcinoma in the gerbils was reported over 10 years ago, but this species has proved an unreliable model for studying H. pylori infection-associated gastric cancer. Helicobacter pylori infection alone appears insufficient to induce gastric cancer in these animals; additional carcinogenic insult is required. The development of invasive adenocarcinoma in inbred mice is rare regardless of the mouse or bacterial strain, and many long-term studies have failed to induce gastric cancer in these animals. Helicobacter pylori infection is also an established causative factor for duodenal ulcer in humans. However, few studies have attempted to develop animal models of H. pylori infection-induced duodenal ulcer. We therefore conclude that both Mongolian gerbils and inbred mice may be inadequate models for studying H. pylori infection-associated gastric cancer and that there is no animal model of H. pylori infection-induced duodenal ulcer.     

Pneumocystis carinii--animal production perspective.

Pneumocystis carinii is an important cause of pneumonia in immunocompromised human patients. The organism is also found as a saprophyte in the lungs of many species of animals. Animal models have been used as a source of P. carinii organisms for study of the disease. The rat model has been especially useful. Initially, the infection was latent in most colonies, and P. carinii pneumonia readily developed when animals were immunosuppressed. Today, many barrier raised rodent colonies are free of adventitious viruses, bacteria, Mycoplasma sp., and parasites, including P. carinii. Variability is now seen in the rat model. The use of cultured organisms to experimentally infect rats and mice prior to immunosuppression has met the need for some investigators, however, latent-infected, barrier-raised and isolator-raised rodents are still required. Colonies specifically infected with P. carinii can provide latent-infected animals and are better protected from potentially interfering organisms than barrier-raised animals. The development of these colonies is feasible as investigators and animal producers work together to define and develop this resource.     

Molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease

SARS coronavirus (SARS-CoV) causes severe acute respiratory tract disease characterized by diffuse alveolar damage and hyaline membrane formation. This pathology often progresses to acute respiratory distress (such as acute respiratory distress syndrome [ARDS]) and atypical pneumonia in humans, with characteristic age-related mortality rates approaching 50% or more in immunosenescent populations. The molecular basis for the extreme virulence of SARS-CoV remains elusive. Since young and aged (1-year-old) mice do not develop severe clinical disease following infection with wild-type SARS-CoV, a mouse-adapted strain of SARS-CoV (called MA15) was developed and was shown to cause lethal infection in these animals. To understand the genetic contributions to the increased pathogenesis of MA15 in rodents, we used reverse genetics and evaluated the virulence of panels of derivative viruses encoding various combinations of mouse-adapted mutations. We found that mutations in the viral spike (S) glycoprotein and, to a much less rigorous extent, in the nsp9 nonstructural protein, were primarily associated with the acquisition of virulence in young animals. The mutations in S likely increase recognition of the mouse angiotensin-converting enzyme 2 (ACE2) receptor not only in MA15 but also in two additional, independently isolated mouse-adapted SARS-CoVs. In contrast to the findings for young animals, mutations to revert to the wild-type sequence in nsp9 and the S glycoprotein were not sufficient to significantly attenuate the virus compared to other combinations of mouse-adapted mutations in 12-month-old mice. This panel of SARS-CoVs provides novel reagents that we have used to further our understanding of differential, age-related pathogenic mechanisms in mouse models of human disease.     

Role of type I IFNs in pulmonary complications of Pneumocystis murina infection

Despite the advent of highly active antiretroviral therapy, pulmonary complications in AIDS are a common clinical problem. Pneumocystis jiroveci infection causes a life-threatening pneumonia, especially in individuals with CD4 T cell deficiencies as occurs in AIDS. Although Pneumocystis sp. is an extracellular fungal pathogen, CD8 T cells are the predominant lymphocyte recruited to the lung in CD4-deficient humans and mice during Pneumocystis pneumonia, and we have found that these CD8 T cells are responsible for subsequent lung damage in CD4 T cell-depleted mice. Comparing CD4 T cell-depleted IFN-alpha receptor knockout (KO) mice to wild-type mice, we found that this CD8 T cell recruitment and lung damage is type I IFN (IFN-alphabeta) dependent. However, in both CD4 competent, wild-type and IFN-alpha receptor (IFNAR) KO mice, Pneumocystis infection leads to an eosinophilic granulocyte influx with bronchial epithelial changes as seen in asthma. This response is delayed in IFNAR KO mice, as is pathogen clearance. Although the inflammation is transient in wild-type animals and resolves upon Pneumocystis clearance, it is more severe and persists through day 35 postinfection in IFNAR KO mice, leading to fibrosis. In addition, IFNAR KO, but not wild-type, mice mount a Pneumocystis-specific IgE response, an indicator of allergic sensitization. Thus, in the absence of IFNAR signaling and CD4 T cells, Pneumocystis-mediated lung damage does not occur, whereas in CD4-competent animals, the absence of IFNAR signaling results in an exacerbated Th2 response, asthma-like symptoms, and fibrosis. Therefore, both CD4 T cell- and type I IFN-mediated mechanisms can determine pulmonary complications from Pneumocystis infection.     

Investigations on the efficacy of monovalent Pseudomonas aeruginosa vaccine for oral administration in Pseudomonas aeruginosa experimental infection

Therapeutic efficacy of Pseudomonas aeruginosa vaccine for oral use (10(10) killed germs/ml), prepared from strain 4922, belonging to serotype XV, by Meitert-Meitert scheme, on 4 experimental models in mice (pneumonia, infected burn, septicaemia and urinary tract infection) was studied in comparison with monovalent Ps. aeruginosa vaccine serotype XV (10(9) killed germs/ml) for subcutaneous use and also with associated administration of the two vaccine variants. Mice immunization by using vaccine for oral use was performed by 0.5 ml vaccine per day, for 10 days and vaccine for subcutaneous use was administrated in a volume of 0.5 ml x 2, at 3 days interval. Mice immunization by using the two vaccine types, in association was concomitantly performed and in the same quantity as for separate immunization. In experimental pneumonia, Ps. aeruginosa vaccine for oral use protected mice in 35% of cases, those with infected burns were protected in 33.3% of cases, those with septicemia--in 96.6% of cases and those with urinary tract infection in 50% of cases. As compared to Ps. aeruginosa vaccine for subcutaneous use, the results obtained by vaccine for oral use are less favourable but associated administration of both vaccine variants led to superior results. Thus, in experimental pneumonia, it was obtained a surviving rate of 65% for animals immunized with both vaccine types, in comparison with 50% for animals immunized with vaccine for subcutaneous use only, and in Ps. aeruginosa infected burn, it was obtained a recovering rate of 79.1% for the animals immunized by using both vaccines, in comparison with 70.8% surviving for animals immunized with vaccine for subcutaneous use. In experimental septicaemia and urinary tract infection, combined use of both vaccine variants determined animals surviving and recovering in percents similar to those obtained by separate administration of vaccine for subcutaneous use (in septicemia--100% protection; in urinary tract infection--75% protection).     

Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-α/β and IFN-γ receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-α/β receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-γ receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.     

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.     

Animal models of the immunology and pathogenesis of human babesiosis

Animal models of human babesiosis have provided a basic understanding of the immunological mechanisms that clear, or occasionally exacerbate, Babesia infection and those pathological processes that cause disease complications. Human Babesia infection can cause asymptomatic infection, mild to moderate disease, or severe disease resulting in organ dysfunction and death. More than 100 Babesia species infect a wide array of wild and domestic animals, and many of the immunologic and pathologic responses to Babesia infection are similar in animals and humans. In this review, we summarize the knowledge gained from animal studies, their limitations, and how animal models or alternative approaches can be further leveraged to improve our understanding of human babesiosis.     

Animal models of psychiatric disease

Animal models of psychiatric diseases are useful tools for screening new drugs and for investigating the mechanisms of those disorders. Despite the difficulties inherent in modelling human psychiatric phenotypes in animals, there has been recent success identifying mutations in mice that give rise to some of the characteristic features of anxiety, depression, schizophrenia, autism, obsessive-compulsive disorder and bipolar disorder. In some cases these models have the additional strength that drugs used to treat the human condition alleviate the symptoms in mice. Robust genetic evidence of the involvement of multiple susceptibility genes in psychiatric disease will enable future studies to move from single-gene models to models with multiple modified loci, with the promise of better representing the complexity of the human diseases.     

Trypanosoma cruzi: roles for perforin-dependent and perforin-independent immune mechanisms in acute resistance

CD8+ T cells have been shown to be required for acute resistance to infection with the protozoan parasite, Trypanosoma cruzi, the causative agent of Chagas' disease. However, to date, the mechanism by which CD8+ T cells mediate protection in vivo has not been determined. While CD8+ T cells can exhibit cytolytic function, they also secrete cytokines such as IFN-gamma, which is known to mediate protection against T. cruzi infections. To determine whether cytolysis is an important effector function in vivo, we have compared outcomes of T. cruzi infection in normal and perforin-deficient mice. Our results indicate that while perforin-dependent cytolytic mechanisms clearly make a major contribution to acute resistance to T. cruzi infection, this contribution may be strain and challenge dose-dependent, since perforin-deficient mice challenged with lower doses of a less virulent strain survived and were subsequently resistant to challenge with virulent organisms. In vivo depletion studies demonstrated that survival of perforin-deficient mice challenged with low doses of T. cruzi requires both CD4+ and CD8+ T cells and is dependent on IFN-gamma secretion. These studies document the participation of both perforin-dependent cytotoxic and perforin-independent, IFN-gamma-dependent immune mechanisms in acute resistance to T. cruzi infection.     

Role of T cells in virus control and disease after infection with pneumonia virus of mice

Infection of mice with pneumonia virus of mice (PVM) is used as a natural host experimental model for studying the pathogenesis of infection with the closely related human respiratory syncytial virus. We analyzed the contribution of T cells to virus control and pathology after PVM infection. Control of a sublethal infection with PVM strain 15 in C57BL/6 mice was accompanied by a 100-fold increase in pulmonary cytotoxic T lymphocytes, 20% of which were specific for PVM. T-cell-deficient mice failed to eliminate PVM and became virus carriers in the absence of the clinical or histopathological signs of pneumonia that occurred after infection of control mice. Mice with limited T-cell numbers did not achieve virus control without weight loss, indicating that T-cell-mediated virus control was closely linked to immunopathology. Both CD4 and CD8 T cells independently contributed to virus elimination and disease. Virus control and disease were similar in the absence of perforin, gamma interferon, or tumor necrosis factor alpha. Interestingly, disease and mortality after lethal high-dose PVM infection were independent of T cells. These data illustrate a key role for T cells in control of PVM infection and demonstrate that both T-cell-dependent and -independent pathways contribute to disease in a viral dose-dependent fashion.     

The biological basis of severe outcomes in Anaplasma phagocytophilum infection

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an acute disease in humans that is also often subclinical. However, 36% are hospitalized, 7% need intensive care, and the case fatality rate is 0.6%. The biological basis for severe disease is not understood. Despite A.?phagocytophilum's mechanisms to subvert neutrophil antimicrobial responses, whether these mechanisms lead to disease is unclear. In animals, inflammatory lesions track with IFNγ and IL-10 expression and infection of Ifng(-/-) mice leads to increased pathogen load but inhibition of inflammation. Suppression of STAT signaling in horses impacts IL-10 and IFN-γ expression, and also suppresses disease severity. Similar inhibition of inflammation with infection of NKT-deficient mice suggests that innate immune responses are key for disease. With severe disease, tissues can demonstrate hemophagocytosis, and measures of macrophage activation/hemophagocytic syndromes (MAS/HPS) support the concept of human granulocytic anaplasmosis as an immunopathologic disease. MAS/HPS are related to defective cytotoxic lymphocytes that ordinarily diminish inflammation. Pilot studies in mice show cytotoxic lymphocyte activation with A.?phagocytophilum infection, yet suppression of cytotoxic responses from both NKT and CD8 cells, consistent with the development of MAS/HPS. Whether severity relates to microbial factors or genetically determined diversity in human immune and inflammatory response needs more investigation.     

The use of tetrathiomolybdate in treating fibrotic, inflammatory, and autoimmune diseases, including the non-obese diabetic mouse model

Tetrathiomolybdate was originally developed for use in Wilson's disease. However, lowering copper levels to below normal levels with tetrathiomolybdate has been found to have efficacy in cancer, probably by turning down signaling by angiogenic cytokines. More recently, we have shown in animals models that tetrathiomolybdate dramatically inhibits pulmonary and liver fibrosis. In other animal models, we have shown that the drug also inhibits liver damage from concanavalin A and acetaminophen, and heart damage from doxorubicin. These studies are briefly reviewed, and we then present data on tetrathiomolybdate's partially protective effect against diabetes in non-obese diabetic mice, an autoimmune model of type I diabetes. Possible mechanisms of tetrathiomolybdate's protective effect are briefly considered.     

Plant models for animal pathogenesis

Several bacteria that are pathogenic to animals also infect plants. Mechanistic studies have proven that some human/animal pathogenic bacteria employ a similar subset of virulence determinants to elicit disease in animals, invertebrates and plants. Therefore, the results of plant infection studies are relevant to animal pathogenesis. This discovery has resulted in the development of convenient, cost-effective, and reliable plant infection models to study the molecular basis of infection by animal pathogens. Plant infection models provide a number of advantages in the study of animal pathogenesis. Using a plant model, mutations in animal pathogenic bacteria can easily be screened for putative virulence factors, a process which if done using existing animal infection models would be time-consuming and tedious. High-throughput screening of plants also provides the potential for unravelling the mechanisms by which plants resist animal pathogenic bacteria, and provides a means to discover novel therapeutic agents such as antibiotics and anti-infective compounds. In this review, we describe the developing technique of using plants as a model system to study Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus pathogenesis, and discuss ways to use this new technology against disease warfare and other types of bioterrorism.     

Immune and pathophysiological responses to different strains of Giardia duodenalis in neonatal mice

Numerous studies have demonstrated various strain differences between Giardia isolates, but little is known about the immunology and pathogenesis of infections. This study aimed to compare host responses to strains of Giardi duodenalis differing in levels of virulence and pathogenicity and, by doing so, elucidate the mechanisms via which pathogenic strains establish infections. Marked differences were found in the infection dynamics, histopathological responses and serum antibody responses of neonatal mice infected with either G. duodenalis strain BRIS/83/HEPU/106 (isolated from a human) or BRIS/95/HEPU/2041 (isolated from a sulphur-crested cockatoo, Cacatua galerita). Infections with the bird strain were more intense (6.7-times greater) and persisted longer (by 14days) than infections with the human strain. The bird strain was more pathogenic and caused greater pathophysiological alteration to the gut mucosa, including increased villous atrophy, hyperplasia of goblet cells and vacuolated epithelial cells. Mice infected with the bird strain produced less serum anti-Giardia IgA and IgM, but more total (non-specific) serum IgA than those infected with the human strain of Giardia. This suggests that avian G. duodenalis strains are infective for mammalian hosts and may contribute to zoonotic infections. Furthermore, infection of mice with BRIS/95/HEPU/2041 serves as a good experimental model to provide further insight into the mechanisms via which G. duodenalis causes disease.     

Detailed Analysis of the African Green Monkey Model of Nipah Virus Disease

Henipaviruses are implicated in severe and frequently fatal pneumonia and encephalitis in humans. There are no approved vaccines or treatments available for human use, and testing of candidates requires the use of well-characterized animal models that mimic human disease. We performed a comprehensive and statistically-powered evaluation of the African green monkey model to define parameters critical to disease progression and the extent to which they correlate with human disease. African green monkeys were inoculated by the intratracheal route with 2.5×10⁴ plaque forming units of the Malaysia strain of Nipah virus. Physiological data captured using telemetry implants and assessed in conjunction with clinical pathology were consistent with shock, and histopathology confirmed widespread tissue involvement associated with systemic vasculitis in animals that succumbed to acute disease. In addition, relapse encephalitis was identified in 100% of animals that survived beyond the acute disease phase. Our data suggest that disease progression in the African green monkey is comparable to the variable outcome of Nipah virus infection in humans.