Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA226681

Local Sample ID:	39
Subject ID:	SU002390
Subject Type:	Mammal
Subject Species:	Myotis lucifugus

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Subject:

Subject ID:	SU002390
Subject Type:	Mammal
Subject Species:	Myotis lucifugus

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
39	SA226681	FL027499	Pd	Treatment

Collection:

Collection ID:	CO002383
Collection Summary:	Bats were housed within mesh cages (Exo-terra Flexarium©; 22 cm x 35 cm x 43 cm) that were placed within temperature/humidity-controlled incubators (Caron© Environmental Chamber model 6041; 90.1 cm x 84.5 cm x 228.9 cm set to 8°C and 98% humidity) to encourage hibernation. Four cages were used to house the bats, separated by species and inoculation group; M. lucifugus (one sham-treatment: 16 individuals/cage, 10 male and 6 female; one Pd-inoculated; E. fuscus (8 individuals/cage, 4 male and 4 female). Bats were infected with a sham inoculum (20 μl, Phosphate Buffered Saline with Tween®20 (PBS-T)) or a Pd inoculum (20 μl, PBST containing 5x105 conidia) on wing and tail membranes. We monitored arousal frequency and sickness behavior using motion-activated infrared video. During the 71-day period bats found unresponsive or extremely sluggish were humanely euthanized (CO2 inhalation after isoflurane anesthesia and cervical dislocation), and were not included in this experiment. Several M. lucifugus showed signs of morbidity around day 55. We therefore, terminated the experiment 71 – 77 days post-inoculation (mid-March) which coincided with early stages of WNS progression. Disease severity was assessed by fungal load by qPCR33, UV fluorescence indicative of hyphae invasion, survival rate, and behavioral changes 32. E. fuscus was chosen as a WNS-resistant control and we found changes in their splenic lipidome to be minor. Tissue samples were collected, flash frozen, and shipped to Georgetown University Medical Center on dry ice and stored at -80°C until analysis.
Sample Type:	Spleen

Treatment:

Treatment ID:	TR002402
Treatment Summary:	Bats were housed within mesh cages (Exo-terra Flexarium©; 22 cm x 35 cm x 43 cm) that were placed within temperature/humidity-controlled incubators (Caron© Environmental Chamber model 6041; 90.1 cm x 84.5 cm x 228.9 cm set to 8°C and 98% humidity) to encourage hibernation. Four cages were used to house the bats, separated by species and inoculation group; M. lucifugus (one sham-treatment: 16 individuals/cage, 10 male and 6 female; one Pd-inoculated; E. fuscus (8 individuals/cage, 4 male and 4 female). Bats were infected with a sham inoculum (20 μl, Phosphate Buffered Saline with Tween®20 (PBS-T)) or a Pd inoculum (20 μl, PBST containing 5x105 conidia) on wing and tail membranes. We monitored arousal frequency and sickness behavior using motion-activated infrared video. During the 71-day period bats found unresponsive or extremely sluggish were humanely euthanized (CO2 inhalation after isoflurane anesthesia and cervical dislocation), and were not included in this experiment. Several M. lucifugus showed signs of morbidity around day 55. We therefore, terminated the experiment 71 – 77 days post-inoculation (mid-March) which coincided with early stages of WNS progression. Disease severity was assessed by fungal load by qPCR33, UV fluorescence indicative of hyphae invasion, survival rate, and behavioral changes 32. E. fuscus was chosen as a WNS-resistant control and we found changes in their splenic lipidome to be minor. Tissue samples were collected, flash frozen, and shipped to Georgetown University Medical Center on dry ice and stored at -80°C until analysis.

Treatment ID:

TR002402

Treatment Summary:

Bats were housed within mesh cages (Exo-terra Flexarium©; 22 cm x 35 cm x 43 cm) that were placed within temperature/humidity-controlled incubators (Caron© Environmental Chamber model 6041; 90.1 cm x 84.5 cm x 228.9 cm set to 8°C and 98% humidity) to encourage hibernation. Four cages were used to house the bats, separated by species and inoculation group; M. lucifugus (one sham-treatment: 16 individuals/cage, 10 male and 6 female; one Pd-inoculated; E. fuscus (8 individuals/cage, 4 male and 4 female). Bats were infected with a sham inoculum (20 μl, Phosphate Buffered Saline with Tween®20 (PBS-T)) or a Pd inoculum (20 μl, PBST containing 5x105 conidia) on wing and tail membranes. We monitored arousal frequency and sickness behavior using motion-activated infrared video. During the 71-day period bats found unresponsive or extremely sluggish were humanely euthanized (CO2 inhalation after isoflurane anesthesia and cervical dislocation), and were not included in this experiment. Several M. lucifugus showed signs of morbidity around day 55. We therefore, terminated the experiment 71 – 77 days post-inoculation (mid-March) which coincided with early stages of WNS progression. Disease severity was assessed by fungal load by qPCR33, UV fluorescence indicative of hyphae invasion, survival rate, and behavioral changes 32. E. fuscus was chosen as a WNS-resistant control and we found changes in their splenic lipidome to be minor. Tissue samples were collected, flash frozen, and shipped to Georgetown University Medical Center on dry ice and stored at -80°C until analysis.

Sample Preparation:

Sampleprep ID:	SP002396
Sampleprep Summary:	Tissue samples were homogenized in 300 μL of extraction buffer (isopropanol [IPA]) containing internal standards for the above lipid classes per the manufacturer’s instructions. A 10 μL aliquot of the tissue/extraction buffer mixture was collected for determining protein concentration. The samples were vortexed for 30 seconds, incubated on ice for 30 min, and then centrifuged for 10 min (10,000 x g, 4°C). The supernatant was transferred to a MS vial for LC-MS analysis. A pooled sample of all aliquots was prepared as a quality control and run every 10 samples in addition to the NIST SRM 1950 plasma mix. The NIST SRM 1950 plasma was prepared by aliquoting 20 µL in 100 μL of chilled IPA containing internal standards. Samples were vortexed for 1 min, incubated on ice for 30 min then incubated at -20°C for 2 hours for complete protein precipitation, then centrifuged for 20 min (10,000 x g, 4°C).

Sampleprep ID:

SP002396

Sampleprep Summary:

Tissue samples were homogenized in 300 μL of extraction buffer (isopropanol [IPA]) containing internal standards for the above lipid classes per the manufacturer’s instructions. A 10 μL aliquot of the tissue/extraction buffer mixture was collected for determining protein concentration. The samples were vortexed for 30 seconds, incubated on ice for 30 min, and then centrifuged for 10 min (10,000 x g, 4°C). The supernatant was transferred to a MS vial for LC-MS analysis. A pooled sample of all aliquots was prepared as a quality control and run every 10 samples in addition to the NIST SRM 1950 plasma mix. The NIST SRM 1950 plasma was prepared by aliquoting 20 µL in 100 μL of chilled IPA containing internal standards. Samples were vortexed for 1 min, incubated on ice for 30 min then incubated at -20°C for 2 hours for complete protein precipitation, then centrifuged for 20 min (10,000 x g, 4°C).

Combined analysis:

Analysis ID	AN003765
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Shimadzu
Column	Waters XBridge Amide (100 x 4.6mm,3.5um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	UNSPECIFIED
Units	peak area

Chromatography:

Chromatography ID:	CH002784
Instrument Name:	Shimadzu
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003508
Analysis ID:	AN003765
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	the qlm file was imported into MultiQuant v 2.0
Ion Mode:	UNSPECIFIED