Modeling the host response to inhalation anthrax

Inhalation anthrax, an often fatal infection, is initiated by endospores of the bacterium Bacillus anthracis, which are introduced into the lung. To better understand the pathogenesis of an inhalation anthrax infection, we propose a two-compartment mathematical model that takes into account the documented early events of such an infection. Anthrax spores, once inhaled, are readily taken up by alveolar phagocytes, which then migrate rather quickly out of the lung and into the thoracic/mediastinal lymph nodes. En route, these spores germinate to become vegetative bacteria. In the lymph nodes, the bacteria kill the host cells and are released into the extracellular environment where they can be disseminated into the blood stream and grow to a very high level, often resulting in the death of the infected person. Using this framework as the basis of our model, we explore the probability of survival of an infected individual. This is dependent on several factors, such as the rate of migration and germination events and treatment with antibiotics.     

Observations on the pathogenicity of Pasteuria penetrans, a parasite of root-knot nematodes

A simple test was used to determine whether or not Pasteuria penetrans spores would attach to 17 species of nematodes. All susceptible individuals had spores attached to their cuticles after 24 h of gentle agitation in suspensions containing 10⁵spores/ml. Spores of P. penetrans from six populations of Meloidogyne only adhered to species of Meloidogyne and they adhered in greatest numbers to the species from which they had been originally isolated. Sonication of spores from infected females increased attachment but the effect was dependent on pH and whether the test was conducted in tap or distilled water. Invasion of tomato roots was reduced by up to 86% when, rather than using healthy juveniles, second-stage juveniles bearing 15 or more spores were added to soil at high densities (1000 or 3000/plant); at low densities (500/plant) invasion was not significantly affected. The rate of development of M. incognita juveniles infected with P. penetrans was slower than that of healthy juveniles. The numbers of second-generation of M. incognita were reduced by 82–93% when juveniles encumbered with 1–15 spores were added to soil instead of those bearing no spores. Pasteuria penetrans populations differed in their aggressiveness and when juveniles encumbered with the same number of spores from two populations were added to soil there were differences in the numbers of females that became infected. The implications of these results for the development of P. penetrans as a biological control agent are discussed.     

Characterization of Bacillus anthracis Persistence In Vivo

Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.     

Airborne pollen and spores’ deposition in alveolar tissues as a tool in drowning forensic diagnosis

We report the results of a histological study of lung samples where an unusual quantity of airborne pollen and fungal spores was found in drowned rats. Pollen and spores were found in lungs of drowned rats but not in the post-mortem submerged ones. Another control group consisting of rats that underwent 60 min exposure to a highly pollen-loaded atmosphere also recorded negative for the presence of pollen or fungal spores. Pollen types coincided with plants growing at the surrounding gardens flowering during the days of the experiment, performed during spring, that were detected by the aerobiological trap located at the city. The pollen observed at the lower airways’ tissues were Chenopodiaceae, Cupressus, Ericaceae, Jasminum, Olea europaea, Plantago, Pinus, Poaceae, Quercus and Urticaceae. Regarding fungal spores Alternaria, Aspergillus, Cladosporium cladosporoides, Cladosporium herbarum, Leptosphaeria, Polythrincium and Phitomyces. Pollen and spores’ penetration into deeper regions of the respiratory tract is an unusual phenomenon not happening in regular breathing conditions. Our results revealed that these particles appeared in a significant number in lung samples of drowned animals probably pushed down from upper airways by the force of water inhalation during drowning. Their presence into alveolar spaces offer a useful forensic evidence in doubtful drowning autopsies, favoured by the characteristic of the sporopollenin (pollen wall) and chitin (fungal spore wall) resistance. Moreover, the presence of these particles in alveoli areas of drowned bodies can help forensics to obtain information about pre-mortem dates and places.

     

The effect of three herbicides on the number of spores of Rhynchosporium secalis on barley stubble and volunteer plants

Single sprays of paraquat, glyphosate or dinoseb-in-oil, were applied to barley stubble or volunteer plants and their effects on the number of spores of Rhynchosporium secalis were studied weekly. Spore concentrations were measured by washing samples of stubble or volunteer plants and counting spores using a haemocytometer. The number of spores recovered from stubbles was relatively small; both paraquat and glyphosate caused some reductions but the results were not consistent. Treatment with paraquat generally caused a transitory increase on volunteer plants; this effect was not found after glyphosate. Both herbicides caused persisting reductions from 4 wk after treatment. Volunteer plants treated with dinoseb-in-oil had fewer spores for 6 wk after treatment, but the plants were not killed and when new growth became infected, spore production recovered to equal that on untreated plants. The fungicide captafol caused some reductions in spore number on stubble but results were not consistent, applied after paraquat it had no effect. It was generally ineffective when applied to volunteer plants; applied after paraquat it tended to increase spore number, but the differences were seldom significant. Spores washed from volunteer plants treated with paraquat or glyphosate were inoculated to pot-grown barley seedlings; neither herbicide had any effect on spore viability. Viable spores were recovered in February from stubble or volunteer plants treated the previous autumn with paraquat or glyphosate respectively.     

Determining deposition sites of inhaled lung particles and their effect on clearance

An essential component of lung defense is clearance of particulates and infectious vectors from the mucus membrane of the tracheobronchial tree and the alveolar regions of the lung. To partition clearance between these areas we determined the bronchial branching pattern, the anatomical sites of particle deposition, and subsequent clearance in the same animal. Using a 2.85-microns particle tagged with 57Co for inhalation and deposition in the sheep lung, we followed clearance via a series of computer-stored gamma-scintillation lung images. The same sheep was reinhaled, and the particle distributions for both inhalations were compared. After the animals were killed, the bronchial branching pattern and length of the bronchial tree were documented. The number of particles depositing in all bronchi down to 1 mm diam was determined by scintillation counting, and the number in respiratory bronchioles and alveoli was microscopically counted. We conclude that particles deposited in bronchi greater than or equal to 1 mm diam clear in 2-4 h postdeposition. Bronchi distal to 1-mm-diam bronchi and alveoli clear evenly over 72 h, and the number of particles equal to the tracheobronchial deposition cleared after 45 h.     

Adiaspiromycosis in striped skunks in Alberta, Canada

Pulmonary adiaspiromycosis was diagnosed in seven of 25 striped skunks (Mephitis mephitis) in east-central Alberta. The infection varied from mild, where only microscopic lesions were seen, to severe, where gross lesions of grayish-white nodules were observed in the lung parenchyma. Mild lesions were restricted to the lung, while severe lesions extended to the tracheobronchial and mediastinal lymph nodes. Histologically, the lesions were characterized by a centrally located fungal spherule, surrounded by granulomatous inflammation. The morphology of the fungal spherules was consistent with that of Emmonsia crescens. By electron microscopy, the fungal cells had an outer thick fibrillar wall and an inner cytoplasm filled with large lipid vacuoles with relatively few mitochondria, ribosomes or glycogen inclusions. The absence of endosporulation and budding suggested that each fungal cell in the lung represented a separate inhaled spore. Infection was by inhalation, nevertheless adiaspores may disseminate to the regional lymph nodes.     

Growth of the ferret tracheobronchial tree

Because the ferret is being used increasingly in inhalation toxicology and lung physiology studies, it is necessary to better understand the airway structure of its tracheobronchial tree. Previously published information does not include dimensions of bronchi and bronchioles in either adult or growing ferrets. The airway structure of interest for calculating inhaled particle deposition patterns includes airway lengths, diameters and branching angles in each generation. Measurements of these dimensions were obtained for several selected airway paths on replica casts. Casts were made in-situ in four male litter mates age 14 hours, 9.5 days, 16.5 days and 56 days. These data demonstrate, that as with human lung growth, body length at a given age is a good predictor of airway lengths and diameters. Airway branch angles do not appear to change significantly during growth. Sufficient measurements were made to provide dimensions of a typical tracheobronchial pathway for this species. This pathway begins with the trachea and ends at the terminal bronchiole. The morphometric data were not sufficient to determine whether or not the number of tracheobronchial generations increase or decrease postnatally.     

Changes in the lymph nodes of rabbits following application of the breakdown products of fluoroplast]

Tracheobronchial, mesenterial and inguinal lymph nodes of 15 mature rabbits which were subjected to inhalation of valatile products of fluoroplast destruction were studied. The action of this drug for 1-40 days activated the reactive centers of follicles. The amount of blasts, plasmic cells, eosinophils, macrophages, lymphocytes increased in regional tracheobronchial and distant mesenterial lymph nodes. The prolonged action during 120 days resulted in decreased blasto- and lymphopoesis in the regional tracheobroncheal lymph nodes, depression of the reticular epithelium, suppression of the phagocytic and plasm cell reactions, decreased nucleic metabolism in the cells. Mesenterial lymph nodes retained their functions. The lymphopoesis in them and nucleic metabolism in the cells were intensified. The blastic, plasmic cell, eosinophilic and mast cell reactions were well pronounced.     

Wind Dispersal of Alternaria alternata, a Cause of Leaf Blight of Cotton

Introducing Alternaria alternata, the cause of blight disease of cotton plants, into a field of young healthy plants growing in rows cross-wind, yielded disease foci which were spread downwind up to 7 m from the infection sources. Only light disease incidence was found in the remainder of the field. When the disease was introduced into a field of mature cotton plants grown in rows cross-wind, randomly scattered disease foci occurred. In mature plantations where rows were parallel to the average wind direction, only limited size disease foci developed downwind, up to 16 m from the source. These foci did not developed further during the season. The number of air-borne spores of A. alternata was significantly increased by the presence of diseased cotton plants, being highest close to the diseased plants. The spores were transferred to a distance of at least 20 m. However, the number of air-borne spores significantly decreased 6 m from the infection source. Periodical trapping of air-borne spores of A. alternata in a cotton growing region for 2 years, revealed that their air dispersal is local, probably at the field level. A. alternata in a cotton growing region for 2 years, revealed that their air dispersal is local, probably at the field level. A. alternata air-borne spores were also trapped in rather low numbers regardless of the presence of infected cotton plants. However, the number of the air-borne spores trapped was dependent mainly on the average wind direction and on the Alternaria blight epidemics occurring in the fields twice a year. It is suggested that A. alternata spores are transferred by wind for short distances but are constantly present in small numbers in the atmosphere throughout the whole year. The two peaks recorded for the number of spores present in the air above cotton crops correlate with the annual two outbreaks of Alternaria blight epidemics. In addition, both wind and plant row direction affect disease development in the fields.     

Sequential pathological studies in the udder of goats intramammarily infected withAspergillus fumigatus

Intramammary inoculation of goats withAspergillus fumigatus spores resulted in the development of mastitis with characteristic gross and microscopic lesions. The mastitis and the lesions were restricted to the infected udder halves only and there was no dissemination of infection to other tissues of the body. The experiment was continued for 45 days. Gross changes in the infected udder were observed up to the 45th day post-infection. The lesions, in general, included variable sized abscesses in the first 15 days followed by development of varying sized greyish-white nodules in the infected udders. Microscopic changes consisted of granulomatous reaction with well developed granulomas in the infected udders. Hyphae and spores ofAspergillus fumigatus could be demonstrated in sections of the infected udders up to 45 days after infection. Reisolation of the fungus consistently was achieved up to 45 days. It is concluded that intramammary inoculation ofAspergillus fumigatus spores in goats leads to chronic granulomatous mastitis.     

Effect of Paranosema locustae (Microsporidia) on the behavioural phases of Locusta migratoria (Orthoptera: Acrididae) in the laboratory

The ability of parasites to modify the behaviour of their hosts is a wide spread phenomenon, but the effects of microsporidian parasites on locust behaviour remain unexplored. Here the frequencies of directional changes (ND) and jumping (NJ) per minute of gregarious locusts infected with 2000 spores of the microsporidian parasite Paranosema locustae were significantly different from those of untreated locusts 10 and 16 days after infection, being similar to values for solitary nymphs. In contrast, the behaviour of locusts inoculated with the lower doses of 200 spores/locust was sometimes like that of solitary nymphs. At other times, behaviour was intermediate between solitary and gregarious, i.e. transitional. The rearing density did not affect the turning and jumping behaviour of infected locusts, and their behaviours were similar to those of solitary locusts at 10–16 days after infection. Our study demonstrates that infection with P. locustae may lead gregarious locusts to change some of their behaviour to that typical of solitary locusts.     

Immunohistochemical and immunocytochemical detection of SchS34 antigen in <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis> spores and spore impacted mouse lungs

The purpose of this study was to evaluate the distribution of a 34 kD antigen isolated from S. chartarum sensu lato in spores and in the mouse lung 48 h after intra-tracheal instillation of spores by immuno-histochemistry. This antigen was localized in spore walls, primarily in the outer and inner wall layers and on the external wall surfaces with modest labelling observed in cytoplasm. Immuno-histochemistry revealed that in spore impacted mouse lung, antigen was again observed in spore walls, along the outside surface of the outer wall and in the intercellular space surrounding spores. In lung granulomas the labelled antigen formed a diffusate, some 2–3× the size of the long axis of spores, with highest concentrations nearest to spores. Collectively, these observations indicated that this protein not only displayed a high degree of specificity with respect to its location in spores and wall fragments, but also that it slowly diffuses into surrounding lungs.     

The role of wounds in the infection of oranges by Penicillium digitatum Sacc

Inocula of spores of Penicillium digitatum in water applied to apparently uninjured skin of oranges do not cause lesions to develop. Addition of citric acid, orange juice, or various extracts of rind had little effect on susceptibility to infection. When spores in water are applied to wounds made between oil vesicles, lesions develop only from wounds that penetrate deeply into the albedo. The flavedo of most oranges seems to be resistant to infection even when damaged, but in a few consignments it showed much less resistance. Increasing the number of conidia in the inoculum caused more lesions to develop but some fruits developed lesions from inocula containing very few spores. The method and timing of spore application to wounds had a considerable effect on the incidence of lesions; emanations from infected fruit had no effect. Lesions developed more rapidly and readily when suspensions of spores in water were applied to wounds in the skin that damaged oil vesicles; wounds as shallow as 0–25 mm allowed lesions to develop.     

Lung dendritic cells rapidly mediate anthrax spore entry through the pulmonary route

Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially because anthrax is an emerging bioterrorism agent. The exact mechanisms leading to a severe clinical form through the inhalational route are still unclear, particularly how immobile spores are captured in the alveoli and transported to the lymph nodes in the early steps of infection. We investigated the roles of alveolar macrophages and lung dendritic cells (LDC) in spore migration. We demonstrate that alveolar macrophages are the first cells to phagocytose alveolar spores, and do so within 10 min. However, interstitial LDCs capture spores present in the alveoli within 30 min without crossing the epithelial barrier suggesting a specific mechanism for rapid alveolus sampling by transepithelial extension. We show that interstitial LDCs constitute the cell population that transports spores into the thoracic lymph nodes from within 30 min to 72 h after intranasal infection. Our results demonstrate that LDCs are central to spore transport immediately after infection. The rapid kinetics of pathogen transport may contribute to the clinical features of inhalational anthrax.     

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

The Effect of Ultraviolet and Solar Radiation and Temperature on Survival of Fungal Propagules

Survival of spores, spore aggregates, sclerotia, and pycnidia of fungi was evaluated after exposure to high temperature in the dark, or exposure to ultraviolet (UV) radiation or sunlight. Under most conditions survival decreased from the most resitant Sclerotium rolfsii to Alternaria macrospora, Mycosphaerella pinodes, Aspergillus niger and Botrytis cinerea, in that order. There was no difference in survival among organisms stored in darkness at 45°C as single spores or aggregated into a mass of spores; or between spores of M. pinodes exposed before and after being released from pycnidia. Exposure to UV reduced survival much more than exposure in darkness. Longevity under UV ranged from more than 7 days for S. rolfsii to approximately 50 min for A. macrospora and 3 min for B. cinerea. Much longer survival occurred for spores of all fungi aggregated together; of spores of M. pinodes irradiated when inside rather than outside pycnidia; and in bigger, darker and older rather than in smaller, paler and younger sclerotia of S. rolfsii. Survival of sclerotia cut into slices and exposed to UV increased with thickness, irrespective of exposure to UV with the outer pigmented or innernonpigmented side. As tested with spore aggregates of A. niger, the rate of survival was linearly proportional to the number of conidia aggregated in each body. Irradiation by sunlight affected survival as described for irradiation by UV. The best protection from the detrimental effects of sun and UV radiation was provided by the infected host tissue.     

Morpho-functional status of the lymph nodes of rats during rehabilitation after exposure to vapors of dimethyl sulfate]

By means of histological methods inferior tracheobronchial lymph nodes have been investigated in 48 Wistar rats in 2 weeks and 3 months after cessation of inhalation of DNSV at maximum possible dose (0.1 mg/m3) for 2 and 14 days. Cell composition of the lymph nodes and cross sections of structural components have been compared. The cross section of the cortex decreases, while that of the medulla increases in comparison with corresponding parameters of the acute experiment. In 2 weeks and 3 months after DMSV effect for 2 days the part (%) of poorly differentiated cells and middle lymphocytes increases, while content of plasma cells in the medullary cords decreases. During rehabilitation after DMSV effect for 14 days a low level plateau is noted (as in the acute experiment). However, in lymphoid nodules++ within content of the poorly differentiated cells and middle lymphocytes in the cortical germinative centers in 2 weeks and 3 months the number of the poorly differentiated and reticular cells increases essentially, as well as mitotically dividing cells (in comparison to the acute experiment). In the medullary cords of the lymph nodes after 3 months of rehabilitation content of immature plasma cells is essentially higher than in 2 weeks of rehabilitation and than in the acute experiment. During rehabilitation the level of cells destruction in the lymph nodes noticeably decreases in comparison to the acute experiment.     

Strength in numbers: high parasite burdens increase transmission of a protozoan parasite of monarch butterflies (<Emphasis Type="Italic">Danaus plexippus</Emphasis>)

Parasites often produce large numbers of offspring within their hosts. High parasite burdens are thought to be important for parasite transmission, but can also lower host fitness. We studied the protozoan Ophryocystis elektroscirrha, a common parasite of monarch butterflies (Danaus plexippus), to quantify the benefits of high parasite burdens for parasite transmission. This parasite is transmitted vertically when females scatter spores onto eggs and host plant leaves during oviposition; spores can also be transmitted between mating adults. Monarch larvae were experimentally infected and emerging adult females were mated and monitored in individual outdoor field cages. We provided females with fresh host plant material daily and quantified their lifespan and lifetime fecundity. Parasite transmission was measured by counting the numbers of parasite spores transferred to eggs and host plant leaves. We also quantified spores transferred from infected females to their mating partners. Infected monarchs had shorter lifespans and lower lifetime fecundity than uninfected monarchs. Among infected females, those with higher parasite loads transmitted more parasite spores to their eggs and to host plant leaves. There was also a trend for females with greater parasite loads to transmit more spores to their mating partners. These results demonstrate that high parasite loads on infected butterflies confer a strong fitness advantage to the parasite by increasing between-host transmission.