effect of animal sera on bacillus anthracis sterne spore germination and vegetative cell growth

ORIGINAL ARTICLE

Effect of animal sera on Bacillus anthracis Sterne sporegermination and vegetative cell growthM.D. Bensman, R.S. Mackie, Z.A. Minter and B.W. Gutting

Dahlgren Division, CBR Concepts and Experimentation Branch (Z21), Naval Surface Warfare Center, Dahlgren, VA, USA

Introduction

Bacillus anthracis is a Gram-positive spore forming bacte-

rium and is the causative agent of inhalational anthrax

disease. Bacillus anthracis is a dangerous biological

weapon, as was seen in 2001 when spores were sent

through the US mail system causing multiple casual-

ties (Frazier et al. 2006; Bush and Perez 2012). Today,

B. anthracis may represent the single greatest biological

warfare threat (MacIntyre et al. 2006).

Understanding host–pathogen interactions, disease

incubation period, infection kinetics and how these mech-

anisms ⁄ kinetics differ among laboratory animals and man

is central to developing strategies to defend against

another attack (Goossens 2009). For example, this infor-

mation can be helpful in assessing overall risk of disease

(Coleman et al. 2008), defining therapeutic windows to

better target postexposure treatments (Yi and Setlow

2010; Weiss et al. 2011) and can also aid in locating the

point of spore release in the environment (Franz 2009;

Kournikakis et al. 2010).

Two critical pathogenic mechanisms of inhalational

anthrax are germination of newly deposited spores and rep-

lication of vegetative bacteria (Frankel et al. 2009; Twenha-

fel 2010; Cote et al. 2011). Spore germination converts the

dormant inert spore into a metabolically active vegetative

bacterium capable of secreting toxin and replicating in the

host to ultimately reach a threshold number of bacteria suf-

ficient to induce clinical disease and death. To counteract

these processes, the host has numerous defence mecha-

nisms (Passalacqua and Bergman 2006; Tournier et al.

2009; Cancino-Rodezno et al. 2010). Thus, disease out-

come rests in the balance between host defences working to

contain and eliminate the infection and the spores evading

the host defences. It has long been recognized that host

serum components can have an effect on spore germina-

tion, replication and spread of bacteria from the lung to the

circulation (Gross et al. 1978; Ferguson et al. 2004). It is

therefore important to compare and contrast how sera

from different species effect spore germination and vegeta-

tive cell growth in an attempt to better understand the

infection process for a given host.

Keywords

Bacillus anthracis, germination, growth, sera,

spores.

Correspondence

Bradford W. Gutting, 4045 Higley Road, Suite

344, Dahlgren, VA 22448, USA. E-mail:

[email protected]

2012 ⁄ 0048: received 10 January 2012,

revised 28 March 2012 and accepted 13 April

2012

doi:10.1111/j.1365-2672.2012.05314.x

Abstract

Aims: The aims of this work were to investigate the effects of sera on

B. anthracis Sterne germination and growth. Sera examined included human,

monkey and rabbit sera, as well as sera from eight other species.

Methods and Results: Standard dilution plate assay (with and without heat

kill) was used as a measure of germination, and spectroscopy was used to mea-

sure growth. In addition, a Coulter Counter particle counter was used to moni-

tor germination and growth based on bacterial size. Spores germinated best in

foetal bovine and monkey sera, moderately with human sera and showed lim-

ited germination in the presence of rabbit or rat sera. Vegetative bacteria grew

best in foetal bovine sera and moderately in rabbit sera. Human and monkey

sera supported little growth of vegetative bacteria.

Conclusion: The data suggested sera can have a significant impact on germina-

tion and growth of Sterne bacteria.

Significance and Impact of the Study: These data should be considered when

conducting in vitro cell culture studies and may aid in interpreting in vivo

infection studies.

Journal of Applied Microbiology ISSN 1364-5072

276 Journal of Applied Microbiology 113, 276–283 ª 2012 The Society for Applied Microbiology

ª 2012 The Authors

In addition to examining in vivo pathogenesis, another

important aspect of modern day research into anthrax

disease is the use of in vitro cell culture systems to study

isolated interactions between B. anthracis and specific

host cells, or in some cases, to study host–pathogen inter-

actions that are impossible to observe using in vivo mod-

els. Here, in vitro culture systems nearly always include

sera to culture media that allow the host cells to survive

out to longer time points (Welkos et al. 2002; Pickering

et al. 2004; Hu et al. 2007; Oliva et al. 2008, 2009; Doz-

morov et al. 2009; Xue et al. 2010; Gut et al. 2011).

Although beneficial (and often required) to the host cell

under study, it is unclear what effect host sera has on the

bacteria and whether or not observations made using in

vitro systems are because of host cell being studied or are

the result of the sera supplement. For example, one com-

mon finding under these conditions is that serum amend-

ments can have a substantial degree of influence on

B. anthracis spore germination and the outcome of the

spore–host cell interaction (Hu et al. 2007; Gut et al.

2011). This has prompted some investigators to vary the

amount of sera in the culture or completely remove it

altogether (Guidi-Rontani et al. 2001; Bergman et al.

2007). In contrast, other studies suggest that the addition

of sera has little outcome (Gut et al. 2011). This high-

lights the importance of gaining a better understanding of

how sera from different host species can support or hin-

der B. anthracis germination and growth, and this infor-

mation may be useful when studying inhalational anthrax

using in vivo or ex vivo tissue culture methods.

In this work, B. anthracis Sterne spores were inoculated

into media amended with various animal sera to deter-

mine how sera affected the rate of spore germination and

growth of vegetative bacteria. Sera from eleven animal

species were examined in total but the work focused on

four species: foetal bovine serum because it is a common

serum used for in vitro studies regardless of what animal

the cells under study originated in, rabbit and non-

human primate sera because these two species are the

current recommended animal models to study inhala-

tional anthrax, and human serum. The data suggest that

the type of animal sera used can have a significant

influence on spore germination rate and the growth of

vegetative bacteria.

Materials and Methods

Bacillus anthracis Sterne strain

Sterne spores were grown under aerated conditions in LB

broth shaking (225 rev min)1) at 37�C for 2 days. There-

after, spores were washed twice with cold PBS, heat-

shocked, titred and stored at )80�C until used.

Sera

All sera preparations contained 10% of animal sera (in

90% Dulbecco’s modified Eagle’s medium (DMEM)) that

were heat inactivated for 30 min at 56�C prior to use.

Individual aliquots of heat-inactivated sera were stored at

)80�C. DMEM was purchased from ATCC. Sera used

included foetal bovine, rabbit, rat, mouse, porcine, guinea

pig, canine and feline sera (all purchased from Equitech-

Bio, Kerrville, TX), as well as, cynomolgus monkey, rhe-

sus monkey and pooled human AB sera (all purchased

from Innovative Research, Novi, MI).

Spore germination assay

To maintain consistency with standard in vitro study pro-

tocols, 10% of the respective sera (in 90% DMEM) were

added to each well of a 24-well tissue culture plate. At

least three separate wells were used in each experiment

(n = 3). Spore aliquots were thawed at room temperature

and 2 · 104 Sterne spores (in 20 ll) were added to each

well. The final volume was 1 ml per well. Plates were then

incubated at 37�C with 5% CO2 until desired time point

was reached (4 or 8 h). Thereafter, an aliquot of each

sample was plated on Tryptic Soy Agar (TSA) plates

(Hardy Diagnostics, Santa Maria, CA) with (65�C for

30 min) or without heat.

For analysis using the Coulter Counter (MultiSizer3;

Beckman Coulter, Brea, CA), 1 · 105 Sterne spores were

added to each well of a 24-well tissue culture plate. Plates

were then incubated at 37�C with 5% CO2 until desired

time point was reached. At the desired time point, sam-

ples were removed, diluted with 9 ml of triple filtered

PBS and 100 ll was ran through the Coulter Counter.

Particles that fell between 0Æ971 and 1Æ557 lm were con-

sidered spores and those falling between 1Æ557 and

5Æ874 lm were vegetative bacteria.

Vegetative cell assay

Vegetative cell cultures of B. anthracis Sterne were

prepared by adding 50 ll of spore stock (1Æ1 · 1010 spor-

es ml)1) to 50 ml Tryptic Soy Broth (TSB) in a 500-ml

baffled flask shaking (225 rev min)1) overnight at 37�C.

Thereafter, log-phase vegetative cultures were produced

by placing 5 ml of the overnight culture into 45 ml of

fresh TSB and shaking at 225 rev min)1 for 2 h at 37�C.

Aliquots of vegetative culture (1 ml) were then removed

from the flask and centrifuged at 5000 g for 2 min to pel-

let bacteria. Supernatants were removed, and pellets were

washed twice with ice-cold DMEM and resuspended in

5 ml of 10% sera ⁄ 90% DMEM at 3Æ3 · 106 CFU ml)1.

Thereafter, solutions were diluted 1 : 10 in 200 ll 10%

M.D. Bensman et al. Sera effect on B. anthracis

No claim to US Government works

Journal of Applied Microbiology 113, 276–283 ª 2012 The Society for Applied Microbiology 277

sera ⁄ 90% DMEM into wells of a 96-well plate. Plates were

incubated at 37�C in a Synergy HT Microplate reader

(Biotek, Winooski, VT) with continuous shaking on med-

ium setting. Optical density measurements at 600 nm

were performed on the entire plate every 20 min for a

total of 600 min.

Statistics

All data are reported as mean ± standard error of the

mean (SEM). Statistical analysis was performed using

KYPLOT using Student’s t-test with P-values < 0Æ05 consid-

ered significant.

Results

Sterne spore germination and growth in various animal

sera

Heat-shocked Sterne spores were added to specific sera

(10% sera, 90% DMEM) and at specified time points (4

and 8 h), spores were removed and plated with or with-

out heat kill. Spores incubated in the presence of foetal

bovine serum or rhesus serum showed the most signifi-

cant germination based on loss of heat-resistant CFU

recovered from the culture (Table 1). As shown, after 4 h,

<0Æ1% of the CFU were heat resistant in the presence of

foetal bovine sera or rhesus sera, and after 8 h, <0Æ001%

of the CFU were heat resistant for either bovine or rhesus

serum (Table 1). It should be noted that by 4 or 8 h

incubation in the presence of foetal bovine or rhesus sera,

there was significant outgrowth of bacteria. As a result,

the number of vegetative bacteria quantified at these time

points greatly exceeded the number of spores originally

placed in the culture (0 h time point) or the number of

spores remaining at the respective time point, which

explains why some spore numbers are exceptionally small

(i.e. 0Æ00008% remaining, Table 1). In contrast to foetal

bovine or rhesus sera, human or rabbit sera appeared to

support limited germination as approximately 32 and

79% spores remained after 8 h, respectively. Spores

showed moderate germination in the presence of human

sera with 19 and 0Æ65% spores remaining after 4 or 8 h,

respectively. Spores in DMEM alone showed 85% spores

remaining at 8 h.

It was noticed during the course of these experiments

that some of the spores may be sticking to the sides and

bottom of the culture plate. This made it impossible to

accurately measure germination in the presence of differ-

ent sera by comparing the number of spores recovered at

4 or 8 h directly with the number of known spores inocu-

lated in each well at the beginning of the incubation. For

example, if 2 · 104 heat-shocked spores were added to a

particular culture well and after 4 h of incubation 1 · 102

heat-resistant CFU were recovered from the well, it is not

known if half the spores germinated (became heat sensi-

tive) or if half the spores were still spores that were stuck

to the plate. For this reason, we report the data in

Table 1 as per cent of recovered CFU that is heat sensitive

and we also used a second independent analytical method

to measure germination – the Coulter Counter.

The Coulter Counter was used to measure germination

in various sera where the number of spores (0Æ971–

1Æ557 lm size particles) was measured over time. In addi-

tion, an increase in larger particles (1Æ557–5Æ874 lm parti-

cles) was used as an initial measure of vegetative growth.

As shown in Fig. 1(a), in the presence of foetal bovine

serum, the spore peak at the beginning of the incubation

(thick line) completely disappears by 4 h (thin line) and

is replaced by a significant vegetative cell peak at 8 h

(solid line, shown rescaled in the inset graph of Fig. 1a).

These data support the dilution plate data in Table 1 and

suggest that in the presence of foetal bovine serum, there

is near complete germination by 4 h. For normal rabbit,

rhesus and human sera, there was little germination

detected at 4 h as shown by the overlap of thick solid line

and thin line in Fig. 1(b–d), but all three showed an

increase in vegetative cells by 8 h suggesting some germi-

nation and growth. It is worth noting the number of veg-

etative cells detected at 8 h using the Coulter Counter. In

the inset figure in Fig. 1(a) (foetal bovine serum), the

y-axis scale ranges from 0 to 3500 particles, which sug-

gests a significant increase in the number of vegetative

cells at 8 h. This is in sharp contrast to data collected

using human sera where the entire vegetative particle

counts can be shown using a scale of 0–140 particles. This

suggests that foetal bovine sera support Sterne outgrowth

better than sera from other species.

To measure more directly what effect sera has on vege-

tative cell growth, log-phase growth Sterne bacteria were

combined with different sera and their growth was mea-

sured over time using spectroscopy. As shown in Fig. 2,

foetal bovine serum provided the best growth conditions

when compared with the other sera tested. As shown,

rhesus and human sera did not support vegetative cell

growth under the experimental conditions used and

rabbit sera supported moderate growth with increased

time.

Discussion

The aim of the current work was to compare and contrast

what effect different animal sera had on Sterne spore ger-

mination and vegetative cell growth. The data suggested

sera can have a significant impact on both events. For

example, when using heat-shocked spores, <0Æ0001% of

Sera effect on B. anthracis M.D. Bensman et al.


ª 2012 The Authors

the CFU recovered after 8 h of incubation had retained

heat resistance in the presence of foetal bovine serum,

whereas nearly 80% of CFU recovered after 8 h of incu-

bation in the presence of rat sera retained their heat resis-

tance. When comparing the sera from human, monkey

and rabbit, nearly 100% of the spores appeared to germi-

nate in the presence of rhesus and cynomolgus sera,

whereas rabbit sera were a poor supporter of germination

(nearly comparable with observations made using serum-

free-DMEM alone) and human sera fell in between. For

replication, limited vegetative cell growth was observed

using human or monkey sera after 10 h of incubation. In

contrast, foetal bovine sera supported the most vegetative

cell replication and rabbit sera fell in between foetal

bovine serum and rhesus ⁄ human sera. These observations

may be important when comparing and contrasting inha-

lational anthrax disease in different animal models and

may be useful in predicting certain aspects of the disease

in humans. In addition, the data should be considered

when designing and conducting in vitro or ex vivo cell

Table 1 Affect of sera on Bacillus anthracis Sterne spore germination

Type of sera

Per cent spores remaining

4 h 8 h

Foetal Bovine 0Æ068 ± 0Æ04 0Æ00008 ± 0Æ00008

Rhesus 0Æ0 ± 0Æ0 0Æ0009 ± 0Æ0005

Cynomolgus nd 0Æ003 ± 0Æ002

Feline nd 0Æ005 ± 0Æ0008

Pig nd 0Æ005 ± 0Æ002

Human A ⁄ B 19Æ3 ± 2Æ97 0Æ65 ± 0Æ062

Canine nd 2Æ78 ± 0Æ66

Guinea Pig nd 24Æ4 ± 4Æ4

Mouse nd 26Æ33 ± 5Æ93

Normal Rabbit 85Æ4 ± 11Æ7 32Æ03 ± 0Æ26

Rat nd 78Æ7 ± 19Æ3

DMEM 86Æ1 ± 16Æ4 84Æ9 ± 14Æ0

nd, not determined; DMEM, Dulbecco’s modified Eagle’s medium.

Per cent spores in sample at specified time point = (heat-resistant

CFU ⁄ total CFU) · 100.

150 4000

3500

3000

2500

2000

1500

1000

500

0

Num

ber

of p

artic

les

0Particle diameter (mm)

1 2 3

140

4 5 6

1301201101009080706050403020100

0 1 2Particle diameter (µm)

Num

ber

of p

artic

les

3 4 5 6

Num

ber

of p

artic

les

1200

1000

800

600

400

200

00 1 2 3 4 5 6

Particle diameter (mm)

1501401301201101009080706050403020100


Num

ber

of p

artic

les

3 4 5 6

700

600

500

400

300

200

100

00 1 2 3 4 5 6

Particle diameter (mm)

Num

ber

of p

artic

les

1501401301201101009080706050403020100


Num

ber

of p

artic

les

3 4 5 6

Particle diameter (mm)0

0

50

100

150

Num

ber

of p

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1 2 3 4 5 6

1501401301201101009080706050403020100


Num

ber

of p

artic

les

3 4 5 6

(a) (b)

(c) (d)

Figure 1 (a–d) Coulter Counter analysis of Bacillus anthracis Sterne spores cultures in the presence of various animal sera. Foetal bovine sera (a),

rabbit sera (b), rhesus sera (c) or human AB sera (d) were incubated with spores for various time points: 0 h (thick line), 4 h (thin line) and 8 h

(inset graph). Particles that fell between 0Æ971 and 1Æ557 lm were considered to be spores and those falling between 1Æ557 and 5Æ874 lm were

interpreted as vegetative bacteria.




culture studies where investigators need to distinguish

between cell-induced affects and culture media-induced

affects.

There are limited human data available to directly

assist decision-makers on aspects of inhalational anthrax

disease in man. As a result, most decisions on inhala-

tional anthrax disease in man are based on animal model

data (FDA 2007, 21 C.F.R. § 314.610, drugs; § 601.91,

biologics) and two animal models often used are the non-

human primate and NZW rabbit. A potential limitation

in the rabbit model is the fact that the disease progresses

much faster in rabbits than that observed in non-human

primate models and what is thought to occur in man and

as a result some pathological changes observed in higher

order species do not have time to develop in the rabbit

(Zaucha et al. 1998). The current work may help to

explain, in part, host-specific disease kinetics. Rabbit sera

supported vegetative cell replication much better than

either human or non-human primate sera (Fig. 2). Faster

bacterial replication in the circulation, coupled with the

smaller size and total blood volume of rabbits compared

with human or non-human primates, suggest B. anthracis

could reach the threshold concentration of bacteria in the

circulation required to induce clinical disease and death

much faster than in man or non-human primates. The

data presented here also suggest faster outgrowth when

starting with spores rather than log-phase bacteria in the

presence of rabbit sera compared with non-human pri-

mate or human sera (Fig. 1). This, again, may help

explain the faster inhalational anthrax disease kinetics in

rabbits because it has been known for some time that

bacterial–serum interactions can have a significant influ-

ence on the outcome of pulmonary infections (Gross

et al. 1978). Thus, the results presented in the current

study suggest vegetative cells may find a more favourable

environment for outgrowth in the rabbit compared with

human or non-human primate and this may explain, in

part, why disease progresses faster in rabbits.

There was a wide range of germination rates observed

when using different sera. Nearly, 100% of the spores

after 4 h of incubation with rhesus or foetal bovine serum

appeared to have germinated, whereas there was no

detectable difference in germination between rabbit sera

and serum-free DMEM after 4 h (Table 1). As germina-

tion is the initial step in disease pathogenesis in vivo and

serum components are known to interact with bacteria in

the lung after they are inhaled, the data presented here

would suggest rabbits may be better suited to survive an

inhaled dose of spores compared with non-human pri-

mates because deposited spores may germinate slower in

rabbits, thereby giving the rabbit a larger window to clear

the inert spore particles. However, the dose-response data

that are available do not appear to support this hypothe-

sis because the LD50 for rabbits and non-human primates

are thought to be very similar, suggesting rabbits and

non-human primates are equally sensitive to inhalational

anthrax disease (Coleman et al. 2008; Twenhafel 2010). It

is worth noting this conclusion is based on high-dose

studies where there are 104–106 spores being deposited

and it is possible that any difference in germination

across species may be swamped simply because of the

number of spores present and saturation of clearance

pathways. In other words, if rabbit serum fails to support

spore germination and this affect helps to render rabbits

resistant to inhalational anthrax, then this would be most

readily observable following low-dose exposures where

low numbers of spores are being deposited. Our prelimin-

ary data have demonstrated that groups of rabbits

exposed to daily low-dose (�1000 spores) aerosols of

fully virulent Ames spores 15 separate times over 3 weeks

survived without any sign of morbidity or mortality

(unpublished observations). In this light, rabbits appear

resistant to lethal infections following multiple low-dose

exposures. The mechanistic reasons why the rabbits

appeared to clear the low-dose infections remain to be

determined, but the data presented in the current work

suggest a lack of conditions conducive for germination

owing to rabbit sera may contribute. It is unknown if

non-human primates or other species can survive a

similar dosing regimen with fully virulent Ames spores.

A hypothesis that could be formed based on data and

discussion above is that rabbits are less sensitive to

acquiring inhalational anthrax disease, but once disease is

established it progresses faster in rabbits compared with

the other species tested. In the other extreme, the germi-

nation and outgrowth data presented here suggest that of

the 11 species tested, foetal bovine serum is the best sup-

porter of germination and the best serum for vegetative

cell growth. This suggests cows may be very sensitive to

anthrax disease compared with the other species. In sup-

port of this hypothesis, a recent naturally occurring out-

Figure 2 Growth kinetics of Bacillus anthracis Sterne vegetative cells

in Dulbecco’s modified Eagle’s medium amended with 10% animal

sera: FBS (•), NRS ( ), Rhesus ( ), HAB (.).



ª 2012 The Authors

break of anthrax in Canada showed several species were

exposed during the event but most of the losses occurred

in cattle (Epp et al. 2006) and an anthrax endemic in

North Dakota appeared to target livestock (Ndiva Mon-

goh et al. 2008). Finally, the data presented here suggested

the effect of human sera on B. anthracis are very similar to

non-human primates. The vegetative growth data for the

two were identical (Fig. 2) and after 8 h of incubation with

spores and the respective sera, <1% of the recovered bacteria

were heat resistant (Table 1). This is in line with the long-

held belief that non-human primate inhalational anthrax

models are most representative of the disease in man.

Although the discussion above tries to connect observa-

tion made in the present work with disease in a specific

host, it is unlikely that a full description of inhalational

anthrax can be made simply by looking at the direct

effects of host sera on spores or vegetative cells because

numerous host cells and tissues have a role in disease

establishment and progression (reviewed by Cote et al.

2011). For this reason, many investigators study anthrax

in tissue culture systems where the addition of sera to

basal culture media (such as DMEM) is common practice

and is often required to keep the eukaryotic cells alive.

A common serum used in cell culture studies is foetal

calf serum regardless of the species origin of the cell

under study. For example, foetal calf serum has been used

with human primary cell cultures (Gold et al. 2004; Doz-

morov et al. 2009), mouse primaries (Pickering et al.

2004; Kang et al. 2005; Cleret et al. 2006; Hu et al. 2006,

2007; Sabet et al. 2006) as well as with immortalized cell

lines (Ireland and Hanna 2002; Gold et al. 2004; Bergman

et al. 2005, 2007; Gutting et al. 2005; Hu et al. 2006; Cote

et al. 2008). One question raised from the current work is

whether or not the use of foetal calf serum introduces

biology that would not normally be seen if the sera used

came from the same species as the cell under study

(human, mouse, other). The data presented in the current

work show rapid germination and growth in foetal calf

serum compared with other sera tested. This observation,

coupled with recent data that showed the outcome of

RAW264.7 cells infected with B. anthracis was dependent

on the germinating efficiency of the spore in culture (Gut

et al. 2011) highlights the potential issue with using foetal

calf serum in culture with mouse or human cells.

Another challenge with in vitro culture studies is that

investigators often want to study intracellular germination

and growth inside immune cells because this is thought

to represent key steps in in vivo pathogenesis. In an

attempt to isolate intracellular events, investigators add

the antibiotic gentamicin to culture to kill extracellular

vegetative cells from replicating, (Gold et al. 2004; Picker-

ing et al. 2004; Kang et al. 2005; Cleret et al. 2006; Sabet

et al. 2006; Cote et al. 2008; Dozmorov et al. 2009) – it

should be noted that this does not stop extracellular ger-

mination, only extracellular outgrowth. However, because

the addition of small molecule antibiotics could introduce

unwanted side effects on the spore and ⁄ or eukaryotic cell,

some investigators opt out of adding antibiotics and have

instead grown ⁄ maintained murine cell lines in foetal calf

serum and then during the experimentation step when

spores are added they switch to a nongerminating med-

ium such as 10% horse serum (Bergman et al. 2005,

2007). The results of the current work suggest that similar

effects could be achieved by switching to mouse sera

because mouse sera were one of the worst supporters of

spore germination (Table 1). This would also introduce

more biological relevance into the culture system, because

the investigators were using murine cell lines.

In summary, the observations made in this work mea-

sured germination and growth of B. anthracis spores in

the presence of sera from different animal species. The

data suggested both spore germination and vegetative cell

growth can be significantly affected by the type of sera.

These observations may be relevant when comparing dis-

ease pathogenesis in different host species, as well as when

conducting in vitro or ex vivo studies using host cells that

are known to interact with a germinating spore or vegeta-

tive cell. As part of follow-on studies, research should

investigate which serum components (e.g. amino acids,

small molecules, complement components, etc.) are

responsible for the differences observed in the present

work to advance our advancing host-specific disease char-

acteristics and also to potentially develop novel targets for

therapeutics.

Acknowledgements

Parts of this work was funded by the Defence Threat

Reduction Agency (CBS.PHYSIO.01.10.SW.PP.005), the

Environmental Protection Agency (DW17922155-01-1)

and the NSWCDD Academic Fellowship Program

(R.S.M.).

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