Duddingtonia flagrans: chlamydospore production during dehydration, capture efficiency for cyathostomins vs Panagrellus redivivus, and the effects of NaHSO4 on poultry litter microbiome diversity and viability
Date
2019
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Duddingtonia flagrans is a nematode trapping-fungus that has shown
promising results as a tool to combat parasitic nematode infections in livestock. The
fungus interrupts the parasitic lifecycle by trapping and killing larval stages on pasture
to prevent re-infection of animals. One barrier to the fungus’ commercial use is
scaling up production of the fungus, and specifically of chlamydospores, which
survive the digestive tract to grow in fecal pats on pasture, thus have potential as a
feed through anthelmintic. The purpose of this study was to evaluate the effect of
dehydration on sporulation of the fungus. Disks of Duddingtonia flagrans type strain
(ATCC® 13423™) were grown on 17% cornmeal agar for 26 days at 30° C, then split
into three groups; dried quickly at 38° C and 37% humidity over 48 hours, dried more
slowly at 24° C and 55% humidity over 10 days, or kept at 30° C and sealed with
parafilm to prevent loss of moisture (control). Half of each dried culture was
resuspended in water, then half of each culture was heated to liquify, homogenized
through vortexing. Spores were then counted in a Neubauer hematocytometer. Both
the 2 day and 10 day drying techniques yielded significantly more spores than the
control (Welch Two Sample t-test p-values of .0359 and .0411, respectively). The
difference in number of spores was insignificant between the two drying techniques,
although a visual representation of the data shows less variability in the measurements
of the slower 10 day drying. ☐ Biocontrol fungus Duddingtonia flagrans may have differing efficacy on
parasitic nematodes of different species, and in the presence of free-living soil
nematodes. D. flagrans was cultured on three petri dishes for each trial set, which were
randomly assigned to have equine parasitic cyathostomin larvae, free-living Panagrellus redivivus, or a mixture of both nematodes added. After 24 hours of interaction the plates were observed and each nematode was categorized as trapped or
not trapped by the fungus. This was repeated for three trial sets. Trapped and not
trapped counts from the trial sets were aggregated for statistical analysis. Chi-squared
and fisher’s exact tests were both conducted to compare the number of cyathostomin
larvae captured at the 24 hour mark in mixed plates to the number captured at 24 hours
in monocultured plates. A one sample proportions test was conducted on the P.
redivivus and on the cyathostomin free/captured ratios, respectively, to determine if
the samples of each group were more or less likely to be captured at that time point,
and to establish a 95% confidence interval of the true population percentage captured
at 24 hours under these conditions. The monoculture cyathostomin capture rate was
53%, and in the mixed cyathostomin and P. redivivus cultures the parasitic
cyathostomin larvae had a capture rate of 56%. Chi-squared and fisher’s exact tests
comparing the two cyathostomin conditions both yielded p-values of 1; no discernable
difference in means. The total capture rate for P. redivivus was 66.14%, and the total
capture rate for cyathostomin larvae was 54.55%. P. redivivus are significantly more
likely to be trapped than untrapped at 24 hours of interaction with the fungus (p<.001),
whereas the cyathostomin larvae are not. D. flagrans does trap different nematode
species at different rates. This should be taken into consideration when selecting it as a
biological control agent for specific parasitic species to be managed. D. flagrans does
not appear to trap parasitic species at differing rates in the presence of free living
nematodes; the pasture nematode content does not need to be taken into consideration. ☐ The purpose of the project was to measure abundance of live/dead populations
of Enterococcus cecorum, Clostridium perfringens, and Staphylococcus aureus in poultry litter following treatment with sodium bisulfate (SB). Sodium bisulfate is a litter treatment that lowers the litter pH and ammonia. This experiment measured
secondary effects on the poultry litter microbiome including commensal and
pathogenic organisms. Broiler litter from a treatment and a control house was sampled
at -1, 2, 24, and 27 days. The treatment house received SB at a rate of 100 lb/1000 ft2
on days -1 and 24. Samples were mixed with sterile phosphate-buffered saline (PBS)
to suspend bacterial cells, and then washed by centrifugation and resuspended in PBS.
Supernatants (500 ml aliquots) were collected and treated with either PMAxx™ or
sterile PBS buffer (control), or autoclaved and PMA treated (heat-killed control) to
monitor PMA binding efficiency. The samples were dark/light incubated for PMA
activation. These treated solutions were centrifuged, DNA was extracted from the
pelleted cells using QIAmp Powerfecal kit. Differential qPCR (40 cycles) comparing
PMA-treated and untreated DNA with species specific primers to target bacteria of
interest was used to differentiate between live/dead cells. DNA from a composite
sample from each house at each timepoint was sequenced using Illumina MiSeq for
comparison. Data from the averages of each time point’s PMA treated and untreated
cycle threshold (Ct) values were compared to create a ΔCt to represent relative dead
levels of bacteria, the PMA treated and PMA treated heat killed data was used to
create a ΔCt to help determine PMA binding efficiency for each target organism.
MiSeq data was normalized to absolute abundance before comparison. The treatment
house had consistently slightly higher levels of dead bacteria overall. S. aureus was
not found in either house. C. perfringens may require further investigation into PMA
binding efficiency. E. cecorum shows a consistent increase in levels of dead bacteria
in the treatment house demonstrating it may be reduced by SB. Some bacterial families present in the survey data for the control house were found to be eliminated in
the treatment house.