Peter J. Didier

Microsporidiosis in mammals

Microsporidia are small, single-celled, obligately intracellular parasites that have caused significant agricultural losses and interference with biomedical research. Interest in the microsporidia is growing, as these organisms are recognized as agents of opportunistic infections in persons with AIDS and in organ transplant recipients. Microsporidiosis is also being recognized in children and travelers, and furthermore, concern exists about the potential of zoonotic and waterborne transmission of microsporidia to humans. This article reviews the basic biology and epidemiology of microsporidiosis in mammals.

Genetic Requirements for the Survival of Tubercle Bacilli in Primates

BACKGROUND Tuberculosis (TB) leads to the death of 1.7 million people annually. The failure of the bacille Calmette-Guérin vaccine, synergy between AIDS and TB, and the emergence of drug resistance have worsened this situation. It is imperative to delineate the mechanisms employed by Mycobacterium tuberculosis to successfully infect and persist in mammalian lungs. METHODS Nonhuman primates (NHPs) are arguably the best animal system to model critical aspects of human TB. We studied genes essential for growth and survival of M. tuberculosis in the lungs of NHPs experimentally exposed to aerosols of an M. tuberculosis transposon mutant library. RESULTS Mutants in 108 M. tuberculosis genes (33.13% of all genes tested) were attenuated for in vivo growth. Comparable studies have reported the attenuation of only approximately 6% of mutants in mice. The M. tuberculosis mutants attenuated for in vivo survival in primates were involved in the transport of various biomolecules, including lipid virulence factors; biosynthesis of cell-wall arabinan and peptidoglycan; DNA repair; sterol metabolism; and mammalian cell entry. CONCLUSIONS Our study highlights the various virulence mechanisms employed by M. tuberculosis to overcome the hostile environment encountered during infection of primates. Prophylactic approaches aimed against bacterial factors that respond to such in vivo stressors have the potential to prevent infection at an early stage, thus likely reducing the extent of transmission of M. tuberculosis.

Efficacy and safety of live attenuated persistent and rapidly cleared Mycobacterium tuberculosis vaccine candidates in non-human primates.

Tuberculosis (TB) remains a global health burden for which safe vaccines are needed. BCG has limitations as a TB vaccine so we have focused on live attenuated Mycobacterium tuberculosis mutants as vaccine candidates. Prior to human studies, however, it is necessary to demonstrate safety in non-human primates (NHP). In this study, we evaluate the safety and efficacy of two live attenuated M. tuberculosis double deletion vaccine strains mc(2)6020 (DeltalysA DeltapanCD) and mc(2)6030 (DeltaRD1 DeltapanCD) in cynomolgus macaques. In murine models, mc(2)6020 is rapidly cleared while mc(2)6030 persists. Both mc(2)6020 and mc(2)6030 were safe and well tolerated in cynomolgus macaques. Following a high-dose intrabronchial challenge with virulent M. tuberculosis, mc(2)6020-vaccinates were afforded a level of protection intermediate between that elicited by BCG vaccination and no vaccination. BCG vaccinates had reduced tuberculosis-associated pathology and improved clinical scores as compared to saline and mc(2)6030 vaccinates, but survival did not differ among the groups.

Infectious Agent and Immune Response Characteristics of Chronic Enterocolitis in Captive Rhesus Macaques

ABSTRACT Chronic enterocolitis is the leading cause of morbidity in colonies of captive rhesus macaques (Macaca mulatta). This studys aim was to identify the common enteric pathogens frequently associated with chronic enterocolitis in normal, immunocompetent rhesus monkeys and to elucidate the influence of this clinical syndrome on the host immune system. We analyzed the fecal specimens from 100 rhesus macaques with or without clinical symptoms of chronic diarrhea. Retrospective analysis revealed an increased incidence of Campylobacter spp. (Campylobacter coli and Campylobacter jejuni), Shigella flexneri, Yersinia enterocolitica, adenovirus, and Strongyloides fulleborni in samples collected from animals with chronic diarrhea (P < 0.05). The presence of additional enteric pathogens, such as Escherichia coli, carrying the eaeA intimin or Stx2c Shiga toxin virulence genes, Balantidium coli, Giardia lamblia, Enterocytozoon bieneusi, and Trichuris trichiura was found in all animals regardless of whether diarrhea was present. In addition, the upregulation of interleukin-1α (IL-1α), IL-3, and tumor necrosis factor alpha cytokine genes, accompanied by an increased presence of activated (CD4+ CD69+) T lymphocytes was found in gut-associated lymphoid tissues collected from animals with chronic enterocolitis and diarrhea in comparison with clinically healthy controls (P < 0.05). These data indicate that chronic enterocolitis and diarrhea are associated, in part, with a variety of enteric pathogens and highlight the importance of defining the microbiological status of nonhuman primates used for infectious disease studies. The data also suggest that chronic colitis in rhesus macaques may have potential as a model of inflammatory bowel disease in humans.

The non‐human primate model of tuberculosis

Non‐human primates (NHPs) are used to model human disease owing to their remarkably similar genomes, physiology, and immune systems. Recently, there has been an increased interest in modeling tuberculosis (TB) in NHPs. Macaques are susceptible to infection with different strains of Mycobacterium tuberculosis (Mtb), producing the full spectrum of disease conditions, including latent infection, chronic progressive infection, and acute TB, depending on the route and dose of infection. Clearly, NHPs are an excellent model of human TB. While the initial aim of the NHP model was to allow preclinical testing of candidate vaccines and drugs, it is now also being used to study pathogenesis and immune correlates of protection. Recent advances in this field are discussed in this review. Key questions such as the effect of hypoxia on the biology of Mtb and the basis of reactivation of latent TB can now be investigated through the use of this model.

Therapeutic strategies for human microsporidia infections

Over the past 20 years, microsporidia have emerged as a cause of infectious diseases in AIDS patients, organ transplant recipients, children, travelers, contact lens wearers and the elderly. Enterocytozoon bieneusi and the Encephalitozoon spp., Encephalitozoon cuniculi, Encephalitozoon hellem and Encephalitozoon intestinalis, are the most frequently identified microsporidia in humans, and are associated with diarrhea and systemic disease. The microsporidia are small, single-celled, obligately intracellular parasites that have been identified in water sources, as well as in wild, domestic and food-producing farm animals, thereby raising concerns for waterborne, foodborne and zoonotic transmission. Current therapies for microsporidiosis include albendazole, a benzimidazole that inhibits microtubule assembly and is effective against several microsporidia, including the Encephalitozoon spp., although it is less effective against Encephalitozoon bieneusi. Fumagillin, an antibiotic and antiangiogenic compound produced by Aspergillus fumigatus, is more broadly effective against Encephalitozoon spp. and E. bieneusi; however, is toxic when administered systemically to mammals. Recent studies are also focusing on compounds that target the microsporidia polyamines (e.g., polyamine analogs), methionine aminopeptidase 2 (e.g., fumagillin-related compounds), chitin inhibitors (e.g., nikkomycins), topoisomerases (e.g., fluoroquinolones) and tubulin (e.g., benzimidazole-related compounds).

Experimental Infection of Rhesus Macaques with West Nile Virus: Level and Duration of Viremia and Kinetics of the Antibody Response after Infection

Reports of transfusion-associated cases of West Nile virus (WNV) infection indicate the need for sensitive screening methods to identify WNV-infected blood products. We experimentally infected 5 rhesus macaques with WNV, to determine the level and duration of viremia, the kinetics of the humoral immune response, and the sensitivity of various assay systems for detecting WNV in blood. All macaques developed subclinical infections with low levels of viremia; nested reverse-transcription polymerase chain reaction was the most sensitive method for detecting virus or viral RNA in blood. Specific WNV antibodies appeared during the second week of infection; the results of an IgM enzyme-linked immunosorbent assay became positive on the ninth or tenth day after infection, followed in 1-2 days by hemagglutination-inhibiting and neutralizing antibodies. Our results suggest that both nucleic acid and serological testing may be needed to determine exposure to WNV and to identify potentially infected blood donors.

PrimaTB STAT-PAK Assay, a Novel, Rapid Lateral-Flow Test for Tuberculosis in Nonhuman Primates

ABSTRACT Tuberculosis (TB) is the most important zoonotic bacterial disease in nonhuman primates (NHP). The current diagnostic method, the intradermal palpebral tuberculin test, has serious shortcomings. We characterized antibody responses in NHP against Mycobacterium tuberculosis to identify immunodominant antigens and develop a rapid serodiagnostic test for TB. A total of 422 NHP were evaluated, including 243 rhesus (Macaca mulatta), 46 cynomolgus (Macaca fascicularis), and 133 African green (Cercopithecus aethiops sabaeus) monkeys at five collaborative centers. Of those, 50 monkeys of the three species were experimentally inoculated with M. tuberculosis. Antibody responses were monitored every 2 to 4 weeks for up to 8 months postinfection by MultiAntigen Print ImmunoAssay with a panel of 12 recombinant antigens. All of the infected monkeys produced antibodies at various levels and with different antigen recognition patterns. ESAT-6 and MPB83 were the most frequently recognized proteins during infection. A combination of selected antigens which detected antibodies in all of the infected monkeys was designed to develop the PrimaTB STAT-PAK assay by lateral-flow technology. Serological evaluation demonstrated high diagnostic sensitivity (90%) and specificity (99%). The highest rate of TB detection was achieved when the skin test was combined with the PrimaTB STAT-PAK kit. This novel immunoassay provides a simple, rapid, and accurate test for TB in NHP.

Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus

Background  Tuberculosis (TB) and AIDS together present a devastating public health challenge. Over 3 million deaths every year are attributed to these twin epidemics. Annually, ∼11 million people are coinfected with HIV and Mycobacterium tuberculosis (Mtb). AIDS is thought to alter the spontaneous rate of latent TB reactivation.

Possible role of glial cells in the onset and progression of Lyme neuroborreliosis

BackgroundLyme neuroborreliosis (LNB) may present as meningitis, cranial neuropathy, acute radiculoneuropathy or, rarely, as encephalomyelitis. We hypothesized that glia, upon exposure to Borrelia burgdorferi, the Lyme disease agent, produce inflammatory mediators that promote the acute cellular infiltration of early LNB. This inflammatory context could potentiate glial and neuronal apoptosis.MethodsWe inoculated live B. burgdorferi into the cisterna magna of rhesus macaques and examined the inflammatory changes induced in the central nervous system (CNS), and dorsal root nerves and ganglia (DRG).ResultsELISA of the cerebrospinal fluid (CSF) showed elevated IL-6, IL-8, CCL2, and CXCL13 as early as one week post-inoculation, accompanied by primarily lymphocytic and monocytic pleocytosis. In contrast, onset of the acquired immune response, evidenced by anti-B. burgdorferi C6 serum antibodies, was first detectable after 3 weeks post-inoculation. CSF cell pellets and CNS tissues were culture-positive for B. burgdorferi. Histopathology revealed signs of acute LNB: severe multifocal leptomeningitis, radiculitis, and DRG inflammatory lesions. Immunofluorescence staining and confocal microscopy detected B. burgdorferi antigen in the CNS and DRG. IL-6 was observed in astrocytes and neurons in the spinal cord, and in neurons in the DRG of infected animals. CCL2 and CXCL13 were found in microglia as well as in endothelial cells, macrophages and T cells. Importantly, the DRG of infected animals showed significant satellite cell and neuronal apoptosis.ConclusionOur results support the notion that innate responses of glia to B. burgdorferi initiate/mediate the inflammation seen in acute LNB, and show that neuronal apoptosis occurs in this context.