Background. This assay detects A. fumigatus DNA in blood. The DNA we detect may originate from viable molds or from molds which are no longer viable (i.e., partially-degraded DNA). Hence, this assay is not equivalent to an assay of fungaemia. Blood samples should be taken from animals either prior to death or within 6 hours of death.

Three (3) methods may be used for submission of blood samples to the research lab. These are:

A. Preferred method (Use an OmniGen Research Mycotic Infection Kit: $25/kit). Each kit contains enough material for sampling and shipping 12 blood samples. Kits are available by calling the laboratory at 1-541-737-1920 or through the Website at www.omnigenresearch.com. Your Prince-Agri Products representative may also have blood sampling kits available.

B. Optional Method 1. Collect blood sample using a vacutainer (blue top) or a syringe using citric acid (ACD) as a preservative*. Remove 0.5 ml of whole blood and combine with 0.5 ml of 100% ethanol or 100% isopropanol (final concentration should be 50% alcohol) and place in a non-crushable plastic tube. Seal blood samples in a secondary container (e.g., Ziplock bag) and ship in a padded envelope via regular mail (or 2-day mail) to the research laboratory (see address below). Include information on sample number, your address, phone number, FAX number and billing address.

C. Optional method 2. Collect blood samples (at least 0.5 ml) using a vacutainer (blue top) or a syringe using citric acid (ACD) as a preservative*. Place sample on ice inside a Styrofoam container and ship to research laboratory using overnight express mail. Include information on sample number, your address, phone number, FAX number and billing address.

*-Note: Other anti-coagulants are acceptable (e.g., heparin or EDTA) but may reduce efficiency of our detection of A. fumigatus DNA.

Tissue samples (Cost: $40/sample):

Background. A. fumigatus can disseminate and colonize tissues including muscle, liver, kidney, lung and brain. Lymph nodes may also collect significant quantities of A. fumigatus DNA. Infected regions of tissues usually do not harbor visible mold.

Sampling and shipping tissues. Our assay requires only 25 mg of tissue; hence, a 1 gram sample is sufficient. Tissue samples should be place in a sealed plastic Ziplock bag, labeled and shipped in a leak-proof secondary container (frozen with ice or dry ice) using overnight delivery. Formalin-preserved samples cannot be analyzed.

Feed samples (Cost: $40/sample):

Background. A. fumigatus is a ubiquitous mold. Our assay detects as little as 3 spores and it is quite likely that it will be detectable in your sample. Our greatest concern rests with samples which test very high. Composite samples from within a feed resource and TMRs rarely test high for A. fumigatus. High-testing samples are usually from regions of feeds exposed to moisture, adequate temperature and oxygen. Heavily-infected feeds show a grey-blue color.

Sampling method. Select areas of feeds (less than 1/2 lb) which may be of concern (surfaces of silage, fill lines, exposed faces of feeds, moisture-damaged areas, augurs). Alternatively, representative samples of feeds from several regions of a feed resource may be submitted. Keep in mind that A. fumigatus has potential to grow quickly and the shipping method, therefore, must ensure that mold growth does not occur in transit. Two types of feed samples/shipping methods may be used:

Shipping method 1. -Fresh, non-dried feeds (e.g., silages, < 1/2 lb). Ship frozen on ice or dry ice using overnight delivery. Ship samples in a Ziploc bag (or equivalent) and place in a secondary container. Please include your name, address, contact information, billing address and details about the sample(s).

Shipping method 2. -Dried feeds (including dried silages: < 1 lb). These may be ground or not ground. Ship via regular mail. Ship samples in a Ziploc bag (or equivalent) and place in a secondary container. Please include your name, address, contact information, billing address and details about the sample(s).

Fecal samples (Cost: $40/sample):

Background. A. fumigatus is a gastrointestinal pathogen. Fecal samples may be useful to determine gastrointestinal burden. However, the research lab at OmniGen Research has not analyzed enough fecal samples to adequately interpret values obtained from fecal analysis. Therefore, fecal samples should be submitted for experimental purposes. Please submit less than 100 g (1/5 lb). Two shipping methods are:

Shipping method 1. -Fresh, non-dried fecal samples (e.g., grab sample: < 100 g). Ship frozen on ice or dry ice using overnight delivery. Ship samples in a Ziploc bag (or equivalent) and place in a sealed secondary container. Please include your name, address, contact information, billing address and details about the sample(s).

Shipping method 2. -Dried fecal samples (< 100 g). These may be ground or not ground. Ship via regular mail. Ship samples in a Ziploc bag (or equivalent) and place in a secondary container. Please include your name, address, contact information, billing address and details about the sample(s).

Analysis of samples:

Feed and fecal samples are dried and ground to pass through a 1 mm screen and sub-sampled. Alcohol-preserved blood samples are processed under vacuum to remove ethanol/isopropanol prior to analysis. DNA is then extracted from samples of feed, tissue (25 mg) or blood (25 µl) using the Qiagen method. DNA samples from feed, tissue and fecal samples are then re-purified using a gel filtration column to remove residual contaminants. Concentrations of A. fumigatus DNA in samples are then determined using quantitative Sybr-Green polymerase chain reaction (PCR). All samples are run with both positive and negative controls and with a standard curve (purified genomic A. fumigatus DNA). Results are tallied, interpreted and then sent back to the owner (usually within 1 week of sample receipt). Comparative values are provided to aid in interpretation. Billing is coordinated independently. Bills for analysis are sent out within 1 month of analysis.

Address for receipt of samples (or blood kit requests):

Dr. Neil Forsberg
OmniGen Research LLC
2001 NW Monroe Suite 203
Corvallis, OR 97330
Phone/FAX: 1-541-738-0400
forsberg@omnigenresearch.com