Sporulation on Blood Agar

From 4-3-15.

I was able to calculate the CFU/mL of the P. larvae spores recovered from the Columbian sheep's blood agar plates and slants. The colonies were grown on MYPGP agar at 37C for three days before they were counted. The spore concentrations aren't as a high as I would have liked (aiming for a 10^8 CFU/mL), but through use of the blood agar the concatenation of spores is about 1 to 2 logs higher. It appears that the bacteria growing on either the slant or the agar didn't seem to make much difference.

Stock #	Source	CFU/mL	CFU/mL
10	Plate	11000000	1.1x10^7
11	Plate	1800000	1.8x10^6
12	Plate	600000	6.0x10^5
13	Slant	800000	8.0x10^5
14	Slant	1600000	1.6x10^6
15	Slant	2100000	2.1x10^6
16	Slant	2800000	2.8x10^6

I plan to use these spore stocks for the upcoming chlorine dioxide experiment.

//EWW

Germinant Receptors in P. larvae - Bioinformatics

Goal: I plan to perform a comparison of the known and well characterized germination receptor genes found in Bacillus subtilis and B. anthracis to the genome of Paenibacillus larvae.

For the initial step:

• I downloaded SRA Toolkit from NCBI here: http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?cmd=show&f=software&m=software&s=software
• The Toolkit was moved to my home directory "Elliott" (above my desktop)
• I searched NCBI SRA (http://www.ncbi.nlm.nih.gov/sra) for Paenibacillus larvae and found 97 hits.
• All the sequence data (from the 97 hits) were downloaded onto my computer using the command line code from my terminal.
• I navigated to the dyn90:bin and used the command ./prefect SRR292149
• The "SRR292149" was the SRA annotation given to each sequence hit
• This was repeated for all 97 hits, the sequence data was stored in .sra format in my NCBI --> Public --> sra directory

Below is a list of each hit and their SRA accession number

Hit #	SRA Accession #
1	ERR274161
2	ERR274160
3	ERR274159
4	ERR274158
5	ERR274157
6	ERR274156
7	ERR274155
8	ERR274154
9	ERR274153
10	ERR274152
11	ERR274151
12	ERR274150
13	ERR274149
14	ERR274148
15	ERR274147
16	ERR274146
17	ERR274145
18	ERR274144
19	ERR274143
20	ERR274142
21	ERR274141
22	ERR274140
23	ERR274139
24	ERR274138
25	ERR274137
26	ERR274136
27	ERR274135
28	ERR274134
29	ERR274133
30	ERR274132
31	ERR274131
32	ERR274130
33	ERR274129
34	ERR274128
35	ERR274127
36	ERR274126
37	ERR274125
38	ERR274220
39	ERR274219
40	ERR274218
41	ERR274217
42	ERR274216
43	ERR274215
44	ERR274214
45	ERR274213
46	ERR274212
47	ERR274211
48	ERR274210
49	ERR274209
50	ERR274208
51	ERR274207
52	ERR274206
53	ERR274205
54	ERR274204
55	ERR274203
56	ERR274202
57	ERR274201
58	ERR274200
59	ERR274199
60	ERR274198
61	ERR274197
62	ERR274196
63	ERR274195
64	ERR274194
65	ERR274193
66	ERR274192
67	ERR274191
68	ERR274190
69	ERR274189
70	ERR274188
71	ERR274187
72	ERR274186
73	ERR274185
74	ERR274184
75	ERR274183
76	ERR274182
77	ERR274181
78	ERR274180
79	ERR274179
80	ERR274178
81	ERR274177
82	ERR274176
83	ERR274175
84	ERR274174
85	ERR274173
86	ERR274172
87	ERR274171
88	ERR274170
89	ERR274169
90	ERR274168
91	ERR274167
92	ERR274166
93	ERR274165
94	ERR274164
95	ERR274163
96	ERR274162
97	SRR292149

//EWW

Chlorine Dioxide - Roach & RFB Lethality

From 4-3-15.

Checked the survival status of the cockroaches and RFB that had been exposed to ClO2 gas. The insects were exposed to the gas for 24 hours, and allowed to recover for 48 hours after that.

ClO2 & Cockroaches

The survival and health of the juvinille Madagascar hissing cockroaches was determined by gentle prodding with a forceps if survival was not apparent through visual examination. All of the five roaches exposed to the 3 grams of chlorine dioxide reagent were very dead and all of the five roaches that were not exposed to the gas were healthy and very active.

ClO2	Survival
3 grams	0/5
None	5/5

It was very interesting to see that the ClO2 seems to effect insects with exoskeletons similarly to how it effected the soft-bodied wax worms by seemingly causing their death. If the ClO2 utilizes the soft bodies of insects basically as a water sink, I wonder what is happening with the hard-shelled cockroaches that causes their demise. This was a relatively high concentration of ClO2 gas used in this experiment and they were young roaches used, however the goal of this experiment was to determine if there would be any effect - and there was. These results could warrant future investigation. Perhaps a titration curve or a pilot study to investigate the gas's effect on adult (dubia) cockroaches.

ClO2 & RFB

I checked the survival of the RFB that were exposed to 100 mg of ClO2 gas. The results were very surprising. In a reverse of what would be expected, all of the RFB not exposed to the gas were dead and those that were were still alive! I am very sure that this was the correct results and that the tubes were simply not switched around accidentally. 

ClO2	Survival
100 mg	25/25
None	0/25

All of the RFB in the control group (no gas) were completely dead and lifeless. All of the RFB exposed to the gas were very lively and active still, as though the gas hadn't effected them at all. I am unsure what caused these surprising and suspicious results. I don't believe that the RFB didn't suffocate in the control group, since the experimental group's cover was heavily parafilmed and would have likely had even less air transfer. Perhaps the ClO2 actually protected or aided in the survival of the RFB when they would have normally perished? Very odd. I will likely repeat this experiment in the near future with a higher ClO2 concentration to see if the results can be replicated....

//EWW

ND Honey Bee Stats

I have received summary data from a Plant Protection Specialist/State Apiary Inspector with the North Dakota Department of Agriculture. A particular set of data that was received was the total amounts of North Dakota apiaries, the amount of them that had been inspected, and the amount of apiaries that were diagnosed with American Foulbrood (AFB) disease for each year since 1979. AFB is caused by the bacterium P. larvae. The data that was received was only summary data, with a single number for cases that year, so statistical analyses was limited. Below is the percent of total registered apiaries in North Dakota that were diagnosed with AFB since 1979:

Raw Data:

Please note, the above graph illustrates the number of ND apiaries that were diagnosed with AFB over the TOTAL number of ND apiaries. This does not take into account how many of the total apiaries that were actually inspected- that data is further below. However, some data from specific years were not complete. There were five years when the total number of apiaries that were inspected was not recorded, and one year when the amount of inspected apiaries diagnosed with AFB was also not recorded. The above graph still includes these years, however they are listed as 0% as values were missing. The specific years and what was not recorded is listed below:

Missing data
Tot apiaries			Apiaries w/ AFB
1979			1992
1982
1983
1984
1985

Raw Data:

Missing data
Tot inspected apiaries			Apiaries w/ AFB
1992			1992

The above data is the amount of apiaries diagnosed with AFB over the amount of ND apiaries inspected. This data provides a more accurate representation of the prevalence of AFB in ND over the years. AFB is a reportable disease, and when it is the hive(s) in the apiary are typically destroyed at the apiarists expense. It is my understanding that the ND state apiary inspectors only visit an apiary if it has been requested, however I am not completely familiar with regulation of apiaries in the state. Overall, it appears that AFB is not a hugely pervasive disease in North Dakota based on the amount that has been diagnosed from those apiaries inspected each year. I can use this percentage data to extrapolate the actual percent of AFB in the entire state of ND, assuming the above data is an accurate representation of the average.

Below is the total amount of registered ND Apiaries. It is surprising to see that the amount actually increased since 1979. It appears that ND is at an all time high for registered apiaries.

//EWW

Wax Worm/P. larvae LD50

From 3-31-15.

It has been seven days since the wax worms were exposed to P. larvae spores both through injection and through their diets. I have been closely monitoring their health and survival. When a wax worms had died in any of the groups it was removed.

LD50 Injection

There were three biological replicates for each treatment, with ten technical replicates each. The below graph was made using Prism 6.0, however it does not include error bars. I wanted to test my skills at using the Prism software and found it to be surprisingly easy to use- you can just copy and paste your data from an Excel spreadsheet (where I had it) to Prism.

Average number of WW that were alive at each time point (n=10)

There was no deaths recorded in both the "No Inject" control group over the course of seven days. There was minimal death observed in the PBS injected group, which I attribute to poor injection skills- this is only about the second time I've injected WW. It is possible that some sort of internal damage was caused by me sticking the needle too far inside the WW to inject. Unfortunately, the groups of wax worms injected with 2000 P. larvae spores showed similar survival to those injected with PBS. This is the same trend that has been seen previously (at least it's consistent!). I likely need a higher concentration of P. larvae spores to inject the WW with in order to cause disease and death... if P. larvae causes disease in WW. If the WW is injected with a high enough concentration then simply the sheer volume of bacteria would be enough to cause the death of the WW. As previously seen from the images I've posted, there are potential signs of disease in the wax worms that have been injected with P. larvae, however it does not lead to their death like what is seen in the B. thur spore groups. The positive control in this experiment made use of the known insect pathogen B. thuringiensis spores. WW were injected with 200,000 B. thur spores and just after the first day about half of the injected WW were dead. This trend is also what has been previously seen. The problem remains, as always, I need to have access to higher concentrations of P. larvae spores.


LD50 Ingestion

Spores added to the container with 5 wax worms:

P. larvae = 10,000 CFU

B. thuringiensis = 1,000,000 CFU

For this study, early instar WW were fed Bee Artificial Diet (BAD) spiked with bacterial spores. A similar overall trend was seen with the WW in this study that was seen in the LD50 Injections study as well. Minimal deaths were observed in the No Diet and No Spores in Diet control groups. The WW that were fed the P. larvae spores had a similar survival that was seen in the two control groups. Once again, a large amount of deaths was recorded in the B. thur group. By day three all the wax worms in this group were dead in all three of the replicates. This isn't overly surprising as the WW were exposed to a very high concentration of B. thur spores. I stopped the experiment after seven days since survival seems to have leveled off in all of the groups, indicating that likely if the spores haven't led to the death of the WW- they never will.

//EWW

Chlorine Dioxide - Roach & RFB Lethality

Chlorine Dioxide - Wax Worm Lethality - 3-30-15

Goal: Perform a pilot study to gauge the efficacy of chlorine dioxide gas's ability to kill cockroaches and red flour beetles (RFB). The gas has previously been show to be effective against soft bodied insects (wax worms), but its effect on insects with exoskeletons has yet to be well characterized. I wish to perform an exposure assay using a relatively high concentration of chlorine dioxide gas. This will give me an idea of whether or not the gas will be lethal at all. However, I need to keep in mind that if lethality isn't observed it doesn't mean the gas did not effect the insects in some other way, only that there may have not been an acute observable effect.

ClO2 & Cockroaches

A number of cockroaches were exposed to ClO2 gas in the 925 mL volume container that had been previously used in the wax worm pilot studies ( 3-30-15). For this experiment, five juvenile Madagascar hissing cockroaches were placed into the container. I did not intend to use the Madagascar roaches, but I didn't notice my mistake in cockroach species until after the gas was added. I intended to use the Blaptica dubia roaches instead as the Madagascar are more valuable and difficult to rear and should not be squandered on a preliminary study such as this!

I modified a 1.5 Eppendorf tube similarly to how PCR tubes were modified in the wax worm pilot study. Six small holes were made on the sides of the Eppendorf tube using a heated inoculating needle. Four more holes were made in the lid of the tube. A total of 3 grams of chlorine dioxide reagent (1.5 grams from each of the two parts A and B) were mixed in the modified Eppendorf tube.

There were only two groups for this pilot experiment- a negative control and an experimental group. Modified Eppendorf tubes were places into each of the containers with roaches, but only the experimental group's was filled with the chlorine dioxide reagent. The containers were incubated at room temperature, protected from light, for 24 hours. After the 24 hours, I will remove the modified Eppendorf tubes and air out the containers for five minutes. The containers will be incubated at room temperature for an additional 24 hours to allow the insects to recover (if they will) before survival is quantified.

ClO2 & RFB

I also exposed ClO2 gas to a large amount of red flour beetles (RFB). The RFB were exposed to the gas inside of a modified 50 mL conical tube. The same modified PCR tube method was used in this experiment as was used in the wax worm exposure experiment. In order to expose the RFB, and in order to keep the RFB from coming into direct contact with the gas reagents, a modification was made to the cover of the 50 mL conical tube. The modified PCR tube was placed inside of the 50 mL tube cover by creating a small hole in it. Images of this modification is seen below. 

The above modification will ensure that the RFB will not come into direct contact with the gas reagents, but still experience the full effect of the gas (ClO2 is heavier than air and will sink to the bottom of the tube once it is generated).

Between 25 - 30 RFB were added to each container using a metal scoopula. No larval stages of RFB were added. A small amount of wheat flour was also added to the tube. An image of what this looked like is seen above. Only a total of 100 mg of chlorine dioxide reagent was used in this experiment as I was limited due to the size of the PCR tube. The reagents were mixed inside of the modified PCR tube. The modified PCR tube was secured using masking tape and the tube was sealed very thoroughly using Parafilm.

For this experiment, only a negative control and experimental treatment group was used. The tubes were incubated at room temperature, protected from light, for 24 hours. After 24 hours, the health of the RFB will be evaluated.

//EWW

Sporulation on Blood Agar

From 3-31-15.

It has been 10 days since the Colombian sheep's blood agar slants had been inoculated with 300 uL of P. larvae 9545 broth culture and incubated at 37C. The agar slants were washed and endospores were recovered.

A 3 mL volume of chilled sterile ddH2O was added to the surface of the agar slant. The surface of the agar was gently rubbed using a sterile inoculating loop to dislodge any of the attached cells. NOTE: be careful when rubbing as to not loosen or break any agar as it will become difficult to pipet and wash later!The volume was carefully transferred to a sterile 50 mL conical tube. The wash was repeated once more with 3 mL. The entire volume of five slants was combined into a single 50 mL tube. The tube and its contents was centrifuged at 15,000 x g for 5 minutes. The remainder of the wash and endospore extraction procedure was the same as previously performed .

P. larvae growth on the blood agar slants after 10 days

The biggest obstacle I encountered from extracting the spores from agar slants was that sometimes a small portion of the agar breaks off during the abrasion step during removal. This bit of agar can clog the pipet or can be easily transferred and become an issue during the wash steps. The agar is nearly invisible and I often times the only reason I noticed them is when they clogged my pipet. Fortunately, the agar is not as heavy as the endospores and will be removed with proper washing techniques. In the future, I will have to take better care as to limit the amount of agar that is dislodged during the initial steps.

Below is an image for future reference of what the endospore pellet looked like in size. Keep in mind, this is from five slants. I have never seen a pellet this size with P. larvae, and it was very easy to see the distinction between endospores and vegetative cells - the endospores were a much darker color whereas the vegetative cells and other debris were white/yellow.

Endospore pellet of P. larvae. This was the size of the darker bottom pellet (the spores).

A total of four P. larvae 9545 endospore stocks were made (each combined from five different slants). The stock was diluted and plated to determine CFU/mL. Microscopic examination revealed a large amount spores, a bit less than what has typically been seen after extracting B. thur spores. However, the spores in P. larvae were significantly more clumped together compared to Bacillus spores that underwent the exact same treatment and method of re-suspension. This clumping of P. larvae spores may be responsible for some of the erratic CFU counts in previous quantification attempts. After a few days of incubating at 37C I will quantify the concentration of spores.

//EWW