Download Aspergillus fumigatus

Aspergillus fumigatus (Af): Exposure to the opportunistic fungal pathogen Af leads to a
pulmonary infection. To recapitulate the natural route of infection, mice will be challenged with Af
via the intra-tracheal route using a non-invasive procedure. Prior to infection mice will be
anesthesized by isoflurane inhalation to effect until changes in breathing pattern are observed
(approx. 2-3 min. in the isoflurane chamber). Mice will receive different doses of live fungal conidia
suspended in 50 l of PBS. In previous studies we have shown that wild type mice can survive
Af infection without the development of detectable illness(15, 17, 24, 25). In experiments where
the use of genetically deficient mice might be needed the animals will be monitored daily for signs
of disease progression (ruffled fur, listless, dehydrated). When necessary animals will be
euthanized by CO2 asphyxiation. For all experimental procedures mice will be subjected to the
minimal discomfort possible.
Nippostrongylus brasiliensis (Nb): The intestinal nematode parasite Nb, is a widely
used model for examining the development and function of the in vivo type 2 immune response.
In the murine model, Nb host exposure begins with subcutaneous inoculation of third stage larvae
(L3) under the skin, the site of natural infection. L3 migrate in the circulation to the lungs as early
as 11 h after invasion of subcutaneous tissues. L3 molts to fourth stage larvae (L4) and remain
in the lungs ≤50 h post-infection before migrating through the trachea to the intestine. Worm
expulsion, mediated in the intestine, usually occurs by days 9-12 after primary inoculation. This
infection and migration pattern is similar to that of several human intestinal roundworm infections,
including Ancylostoma duodenale (Old World hookworm) and Necator americanus (New World
hookworm), and recent studies suggest that the immune response to these hookworms has many
similar properties to type 2 responses identified in experimental mouse models including the
immune response to Nb(2). To model the natural route of infection Infectious (100-500) L3 Nb will
be injected subcutaneously to establish infection. For the studies in this PPG lung tissue will be
collected at specific time points after inoculation.
Influenza A Virus: Studies with influenza A virus will be done using the mouse-adapted
(H1N1) PR8 strain that has been used in many similar studies. Animals will be anesthetized with
avertin, and inoculated intranasally with 200 pfu in 50 μl PBS. Virus stocks are prepared in
embryonated chicken eggs, and virus concentration is measured by focus forming assay done on
MDCK cells, with foci visualized by immunostaining. Stocks are maintained at -80°C, at
concentrations on the order of 106 pfu/ml. All virus stocks are plaque-purified, and tested for LPS
contamination before use.
Streptococcus pneumonia (Sp): Type 3 Sp (ATCC 6303 clinical isolate with capsular
serotype 3) will be used. This serotype is chosen because it is virulent in mice and man. Bacteria
will be grown in Todd-Hewitt broth with yeast extract at 37°C until log phase. The concentration
of bacteria in broth is determined by measuring the absorbance at 600 nm and then plotting the
OD on a standard curve against known colony counts. The bacterial culture is then diluted to a
concentration of 4 x 10 4 cfu/ml, and 25 μl aliquots delivered intranasally will be administered to
anesthetized animals. Secondary bacterial challenge of influenza-infected mice will be done in
the window of maximal susceptibility, from day 5 to day 7 post-influenza infection. Bacterial load
is determined by plating serial 10-fold dilutions of culture or lung homogenates onto blood agar
plates incubated overnight at 37°C in 5% CO2.
Toxoplasma gondii (Tg): For studies with T. gondii, mice are infected intraperitoneally
with either a uracil auxotrophic carbamoyl phosphate synthase (CPS) mutant strain (2 million
parasites per mouse) or a TdTomato-expressing Prugiaud strain (500 parasites per mouse) of T.
gondii. All strains are maintained and propagated in confluent human foreskin fibroblast cultures.
Upon 60–80% lysis of the HFF monolayer, T. gondii tachyzoites are released by passing through
25G needle twice and spun down by centrifugation at 2,000 × g for 10 min. For the CPS strain,
the pellet was resuspended in PBS and gamma (γ) irradiated at 15,000 rads before priming of
mouse. For lung studies the TdTomato-expressing Prugiaud strain is used, this is a virulent strain
and infects the lung starting at day 7 after i.p administration. All T. gondii strains were routinely
monitored with MycoSensor PCR assay Kit (Agilent Technologies) and were maintained free of
Mycoplasma.