Use of selective media for direct isolation of Francisella ... · Francisella species, subspecies and biovar . Figures Figure 1: Segmentation of media Figure 2: Isolate 1 - Medium

Department for Farm Animals and Veterinary Public Health

University of Veterinary Medicine Vienna

Institute for Veterinary Public Health

(Head: Univ.-Prof. Dr. Josef Köfer)

Use of selective media for direct isolation of Francisella tularensis

from European brown hares

MASTER THESIS

for obtaining the degree

Master of Science (MSc.)

of the University of Veterinary Medicine Vienna

submitted by

Romana Posch

Vienna, July 2012

This work was performed at the Bacteriology Laboratory of the Institute for Veterinary Disease

Control of the Austrian Agency for Health and Food Safety (AGES), Mödling.

Supervisor: Ao.Univ.-Prof. Dr. Friedrich Schmoll

External Supervisor: Dr. Erwin Hofer

Reviewer: Univ.-Prof. Dr. Chris Walzer

ACKNOWLEDGMENTS

I wish to acknowledge the following persons, without them these pages would not be:

I would like to start to thank Prof. Friedrich Schmoll for the opportunity to work on this project and for supervision, critique and support.

Special thanks to Dr. Erwin Hofer of the Austrian Agency for Health and Food Safety (AGES) for his continuous support, patience and supervision and for sharing his knowledge about tularemia.

To Dipl. Tzt. Annika Posautz and her colleagues of the Research Institute of Wildlife and Ecology, Vienna, for providing the frozen tissue samples and for their support. To Dipl.-Ing. Helga Plicka of the Armament and Defence Technology Agency for performing real-time PCR. To Andrea Steffel and Sigrid Träger of AGES for excellent technical assistance. I also want to thank my laboratory colleagues Karin Knoll, Barbara Pohl and Mag. Gabriele Romanek for the good times, for the bad times and especially for the fun times.

The Institute for Veterinary Disease Control, Moedling, of the Austrian Agency for Health and Food Safety (AGES) for funding this project. My family and my friends for their understanding, motivation, continued support, and encouragement.

TABLE OF CONTENTS

1 Introduction and Objective of this study ...................................................................................... 1

1.1 Introduction .............................................................................................................................. 1

1.1.1 Francisellaceae ........................................................................................................... 1

1.1.2 Francisella tularensis ................................................................................................ 2

1.1.3 Incidence of tularemia in Austria .............................................................................. 2

1.1.4 Detection and diagnosis ............................................................................................. 3

1.1.4.1 Culture................................................................................................................ 3

1.1.4.2 Biochemical differentiation................................................................................ 5

1.1.4.3 Molecular detection ........................................................................................... 5

1.2 Objective of this study .............................................................................................................. 6

2 Materials and Methods .................................................................................................................. 7

2.1 Materials ................................................................................................................................... 7

2.1.1 Samples ...................................................................................................................... 7

2.1.1.1 Tissue samples from the FIWI frozen at – 80°C .................................................. 7

2.1.1.2 Tissue samples of unfrozen European brown hares ............................................. 8

2.1.1.3 Recovery of F. tularensis in frozen tissue samples .............................................. 8

2.1.2 Francisella tularensis ssp. holarctica control strains ................................................ 9

2.1.3 Culture Media ............................................................................................................ 9

2.1.4 Media and Materials for biochemical differentiation .............................................. 12

2.2 Methods .................................................................................................................................. 13

2.2.1 Direct Isolation of F. tularensis ............................................................................... 13

2.2.2 Culture diagnosis of tularemia and biochemical characterisation ........................... 14

2.2.3 Molecular methods .................................................................................................. 14

2.2.3.1 PCR template preparation ................................................................................... 14

2.2.3.2 Detection of F. tularensis through LightCycler real-time PCR .......................... 15

2.2.3.3 Discrimination of subspecies by real-time PCR ................................................. 16

2.2.4 Serial dilution .......................................................................................................... 11

3 Results ............................................................................................................................................ 18

3.1 Overview of investigated European brown hares ................................................................... 18

3.1.1 Tissue samples frozen at – 80°C .............................................................................. 18

3.1.2 Tissue samples of unfrozen European brown hares ................................................. 21

3.1.3 Recovery of F. tularensis in frozen tissue samples ................................................. 21

3.2 Isolation of Francisella tularensis in European brown hares ................................................. 22

3.3 Biochemical characterization ................................................................................................. 42

3.4 PCR......................................................................................................................................... 45

3.4.1 Detection of Francisella tularensis by PCR ............................................................ 45

3.4.2 Real-time PCR assay diagnostic of F. tularensis subsp. holarctica ........................ 46

3.5 Contaminants .......................................................................................................................... 47

3.5.1 Enterococcus faecalis .............................................................................................. 47

3.5.2 Bacillus sp. ............................................................................................................... 49

3.5.3 Yeast sp. ................................................................................................................... 50

4 Discussion ...................................................................................................................................... 53

5 Zusammenfassung ........................................................................................................................ 56

6 Summary ....................................................................................................................................... 58

7 References ..................................................................................................................................... 60

LIST OF TABLES UND FIGURES

Tables

Table 1: Francisella tularensis – basic markers for identification of subspecies and biovar

Table 2: Evaluation of frozen tissue samples of 219 European brown hares from Austria

Table 3: Austrian European brown hares differentiated into hunted and found dead

Table 4: Evaluation of frozen tissue samples of 162 European brown hares from Germany

Table 5: Characteristics of diagnostic value in identifying the genus Francisella and its

species, subspecies and biovar

Figures

Figure 1: Segmentation of media

Figure 2: Isolate 1 - Medium C with a F. tularensis colony

Figure 3: Isolate 2 - F. tularensis on Medium A, Medium B and Medium C

Figure 4: Isolate 3 - Growth of F. tularensis on all quadrants

Figure 5: Isolate 4 - F. tularensis colony visible on Medium C

Figure 6: Isolate 5 - Growth of F. tularensis on Medium A, Medium B and Medium C


Figure 8: Isolate 7 - Growth of F. tularensis on selective media


Figure 10: Isolate 9 - F. tularensis after 3 days of incubation

Figure 11: Isolate 10 - Medium A, Medium B and Medium C after 5 days of incubation of

sliced portions

Figure 12: F. tularensis on Legionella medium with Vancomycin and Colistin

Figure 13: Isolate 11 - Medium A, Medium B and Medium C after 8 days of incubation of

sliced portions

Figure 14: Fermentation of glucose and glycerine

Figure 15: Sensitivity to Erythromycin

Figure 16: Detection of F. tularensis using real-time PCR assay diagnostic

Figure 17: Real-time PCR assay diagnostic of F. tularensis subsp. holarctica

Figure 18: Effects of Enterococcus faecalis on Medium A

Figure 19: Comparison of growth of stock solution from Enterococcus faecalis

Figure 20: Effects of Bacillus sp. on Medium B

Figure 21: Growth of stock solution from Bacillus sp. on Medium A, Medium B,

Medium C and COS medium

Figure 22: Growth of Yeast sp. on Medium A

Figure 23: Growth of Yeast sp. in dilution 1:1,000

ABBREVIATIONS

AGES Austrian Agency for Health and Food Safety

CALTA colistin, ampicillin, lincomycin, trimethoprim, amphotericin

CDC Centers for Disease Control and Prevention, Atlanta, USA

CFU Colony Forming Units

CHAB Cystine heart agar with 9 % chocolatized sheep blood

CO2 carbon dioxide

COS Columbia Agar with 5 % sheep blood

CTA Cystine Trypticase Agar

DNA Deoxyribonucleic acid E. coli Escherichia coli

F. tularensis Francisella tularensis

FIWI Research Institute of Wildlife and Ecology

g gram

g gravitational force

IVET Institute for Veterinary Disease Control, Moedling

kDa kilo Dalton

min minute

ml millilitre

PACCV polymyxin B, amphotericin B, cefepime, cycloheximide, vancomycin

PBS Phosphate buffered saline

PCR polymerase chain reaction

rRNA ribosomal ribonucleic acid

sp. species

subsp. subspecies

Tm melting point

µl microlitre

°C degrees Celsius

1

1 Introduction and Objective of this study

1.1 Introduction

Tularemia (also known as rabbit fever, deer fly fever, Ohara’s disease) is a zoonotic disease

caused by the small, gram-negative rod Francisella (F.) tularensis. Tularemia occurs endemically

in most countries of the northern hemisphere. Sporadic cases of tularemia, as well as larger or

smaller outbreaks have been recorded in North America, certain parts of Asia and in most

countries of Europe (GURYCOVÁ and VÝROSTEKOVÁ, 1998b).

The true reservoir of F. tularensis is unknown, but transmission to humans is mostly related to

lagomorphs, rodents, arthropods or water (FRITZSCH and SPLETTSTOESSER, 2010).

Outbreaks of the disease in humans often parallel outbreaks of tularemia in wild animals.

Individuals who spend time in endemic areas, such as farmers, hunters, walkers, and forest

workers, are most at risk of contracting tularemia (ELLIS et al., 2002).

1.1.1 Francisellaceae

Francisella belongs to the Gammaproteobacteria. The family comprises closely related

organisms within the single genus Francisella.

The two recognized species, F. tularensis and F. philomiragia, show a 16S rRNA gene sequence

similarity of ≥ 98.3 %.

The family is distinguishable from other families based on a unique set of phenotypic

characteristics that include a coccoidal morphology, Gram negativity, acid but no gas production

from a limited number of carbohydrates, requirement for cysteine, and a unique fatty acid

composition.

Cells of Francisella typically appear as short, rod-shaped or coccoid cells, 0.2-0.7 x 0.2 µm in

size. They are small, singly occurring, nonmotile, and nonsporulating (SJÖSTEDT, 2005).

2

1.1.2 Francisella tularensis

The species F. tularensis recognizes four subspecies (subsp.): F. tularensis subsp. tularensis,

F. tularensis subsp. holarctica, F. tularensis subsp. mediaasiatica and F. tularensis subsp.

novicida (JOHANSSON et al., 2000b).

F. tularensis subsp. holarctica is comprised of the following three biovars: I Erys (erythromycin

susceptible), II Eryr (erythromycin resistant), and japonica (OLSUFJEV and

MESHCHERYAKOVA, 1983).

F. tularensis subsp. tularensis (type A) is restricted almost completely to North America. In

Europe, isolates have been reported rarely so far (GURYCOVÁ, 1998a).

The infective dose in humans is extremely low: 10 bacteria when injected subcutaneously

(SASLAW et al., 1961) and 25 when given as an aerosol (McCRUMB, 1961).

Consequently, F. tularensis is recognized as a potential biological warfare agent (DENNIS et al.,

2001).

F. tularensis subsp. holarctica (type B) occurs over the Eurasian and American continent. It is

much less virulent than type A and only rarely lethal to humans (TÄRNVIK et al., 2004).

In Austria, erythromycin-resistant F. tularensis subsp. holarctica strains have been isolated from

hares, mice, rabbits, ticks, water and human specimens (TOMASO et al., 2005).

1.1.3 Incidence of tularemia in Austria

In the endemic area of tularemia in the northeast of Austria, which is closely linked to the

endemic areas in Czech Republic, Slovakia and Hungary, less than 10 human cases of tularemia

are officially notified annually during the last decade. During the outbreak in European brown

3

hares during the hunting season in late autumn, an accumulation of cases occurred in 1997/98

with 35 notified human cases (DEUTZ et al., 2009; HOFER, 2002).

During this last outbreak of tularemia 234 European brown hares were analysed. 87 (37.2 %)

hares were positive, of which tularemia was detected in culture directly in 66 animals,

serologically in 13 animals and through immunofluorescence in 8 animals. Besides

splenomegaly, in most cases tularemia-positive hares showed macroscopic lesions like

necrotising pneumonia (37 animals), fibroid pericarditis (21 animals), hepatic necrosis

(15 animals), necrotising nephritis (5 animals) and orchitis (8 animals) (STEINECK and HOFER,

1999).

Ten years after the above mentioned outbreak in European brown hares, 1152 red foxes were

investigated for tularemia during a screening program for rabies. Mandibular lymph nodes were

used for culture. 15 animals (1.3 %) were positive, which indicated that the known natural foci of

tularemia were still active (HOFER et al., 2010).

This raised the question if European brown hares harbour F. tularensis in inter-epizootic years. In

2008, 149 apparently healthy European brown hares were shot from two known endemic districts

and tissue samples were investigated through direct-culture for tularemia. In 5 hares (3.4 %) the

culture was positive. 2 of 9 European brown hares (22 %) which were found dead were also

cultural positive (BEIGLBÖCK, 2011).

Investigations of red foxes and European brown hares from 2007 to 2009 showed that infections

with low bacterial counts in tissues can only be detected direct-cultural if growth of contaminants

is suppressed completely through selective media. Tissues have to be sliced and the sliced

portions must be smeared on the selective medium (HOFER et al., 2010).

4

1.1.4 Detection and Diagnosis

1.1.4.1 Culture

F. tularensis is a fastidious organism which requires enriched medium for growth. The World

Organisation for Animal Health recommends the following media (OIE, 2008): Francis medium,

McCoy and Chapin medium, modified Thayer-Martin agar (Glucose cysteine agar – medium

base supplemented with haemoglobin and Iso VitaleX, GCA agar with thiamine), and Cystine

heart agar (DIFCO) with 5% rabbit blood, and penicillin (100,000 units), Polymyxin B sulphate

(100,000 units) and cycloheximide (0.1 ml of a 1 % stock solution) per litre.

Cystine Heart Agar (DIFCO) with 9 % sheep blood, 100 mg/l ampicillin and 100 mg/l

polymyxin B is also used as selective medium for samples from wildlife (HOFER et al., 2010).

The selective medium for the isolation of F. tularensis from environmental samples has been

described previously (PETERSEN et al., 2009). The media formulation consists of cysteine heart

agar with 9 % chocolatized sheep blood (CHAB), supplemented with 13 mg polymyxin B,

2.5 mg amphotericin B, 4.0 mg cefepime, 100 mg cycloheximide and 4.0 mg vancomycin

(CHAB + PACCV).

Thioglycollate-blood-glucose agar (TBGA) is also used for primary cultivation

(VÝROSTEKOVÁ et al., 2002).

For human samples a modified Thayer-Martin medium is described for the culture of

F. tularensis (JOHANSSON et al., 2000a). This medium is supplemented with 7.5 mg colistin ,

2.5 mg amphotericin B, 0.5 mg lincomycin, 4 mg trimethoprim, and 10 mg ampicillin (per litre).

Legionella B.C.Y.E. alpha agar is described for the primary isolation and cultivation of

F. tularensis in human samples (SCHOBER et al., 2002).

5

F. tularensis needs incubation at 37°C in ambient air. The appearance of individual colonies may

require 2 to 4 days of incubation. On CHAB, colonies are 2 to 4 mm in size, greenish-white,

round, smooth, and slightly mucoid, while on media containing whole blood there is usually a

small zone of alpha-hemolysis surrounding colonies (ELLIS et al., 2002).

1.1.4.2 Biochemical differentiation

F. tularensis strains are classified by basic biological properties: fermentation of glycerol,

glucose and citrulline ureidase activity, susceptibility to erythromycin (GURYCOVÁ, 1998c).

If the organism appears to be F. tularensis based on morphology and growth characteristics, you

should not attempt identifying it using commercial identification systems due to the probability of

misidentification and the risk of laboratory infections.

Table 1: Francisella tularensis – basic markers for identification of subspecies and biovar (source:

GURYCOVÁ, 1998c).

Subspecies Biovars

Pathogenity

for man and

rabbits

Fermentation

of

glycerol glucose

Sensitivity

to

erythromycin

tularensis high + + +

holarctica I EryS

II EryR

japonica

moderate

moderate

moderate

-

-

+

+

+

+

+

-

+

mediaasiatica moderate + - +

1.1.4.3 Molecular detection

PCR can be a valuable diagnostic tool when organisms are non-cultivable or when a culture is not

recommended due to biosafety concerns. The majority of PCR tests for F. tularensis have been

conventional PCR assays targeted at the genes fopA or tul4 encoding the outer membrane

6

proteins (WHO, 2007). The tul4 PCR assay has been validated with specimens from wounds

from patients with ulceroglandular tularemia (JOHANSSON et al., 2000a).

1.2 Objective of this study

The aim of this study was to test two selective media developed for the direct isolation of

F. tularensis from human and environmental samples and to examine if these two selective media

are suitable for direct culture of tissue samples from European brown hares. Further, the ability to

inhibit interfering contaminants and the increase of diagnostic recovery from veterinary samples

were proven.

7

2 Materials and Methods

2.1 Materials

2.1.1 Samples

In this work, tissue samples of European brown hares were investigated. Stated below, the

different kinds of samples are listed.

2.1.1.1 Tissue samples from the FIWI frozen at – 80°C

Frozen samples of European brown hares from Austria

219 European brown hares of the 192 shot down and 27 found dead from 2010 to 2011 were sent

to the FIWI and underwent post-mortem examination. Small pieces of tissue samples from heart,

kidney, lung and spleen were collected and frozen in 2 ml cryo tubes at -80°C for further

processing.

Frozen samples of European brown hares from Germany

An island population of 162 European brown hares predominantly shot between 2010 and 2011

in Germany underwent post-mortem examination on the island and about two days later samples

from heart, kidney, lung and spleen were collected in 2 ml cryo tubes with storage at room

temperature. After transportation from Germany back to the FIWI without cooling, the tubes

were frozen at – 80°C for further processing.

In February 2012, a total of 381 European brown hares, four tissue samples of each, was sent

without defrosting to the AGES/IVET Moedling for examination.

2.1.1.2 Tissue samples of unfrozen European brown hares

European brown hares directly sent to IVET Moedling from hunters or from the FIWI were also

included in this study.

8

European brown hare 1/2012

In March 2012, one European brown hare found dead in the south of Styria was sent to the

Department of Pathology of IVET by a huntsman for investigation. After macroscopic

examination a piece of lung, which showed no pathological abnormalities, and a piece of an

enlarged spleen were, under the suspicion of tularemia, sent to the Bacteriology laboratory.


In March 2012, another European brown hare was sent to the IVET, Department Pathology. This

animal was found dead in the north of Lower Austria. Tissue samples of lung, spleen and heart

were taken from the brown hare and sent to the Section Bacteriology under the suspicion of

tularemia.


In April 2012, a piece of lung, kidney, spleen and testes from a European brown hare were sent

from the FIWI to the Section Bacteriology of IVET Moedling with the request for examination

for tularemia. Splenomegaly, purulent inflammation of testes and a lung with disseminated

whitish nodules were present.

2.1.1.3 Recovery of F. tularensis in frozen tissue samples


In December 2011, F. tularensis ssp. holarctica biovar II was isolated from samples of lung,

spleen and lymph node of a European brown hare from Styria. After isolation of the pathogen and

confirmation, the samples were frozen at -80°C. At the time of the freezing, the tissue samples

were about 10 days old and had until then been stored in the refrigerator.

In May 2012, the frozen tissue samples were cultured again to check the selective media and to

show that recovery after freezing at – 80°C is possible.

All fresh tissue samples handled during this work were cultured within three days and cooled at

2-8°C in the meantime.

9

2.1.2 Francisella tularensis ssp. holarctica control strains

F. tularensis ssp. holarctica biovar I and F. tularensis ssp. holarctica biovar II control strains,

confirmed biochemically and with molecular methods, were used as control for media and the

conditions of incubation.

2.1.3 Culture Media

Medium A: modified CHAB + PACCV according to PETERSEN et al., 2009

Cystine Heart Agar (BD, Cat. No. 247100)

Deionised water

Aqua bidest. “Fresenius”

Defibrinated sheep blood

Polymyxin B (Sigma, Cat.No. P-1004)

Amphotericin B (Sigma, Cat.No. A4888)

Cefepime (Molekula, Cat.No. M27655146)

Vancomycin (Sigma, Cat.No. V2002)

Due to toxic side effects cyclohexamide was not used as supplement during this work.

51g Cystine Heart Agar were dissolved in 1 litre deionised water and soaked for 15 min. Then the

suspension was autoclaved at 121°C for 15 min and afterwards 9 % sheep blood was added and

heated for 7 to 10 min to 80°C. After cooling down the agar (45-47°C), 100 ml defibrinated

sheep blood were added and 13 mg polymyxin B, 2.5 mg amphotericin B, 4 mg cefepime and

4 mg vancomycin which were dissolved first in 5 ml Aqua bidest were added. After stirring,

20 ml volumes of medium were filled into sterile petri dishes.

Medium B: Thayer Martin medium + CALTA according to JOHANSSON et al., 2000

Instead of Thayer Martin medium base, cystine heart agar was used as medium base.

10

Cystine Heart Agar (BD, Cat. No. 247100)

Deionised water



Ampicillin (Sigma, Cat.No. A-9518)

Amphotericin B (Sigma Cat.No. A4888)

Colistin (Sigma Cat.No. C4461)

Lincomycin (Sigma Cat.No. 62143)

Trimethoprim (Sigma Cat.No. T7883)

51 g Cystine Heart Agar were dissolved in 1 litre deionised water and soaked for 15 min. Then

the suspension was autoclaved at 121°C for 15 min and afterwards cooled down to 45-47°C.

100 ml defibrinated sheep blood and 10 mg ampicillin, 2.5 mg amphotericin B, 7.5 mg colistin,

0.5 lincomycin and 4 mg trimethoprim dissolved in 5 ml aqua bidest first, were added. After

stirring, 20 ml volumes of medium were filled into sterile petri dishes.

Medium C: Cystine heart agar with ampicillin and polymyxin according to OLSUFJEV et al.,

1984

Cystine Heart Agar (BD, Order No. 247100)


Deionised water


Ampicillin (Sigma, Cat. No. A-8518)

Polymyxin B (Sigma, Cat. No. P-1004)

51 g Cystine Heart Agar were dissolved in 1 litre deionised water and soaked for 15 min. Then

the suspension was autoclaved at 121°C for 15 min and afterwards cooled down to 45-47°C.

100 ml defibrinated sheep blood, and 100 mg ampicillin and 100 mg polymyxin B dissolved in

11

5 ml aqua bidest. first, were added. After stirring, 20 ml volumes of medium were filled into

sterile petri dishes.

COS: Columbia Agar with 5 % sheep blood

Columbia Agar, Base medium (BD BBLTM, Cat. No. 211124)

Deionised water


42.5 g Columbia Agar were dissolved in 1 litre deionised water and soaked for 15 min. Then the

suspension was autoclaved at 121°C for 15 min and afterwards cooled down to 45-47°C. 50 ml

defibrinated sheep blood were added. After stirring, 20 ml volumes of medium were filled into

sterile petri dishes.

SAB: Sabouraud Agar

Sabouraud Maltose 4 % agar (Merck, Cat.No. 1.105439.0500)


Penicillin G sodium salt (Sigma, Cat. No. P3032)

Streptomycin sulfate salt (Sigma, Cat. No. S-6501)

Deionised water

65 g Sabouraud Maltose agar were dissolved in 1 litre deionised water and soaked for 15 min.

Then the suspension was autoclaved at 121°C for 15 min and afterwards cooled down to 50°C.

40 mg streptomycin sulfate salt and 0.2 ml penicillin were dissolved in 4 ml aqua bidest.

“Fresenius” and added to the suspension. After stirring, 25 ml volumes of medium were filled

into sterile petri dishes.

12

2.1.4 Media and Material for biochemical differentiation

CTA Medium: Cystine Trypticase Agar

CTA Agar (BD BBLTM, Cat. No. 211094)

Deionised water


Glycerine (Merck Cat. No. 1.04094.0500)

Glucose (Fluka Cat. No. 49159)

Horse Serum (Invitrogen)

5g CTA Agar were dissolved in 125 ml deionised water and then autoclaved at 118° for 15 min.

When the medium was cooled down to 47°C, 6.25 ml inactivated (30 min at 56°C) horse serum

were added. Then 1.25 ml Glycerine or 1.25 g Glucose were added under sterile conditions and

then 10 ml volumes of medium were filled into tubes and then tubes were kept in sloping position

until agar was solidified.

Erythromycin sensitivity testing

Erythromycin E 15µg, CT0020, LOT 1150292, Oxoid Ltd.

Oxoid Antimicrobial Susceptibility Test Discs were 6 mm filter paper discs bearing an alpha-

numeric code identifying the antimicrobial agent and concentration, printed on both sides.

Francisella tularensis agglutination testing

Francisella tularensis – antiserum; tube or slidetest, one 5 ml vial

Difco/BBL, Cat. No. 241050

13

2.2 Methods

2.2.1 Direct Isolation of F. tularensis

Francisella tularensis strains were isolated from tissue samples cultured on Medium A,

Medium B and Medium C by direct culture. Therefore tissues were sliced with sterile scissors.

Using sterile forceps, the sliced portions were smeared directly on the culture media.

Tissue samples of four European brown hares, which were sent routinely to the IVET after

finding or hunting, were additionally streaked using a sterile loop.

Whole plates, half plates, or quarter plates were used depending on the circumstances.

In the case of quarter plates, quarters are later termed as follows:

1st quadrant: top left quarter

2nd quadrant: top right quarter

3rd quadrant: bottom left quarter

4th quadrant: bottom right quarter.

Media were always inoculated in the following order:

1. Medium C 2. Medium B 3. Medium A

Media were incubated at 37°C under ambient air at least 7 days and checked for characteristic

growth of F. tularensis. Suspect colonies were sub-cultured on selective media for further testing.

All culture media were checked for contamination and batch controls were done with the control

strains for each isolation attempt. Contaminated plates were discarded.

1st quadrant 2nd quadrant 3rd quadrant 4th quadrant

Figure 1: Segmentation of media

14

2.2.2 Culture diagnosis of tularemia and biochemical characterization

Suspicion of F. tularensis through suspect colonies was confirmed by slide agglutination with a

commercial antiserum. Therefore, one drop of antiserum was put on a glass slide and one

individual or more colonies of a fresh F. tularensis subculture were taken with a loop and mixed

with the antiserum on the slide. This interaction produced clumping, visible with the naked eye.

Positive slide agglutination was confirmatory for F. tularensis.

Subspecies verification was done by testing the fermentation of glycerine and glucose. Therefore,

colonies from a pure culture were taken with a sterile loop and CTA media, one supplemented

with glycerine and one with glucose, were inoculated and incubated at 37°C for at least 2 days.

The change of colour from pink to yellow showed a positive reaction meaning the ability to

ferment glycerine or glucose. Fermentation of glucose and non-fermentation of glycerine was

confirmatory for subspecies holarctica.

To discriminate between biovar I and II the antibiotic susceptibility for erythromycin was tested.

Used was a semi quantitative agar diffusion test method for in vitro susceptibility testing.

Therefore, Medium A, Medium B or Medium C was densely inoculated with a colony of a F.

tularensis strain and a 15 µg Erythromycin antibiotic disc was placed on the agar plate. Then the

antibiotic diffused through the agar to form a gradient. After 48 hours of incubation at 37°C,

inhibition around the disc was proven in comparison to the biovar I control strain.

2.2.3 Molecular methods

2.2.3.1 PCR template preparation

Colonies of a pure F. tularensis culture were taken with a sterile bacteriological loop and put into

a 1.5 ml safe-lock tube. Afterwards, 200 µl PBS were added. Before DNA extraction bacteria

were killed by incubating the suspension at 95°C for 10 minutes.

15

The extraction of DNA was done with the High Pure PCR Template Preparation Kit (Roche,

Cat. No. 11796828).

First of all, 15µl lysozyme solution was added and incubated for 15 min at 37°C. Then 200 µl

tissue lysis buffer and 40 µl reconstituted proteinase K solution was added and the whole content

was mixed and then incubated for 10 min at 70°C. After incubation the sample was mixed with

100 µl isopropanol. Then one filter tube was inserted into one collection tube and the entire

sample was pipetted into the upper buffer reservoir of the filter tube and centrifuged for 1 min at

8000 x g. After centrifugation, the filter tube was put into a new collection tube and 500 µl

inhibitor removal buffer were pipetted into the filter tube.

Another centrifugation step (1 min at 8000 x g) followed and then flow through and collection

tube were discarded and a new collection tube was used. For washing the sample, 500 µl wash

buffer were added and the centrifugation step was repeated, followed by a repeated washing step

with 500 µl wash buffer and centrifugation. Then the filter tube was put in a new collection tube.

Afterwards, the collection tube was discarded and a new clean, sterile 1.5 ml microcentrifuge

tube was used. For eluting the nucleic acid, 200 µl of prewarmed (70°C) elution buffer were

added to the filter tube and centrifuged for 1 min at 8000 x g. The microcentrifuge tube

containing the eluted nucleic acids was then stored at 2 to 8°C for later analysis.

2.2.3.2 Detection of F. tularensis through LightCycler real-time PCR

For the amplification of F. tularensis primers specific for sequences of the gene encoding a

17-kDa lipoprotein, TUL4 were used (SJÖSTEDT et al., 1990).

For a total reaction volume of 20 µl for one sample, 12.5 µl PCR grade water (Qiagen), 0.5 µl

primer mix 9/440 4/20pmol (VBC Genomics) (FT-1 5’-CATTAGCTGTCCACTTACCG-3’ and

Tul4-rev 5’-AGGAAGTGTAAGATTACAATGGC-3’), 1 µl probe mix 441/442 10 pmol (VBC

Genomics) (Tul4-Pr1 5’-CCCAAGTTTTATCGTTCTTCTCAGCATAC-Fluorescein-3’ and

Tul4-Pr2 5’-LCRed640-AGTAATTGGGAAGCTTGTATCATGGCACTTAGAAC-3’-PO4), 4 µl

of Light Cycler 480 Genotyping Master (Roche, Cat. No. 04 707 524 001) and 2 µl of template

DNA were used for amplification.

16

Amplification was carried out in Light Cycler 480 (Roche). The PCR cycling protocol started

with denaturation at 95°C for 10 min, was followed by 45 cycles of 95°C for 10 sec and 58°C for

20 sec and 72°C for 30 sec. Subsequently, melting curves were generated by slowly raising the

temperature from 45 to 95°C, during which time fluorescence was measured at frequent intervals

and analysed using LightCycler® 480 software (Roche).

2.2.3.3 Discrimination of subspecies by real-time PCR

To discriminate Francisella tularensis ssp. tularensis and Francisella tularensis ssp. holarctica, a

real-time PCR assay with melting point analysis based on a denoted targeted F. tularensis DNA

marker (Ft-M19) was performed (BYSTRÖM et al., 2005). This assay detects strains of

F. tularensis subsp. holarctica by identification of a 30-bp deletion unique to the subspecies at a

genomic locus designated Ft-M19 (JOHANSSON et al., 2004).

For a total reaction volume of 20 µl for one sample, 7µl PCR grade water (Qiagen), 1µl of primer

mix 387/509 20pmol (VBC Genomics) (Ft30-r 5’-CCAGTACAAACTCAATTTGGTTATC

ATC-3’ and Ft30_diff 5’-GTTTCAGAATTCATTTTTGTCCGTAA-3’), 10 µl of Light Cycler

480 SYBR green I PCR master mix (Roche, Cat. No. 04 707 516 001) and 2 µl of template DNA

were used for amplification.

Amplification was carried out in Light Cycler 480 (Roche). The PCR cycling protocol started

with denaturation at 95°C for 10 min, was followed by 45 cycles of 95°C for 10 sec and 60°C for

10 sec and 72°C for 15 sec. After the final cycle, a melting point analysis was performed and

analysed using LightCycler® 480 software (Roche).

17

2.2.4 Serial dilution

Most interfering contaminants on growth of F. tularensis were sub-cultured on COS and a stock

solution of 0.5 McFarland was prepared in 9 ml Sodium Chloride. Then ten-fold serial dilution

steps were conducted stopping at a dilution of 10-10. Out of each dilution, 100 µl were plated on

Medium A, Medium B, Medium C and COS (yeasts were also plated on SAB). COS and SAB

were additionally done for a better comparison of media and its ability of inhibition. Media were

incubated at 37°C for 7 days to simulate tularemia diagnostic. Afterwards, the colonies were

counted and CFU per 1 ml stock solution were calculated.

Calculation for living bacteria in the original culture was done by multiplying the counted

colonies with the dilution factor (eg. counted CFU: 40, dilution factor: 1:100,000 → 4.0 million

CFU in 1 ml stock solution).

18

3 Results

3.1 Overview of investigated European brown hares

3.1.1 Tissue samples frozen at – 80°C

Frozen tissue samples of European brown hares from Austria

Tissue samples of heart, lung, kidney and spleen of 219 European brown hares from Austria were

investigated for F. tularensis and selectivity of Medium A, Medium B and Medium C were

compared (Table 2).

F. tularensis was found in 7 hares (3.2 %) of the 219 investigated hares, whereby in tissue

samples of 5 European brown hares F. tularensis was isolated in Medium A, B and C, whereas

two European brown hares showed the growth of F. tularensis only on Medium C in the form of

a single colony only in one tissue sample.

172 of 219 (78.5 %) European brown hares were tested negative for F. tularensis and media of

these 172 hares showed no influencing growth of contaminants.

Tissue samples of 31 (14.2 %) European brown hares were only partly evaluable, which means

that not all tissue samples from a European brown hare were evaluable due to influencing

contaminants, but at least two tissue samples were free of contaminants.

9 European brown hares (4.1 %) were, due to excessive growth of contaminants, not evaluable. In

these cases, Medium A, Medium B and Medium C were not able to suppress contaminants.

19

Table 2: Evaluation of tissue samples of 219 European brown hares from Austria

A further distinguishing characteristic was if the Austrian European brown hares were hunted for

a project study or found dead (Table 3). In this work, 192 European brown hares hunted for a

study and 27 European brown hares found dead were investigated.

In 3 hares (1.6 %) of the 192 killed European brown hares F. tularensis was found, whereas in 4

hares (14.8 %) of the found dead European brown hares tularemia was detected.

Table 3: Austrian European brown hares differentiated into hunted and found dead

0

30

60

90

120

150

180

Euro

pean

bro

wn

hare

s

evaluable

partly evaluable

not evaluable

F. tularensis

0

40

80

120

160

killed found dead

Euro

pean

bro

wn

hare

s

evaluablepartly evaluablenot evaluableF. tularensis

20

Frozen tissue samples of European brown hares from Germany

Samples of heart, lung, kidney and spleen of 162 European brown hares from Germany were

investigated for F. tularensis (Table 4) and the selectivity of Medium A, Medium B and

Medium C was tested.

In all of the 162 European brown hares, F. tularensis was not found.

49 of 162 (29.7 %) European brown hares were tested negative for F. tularensis and media were

evaluable with no influencing contaminants.

Tissue samples of 42 (25.3 %) European brown hares were only partly evaluable, which means

that not all tissue samples from a European brown hare were evaluable due to influencing

contaminants, but at least two tissue samples were free of contaminants.

71 (45 %) European brown hares were, due to excessive growth of contaminants, not evaluable.

In these cases, Medium A, Medium B and Medium C were not able to suppress contaminants.

Table 4: Evaluation of frozen tissue samples of 162 European brown hares from Germany

0

10

20

30

40

50

60

70

Euro

pean

bro

wn

hare

s

evaluable

partly evaluable

not evaluable

F.tularensis

21

3.1.2 Tissue samples of unfrozen European brown hares

In tissue samples of 2 European brown hares, which were found dead and were sent directly from

hunters to IVET Moedling, F. tularensis was found on Medium A, Medium B and Medium C.

In tissue samples of a European brown hare, which was sent from the FIWI to IVET Moedling,

F. tularensis was also found.

3.1.3 Recovery of F. tularensis in frozen tissue samples

Recovery of F. tularensis in tissues frozen at – 80°C of a previously F. tularensis positive

European brown hare was performed and results were comparable with data from direct culture

without freezing. All of the three selective media were suitable for isolation of F. tularensis.

22

3.2 Isolation of Francisella tularensis in European brown hares

In this chapter, all F. tularensis positive European brown hares during this work are listed and

described in relation to the capability of Medium A, Medium B and Medium C and their ability

to suppress contaminants.

Biochemical and molecular characterization is described in later chapters.

The first seven isolates of F. tularensis were from frozen (at -80°C) samples of European brown

hares from Austria. Afterwards, three isolates of unfrozen samples of European brown hares are

described. The last mentioned case of tularemia was the successful recovery of F. tularensis out

of frozen tissue samples.

Isolate 1

Francisella tularensis was found in a European brown hare shot in Lower Austria.

Interestingly, the single colony was isolated from the frozen piece of tissue of the kidney,

although the kidney showed no macroscopic abnormalities.

Medium C was the only medium where growth of a single F. tularensis colony occurred

(Figure 2). This medium was able to inhibit almost all interfering contaminants. The other two

selective media showed overgrowing growth of contaminants namely Pseudomonas sp. and

E. coli as postmortal contaminants. Contaminated plates were thrown away after three days

because in this case, F. tularensis would never have had a chance of growing.

On Medium C, the single colony of F. tularensis was not visible until the third day. After the

weekend, at day six, the colony was clearly visible and with its grey-bluish shiny colour there

was no doubt that it was a Francisella tularensis strain.

23

A sub-culture of the single F. tularensis colony was cultivated to have enough bacterial material

for further tests.

Figure 2: Isolate 1 - Medium C with a F. tularensis

single colony in the 3rd quadrant (see arrow), 10 days

after smearing the tissue samples on the medium Author’s note: the dent within the colony comes from sub-

culturing before the picture was taken

Summarized Medium C was the medium most suitable for the isolation of F. tularensis and

suppressing contaminants.

Isolate 2

The second F. tularensis isolate originated from a European brown hare found dead in Lower

Austria.

First-time inspection of selective media was done after three days (Figure 3, a). The comparison

of selective media showed a significant difference in the rate of growth. Whereas after three days

of incubation, a faint growth was seen on Medium C, on Medium A growth and also clearly

single colonies of F. tularensis were visible already. Medium B showed a slight growth of grey,

shiny colonies.

After eight days of incubation (Figure 3, b), all media showed a growth of colonies typical for

F. tularensis.

24

It was noticeable that after 6 to 8 days of incubation at aerobic atmosphere, colonies on

Medium B and Medium C started to develop haemolysis.

a)

b)

Figure 3: Isolate 2 - F. tularensis on Medium A (left page), Medium B (middle) and Medium C (right

page); 1st quadrant = lung, 3rd quadrant = kidney after a) 3 days of incubation and b) 8 days of incubation

Summarized F. tularensis was visible moderately in the kidney and low-grade in the lung. No

growth of F. tularensis was seen where sliced portions of heart and spleen were smeared on the

media. No contaminants were observable, indicating the high selectivity of Medium A,

Medium B and Medium C.

25

Isolate 3

F. tularensis was isolated from tissue samples of a European brown hare found dead in Lower

Austria.

After three days of smearing the sliced portions on selective media massive growth of

F. tularensis was visible, but no contaminants (Figure 4, a).

On Medium A, an almost equal growth of colonies on the four quadrants occurred.

In heart (2nd quadrant) and kidney (4th quadrant), a poorer growth on Medium B and Medium C

was visible, whereas growth of colonies of spleen (3rd quadrant) and lung (1st quadrant) were

almost the same as on Medium A.

The second assessment on day six showed equal growth of F. tularensis colonies (Table 4, b).

As previously mentioned, from day six on, haemolysis occurred on Medium B and C.

For further analysis subcultures were done.

26

a)

b)

Figure 4: Isolate 3 - Growth of F. tularensis on all quadrants; Medium A (left page), Medium B (middle)

and Medium C (right page) after a) 3 days and b) 6 days of incubation

An acute infection of tularemia was determined based on the quick and high grade growth of

F. tularensis without contaminants on Medium A, Medium B and Medium C.

Isolate 4

The fourth F. tularensis isolate originated from a European brown hare shot in Lower Austria.

First-time inspection of selective media was done after four days. On Medium A and Medium B,

only contaminants were visible. Medium C showed contaminants in the 3rd and 4th quadrants and

no growth in the other two quadrants.

27

After seven days, the second assessment was done. On that day, a small, single colony of about

1 mm in the 2nd quadrant where the sliced portion of the heart tissue was smeared on Medium C

was visible (Figure 5, see arrow). Due to its appearance like colour and slow growth, it was

suspected of F. tularensis. Subcultures were made for further characterisation.

Assuming it was a chronic infection with only a few pathogens inside the organs and a lot of

contaminants, F. tularensis required extraordinary time for growth and occurred only on

Medium C, which was always the first medium of inoculation. Therefore, it would be possible

that due to the fact of low content of F. tularensis the one existing microorganism was smeared

on the first medium and no further pathogens were available for the other two selective media.

Figure 5: Isolate 4 - F. tularensis colony visible on Medium C (see arrow); Medium A (left page),

Medium B (middle) and Medium C (right page) after 7 days of incubation of sliced portions of lung =

1st quadrant, heart = 2nd quadrant, kidney = 3rd quadrant and spleen = 4th quadrant

Predominant contaminants on Medium A belonged to the genus Enterococcus and Yeast. On

Medium B, contaminants like Bacillus sp. were visible and on Medium C, two colonies of yeasts

were seen.

Medium C was the medium most suitable for the isolation of F. tularensis and suppressing

contaminants.

28

Isolate 5

The isolate originated from a European brown hare found dead in Lower Austria.

Sliced portions of heart, lung, spleen and kidney were smeared on the three selective media. After

five days, the first evaluation was done (Figure 6, a).

On Medium A, the quadrants of lung, heart, kidney and spleen showed white-greenish colonies

with a mean diameter of 2 mm of a single colony. Noticeable was the growth of F. tularensis in

the quadrant where the lung was smeared because two white colonies of contaminants inhibited

the growth of F. tularensis in the immediate surroundings (Figure 6, a, see arrow). Colonies of

F. tularensis were very small and became larger with a higher distance. Assuming these

contaminants consumed too many required essential nutrients for F. tularensis, its growth was

therefore delayed. Otherwise it would be possible that contaminants are like bacteriocins and

produce substances, which inhibit the growth of F. tularensis. Contaminants on Medium A were

identified as Enterococcus faecalis biochemically (API 20 Strep V7.0, Profile 5143711).

At the first evaluation of Medium B, grey, shiny colonies were visible on three quadrants. Where

the spleen was smeared on the medium, only a supersize Bacillus sp. colony which acted as

contaminant was visible. In view of the fact that only few F. tularensis colonies were inside the

spleen, the contaminant was predominant and the requested pathogen was suppressed. But

Bacillus sp. seemed to have no significant influence on the growth of F. tularensis because some

colonies grew near the contaminant with the same size as at a distance (Figure 6, a, see arrow). In

comparison to Medium A, single colonies on Medium B were smaller with a diameter of about

0.3-0.5 mm.

Medium C showed grey shiny colonies typical for F. tularensis on all quadrants. The average

diameter of a tiny, single colony was 0.2-0.5 mm and therefore similar to the growth on

Medium B. The high selectivity of Medium C was shown because no bacterial growth of

contaminants occurred in contrast to Medium A and Medium B.

29

The second evaluation after seven days showed mainly the same as two days earlier, except for

the growth of colonies (Figure 6, b). Single colonies on Medium A reached an average diameter

up to 4 mm. On Medium B and Medium C, single colonies were about 0.5-1 mm.

Further details of the different contaminants and their behaviour of growth on the different

selective media is shown in a separate chapter.

a)

b)

Figure 6: Isolate 5 - Growth of F. tularensis on Medium A (left page), Medium B (middle) and

Medium C (right page) after a) 5 days and b) 7 days of incubation of sliced portions; lung = 1st quadrant,

heart = 2nd quadrant, kidney =3rd quadrant and spleen = 4th quadrant

F. tularensis was visible low grade in heart, lung, spleen and kidney. With the exception of some

colonies of contaminants, Medium A, Medium B and Medium C showed high selectivity,

30

therefore Medium A and Medium B, developed for human- and environmental samples, were

suitable for direct-culture of frozen (- 80°C) tissue samples from European brown hares to isolate

F. tularensis.

Isolate 6

The isolate originated from a European brown hare found dead in Lower Austria.

Five days after smearing the sliced portions on Medium A, B and C, characteristic colonies for

F. tularensis occurred (Figure 7, a).

On Medium A, nearly a pure culture with F. tularensis was visible with the exception of two

colonies of contaminants on the 3rd quadrant (kidney). Contaminants were not influencing the

growth of the pathogen.

On medium B, significant growth of F. tularensis was seen on quadrant two and three (heart and

kidney). The first quadrant (heart) showed very small characteristic colonies left from the

haemolysing contaminants (Figure 7, a, see arrow). In the 4th quadrant, no F. tularensis colonies

were seen. Probably growth was suppressed by contaminants.

Medium C showed a similar picture like Medium B because F. tularensis growth was visible

where heart and kidney were smeared on the medium but also some small colonies were seen in

the 4th quadrant (spleen). On the 1st quadrant, a huge colony of a contaminant was visible but also

tiny colonies of F. tularensis in the contaminants surroundings (barely visible in Figure 7, a).

After seven days of incubation, the colonies got bigger and started to develop haemolysis on

Medium B and Medium C (Figure 7, b).

31

a)

b)


Medium C (right page) after a) 5 days and b) 7 days of incubation of sliced portions of lung = 1st quadrant,

heart = 2nd quadrant, kidney = 3rd quadrant and spleen =4th quadrant

Medium A was the most suitable medium for isolation F. tularensis in all tissue samples with

nearly complete inhibition of all contaminants.

Isolate 7

The isolate originated from a European brown hare hunted in Lower Austria.

Three days after smearing the sliced portions of heart, lung, spleen and kidney on Medium A,

Medium B and Medium C, characteristic colonies for F. tularensis occurred on the 1st and the

32

2nd quadrant (Figure 8, a). On Medium A, colonies of F. tularensis were a bit larger than on

Medium B and Medium C.

After six days of incubation, the colonies got bigger and started to develop haemolysis on

Medium B and Medium C (Figure 8, b).

a)

b)

Figure 8: Isolate 7 - Growth of F. tularensis on selective media; Medium A (left page), Medium B

(middle) and Medium C (right page) after a) 3 days and b) 6 days of incubation of sliced portions; lung =

1st quadrant, heart =2nd quadrant, kidney =3rd quadrant and spleen = 4th quadrant

Summarized F. tularensis was visible moderately in lung and low-grade in the heart. No growth

of F. tularensis was seen where sliced portions of kidney and spleen were smeared on the media.

With exception of the only contaminant, Medium A, Medium B and Medium C showed high

selectivity.

33

Isolate 8 – European brown hare 1/2012

This isolate originated from a European brown hare found dead in the district Leibnitz in the

south of Styria. Two days after finding, post-mortem examination was done by the IVET /Section

Pathology where samples of lung and spleen were taken and sent to the section Bacteriology for

investigation.

Because of the adequate size of samples, two different methods were used for the culture.

Firstly, the tissue samples were sliced and then the sliced portions were smeared on the upper half

of Medium A, Medium B and Medium C. Secondly, sterile loops were used for taking material

from the section plane of tissue samples, and then the lower half of media were streaked.

After 3 days, the first inspection was done. On that day, only tiny colonies were visible on

Medium A, where the lung was directly smeared on the medium and on Medium C, where the

spleen was directly smeared on the medium. On all other halves and media, no growth was

visible.

One day later, on all media typical colonies for F. tularensis were visible on the upper half of

plates with the exception of Medium B, where the lung was smeared on the medium. On both

Medium A, growth was also visible where tissues were plated with a loop.

After 5 days of incubation also Medium B where the lung was smeared showed growth

(Figure 9, a).

On Medium A and Medium C was a high-grade growth, whereas on Medium B only few colonies

were present. F. tularensis was also culturable by using a loop on Medium A (Figure 9, a, b).

On Medium A, an influencing contaminant for growth of F. tularensis was observed

(Figure 9, a,b). In the immediate vicinity, no growth of F. tularensis was seen and with increasing

distance colonies got larger.

34

a)

b)


Medium C (right page) after 5 days of incubation of sliced portions of a) lung and b) spleen; upper half of

media = sliced portions of tissues; lower half of media = streaked tissues with loop (Author’s note: The single colony of fungus on b) Medium C did not come from the tissue sample because growing

of the colony occurred beside the area of smearing. Therefore it was unfortunately a contamination from

environment)

F. tularensis was visible in lung and spleen on the three selective media. Medium A, Medium B

and Medium C showed high selectivity with the exception of a colony from a contaminant on

Medium A with the effect of tiny growth in its surrounding, and on Medium C, a fungus grew.

35


A European brown hare found dead in the district of Hollabrunn was sent to IVET Moedling for

post-mortem examination. In the section Bacteriology, tissue samples from lung, spleen and heart

were investigated with suspicion of tularemia.

Because of the adequate size of samples, the previously mentioned two different methods were

used to culture the tissue samples.

Firstly, the tissue samples were sliced and then the sliced portion of each tissue was smeared on

the upper half of Medium A, Medium B and Medium C. Secondly, a sterile loop was used for

taking material from the section plane of each tissue sample and then the lower half of media

were streaked.

The first evaluation after 3 days of incubation showed a high-grade growth of colonies typical for

F. tularensis on Medium A, Medium B and Medium C in the upper half of the media where the

sliced portions were smeared (Figure 10). Due to the obviously septicaemic happenings, growth

was massively and the evaluation of the growth of a single colony on the upper half of media

was, with the exception of the heart, impossible. On Medium A, where the heart was smeared on

the medium, a single colony had a diameter of about 0.9 -1.0 mm.

The method using a sterile loop for inoculation showed more visible single colonies, because less

tissue and therefore less pathogen was transferred with the loop to the media and colonies had

more space for growing. On Medium A, a single colony had a mean diameter of 1 mm. Colonies

on Medium B and Medium C showed a diameter between 0.2-0.5 mm. No colonies were visible

on Medium B and Medium C, where the heart was smeared by using a loop (Figure 10, right

panel).

36

Figure 10: Isolate 9 - F. tularensis after 3 days of incubation; Lung (left panel) and heart (right panel).

Medium B (left) and Medium C (right) in the upper row and Medium A is shown in below Medium B and

Medium C

F. tularensis was present massively on Medium A, Medium B and Medium C without the growth

of contaminants which militates for an acute, septicaemic event. In such a case, contaminants

only play a minor part and all three media showed excellent conditions of growth.

37


The isolate originated from a European brown hare found dead in the district of Wr.Neustadt-

Land of Lower Austria.

Tissue samples of testes, lung, kidney and spleen were plated on media using two different

methods. Firstly, the sliced portions were smeared on the upper half of Medium A, Medium B

and Medium C and afterwards, a sterile loop was taken and material of tissue from the section

plane was streaked to the lower half of media.

After five days the first evaluation was done. On all media, growth was visible, but not all

showed the growth typical for F. tularensis (Figure 11). The highest occurrence of F. tularensis

was seen in the sample of the lung (Figure 11, b). Massive growth was present in both halves on

Medium B and Medium C. Medium A showed growth of contaminants.

In general, Medium B and Medium C showed a high-grade growth of colonies typical for

F. tularensis on the upper half of media. On the lower half, weaker growth, with exception of the

lung, was observed. Single colonies of F. tularensis on Medium B and Medium C had a mean

diameter of 0.8-1.0 mm.

On Medium A, growth of shiny, greenish-white colonies were only seen where the loop was used

for inoculation (Figure 11, a and b, see arrows) with the size between 1.5-2 mm. Apart from these

colonies, only contaminants were growing.

Further examination of contaminants on Medium A turned out to be Enterococcus sp. These

contaminants showed a suppressive effect because F. tularensis colonies only grew at a distance

and significant poorer close to the contaminant. Enterococcus sp. was the predominant pathogen

on the upper half of Medium A, so that the impression was created that this medium was optimal

in its media formulation for enterococci.

38

The second interfering contaminant was noticed on Medium B, where a sliced portion was

smeared directly on the media (Figure not shown). Further tests identified this contaminant as

Bacillus sp. This contaminant was able to slow down the growth of F. tularensis.

a)

b)

Figure 11: Isolate 10 - Medium A (left page), Medium B (middle) and Medium C (right page) after

5 days of incubation of sliced portions in the upper half of media and inoculation with a loop in the lower

half of media from a) testes and b) lung

Out of interest, sliced portions of tissue samples were additionally smeared and streaked on

Legionella Agar with Vancomycin and Colistin, BD (Figure 12) to prove its suitability for use for

the detection of tularemia. Despite the contaminants (yellow colonies), F. tularensis was able to

grow in form of grey, shiny colonies (Figure 12, upper half). The lower half of the medium

displayed colonies of F. tularensis with a diameter of about 3-4 mm.

39

Figure 12: F. tularensis on Legionella medium with Vancomycin and Colistin after 5 days of incubation.

Sliced portion of testes were smeared on the upper half of medium and streaked with a loop on the lower

half of medium

Medium A, Medium B and Medium C were suitable for isolation of F. tularensis because two

methods of smearing the tissue samples on the media were chosen and, therefore, on all media

F. tularensis was found.

40


In 2011, a European brown hare was sent to IVET Moedling. Tissue samples of lung, lymph node

and spleen showed an infection with F. tularensis subsp. holarctica biovar II, which was

confirmed with biochemistry and molecular biology. After confirmation, tissue samples were

frozen at – 70°C. Between the processing of the samples and the confirmation 10 days passed,

and during this time, the tissue samples were stored in the refrigerator.

Six month later, the tissue samples were thawed, and sliced portions of tissues were smeared on

Medium A, Medium B and Medium C to check the selective media and to determine if recovery

after freezing at – 70°C is possible. The sliced portions were also streaked on the selective media

using sterile loops.

The first evaluation was done after four days of incubation. At that time, only colonies suspicious

for F. tularensis grew on Medium A and Medium C, where the sliced portion of lung was

smeared. The remaining media showed very occasional contaminants or no growth.

After eight days of incubation, the last evaluation was done (Figure 13). By this time, colonies of

F. tularensis were clearly visible on Medium A and Medium C. The size of a F. tularensis single

colony on Medium A was of about 3 mm, whereas single colonies on Medium C showed a mean

diameter of 1.5 mm (Figure 13, a). Now on Medium B, colonies typical for F. tularensis were

visible, too. On this medium, single colonies had a mean diameter of about 1 mm (Figure 13, see

arrows).

On Medium A, Medium B and Medium C, where tissue samples of lymph node and spleen were

smeared on media, no growth of F. tularensis occurred (figure not shown).

In view of the result of the first examination six month before, the careless storage of samples

between sample processing and freezing was found guilty.

41

Figure 13: Isolate 11 - Medium A (left page), Medium B (middle) and Medium C (right page) after eight

days of incubation of sliced portions in the upper half of media and inoculation with a loop in the lower

half of media of the lung

In summary, it can be stated that recovery of F. tularensis in the lung was successful. On

Medium A, Medium B and Medium C, growth of F. tularensis was observed. All in all, the three

selective media showed a good selectivity because contaminants occurred very occasionally.

Although initial storage conditions were very bad, a recovery after storage at - 70°C was

possible.

42

3.3 Biochemical characterisation

All 11 isolates were further tested biochemically and thereby subspecies and biovar were

determined.

Before the performance of the different tests, subcultures were cultivated to get pure cultures and

to have enough colonies for biochemical testing. One subculture was done per positive European

brown hare. Therefore, a single colony of F. tularensis was taken with a sterile loop from

Medium A, Medium B or Medium C and smeared on a new fresh selective medium with a

followed dilution step.

Confirmation of the species F. tularensis was done using a commercial antiserum for

agglutination. All 11 strains showed a good visible positive agglutination within seconds.

For the determination of the subspecies, cystine trypticase agar supplemented with 5 % horse

serum with glycerine (1 %) or glucose (1 %) was used. All strains showed the ability to ferment

glucose, but not to ferment glycerine. Therefore all strains belonged to the subspecies holarctica.

Figure 14: Fermentation of glucose and glycerine; acid production from glucose (left); no acid production

from glycerine (right)

43

Biovar-typing was carried out with the erythromycin susceptibility testing. Therefore, a disc

containing 15µg erythromycin was used on an agar diffusion test.

All isolated strains from European brown hares showed resistance to erythromycin, which is

indicative for biovar II. So the correct designation for all isolated strains was F. tularensis subsp.

holarctica biovar II.

Figure 15: Sensitivity to Erythromycin; resistance to erythromycin of control strain F. tularensis subsp.

holarctica biovar II (left panel) and sensitivity to erythromycin of control strain F. tularensis subsp.

holarctica biovar I (right panel) after 72 hours of incubation at 37°C on Medium C

The following table gives an overview about biochemical characteristics of the isolated strains

from 11 European brown hares.

44

Table 5: Characteristics of diagnostic value in identifying the genus Francisella and its species,

subspecies and biovar

Consecutive

number

Internal

description

Agglutination of

F.tularensis

antiserum

Acid production from:

glucose glycerine

Erythromycin

Isolate 1 FIWI 368 + + - resistant







Isolate 8 12023273 + + - resistant




45

3.4 PCR

3.4.1 Detection of Francisella tularensis by PCR

PCR was performed with bacterial DNA samples from all 11 isolates and a F. tularensis positive

control. Water was used as negative control.

Successful amplification of the 17-kDa lipoprotein gene was found for 11 (100%) of the samples

and the positive control (Figure 16, see arrows).

Figure 16: Detection of F. tularensis using real-time PCR assay diagnostic; curves show amplified DNA

from Austrian European brown hares and a control strain of F. tularensis. The relative fluorescence is

indicated on the y axis, and the numbers of cycles are indicated on the x axis

Through this assay, the species Francisella tularensis was confirmed molecular biologically for

all 11 isolates.

Isolates

Positive control

Negative control

46

Control strains

(subsp. tularensis)

Isolates and control strains

(subsp. holarctica)

3.4.2 Real-time PCR assay diagnostic of F. tularensis subsp. holarctica

PCR was performed with bacterial DNA samples from all 11 isolates, control strains of

F. tularensis subsp. tularensis and control strains of F. tularensis subsp. holarctica. Water was

used as negative control.

All 11 isolates showed melting points of 74.0°C which is indicative for F. tularensis subsp.

holarctica (Figure 17, see arrow). Control strains of F. tularensis subsp. tularensis showed

melting points of 72.0°C. These melting points are corresponding to the different subspecies.

Figure 17: Real-time PCR assay diagnostic of F. tularensis subsp. holarctica; The peaks show the

melting point (Tm) of amplified DNA from Austrian isolates of European brown hares and control strains

of F. tularensis subsp. holarctica, and F. tularensis subsp. tularensis. The rate of change of the relative

fluorescence units with time, -d(RFU)/dT, is indicated on the y axis, and the temperature is indicated on

the x axis

Using this PCR assay, subspecies of F. tularensis can easily be distinguished.

Water

47

3.5 Contaminants

This chapter deals with contaminants which were isolated from tissue samples of European

brown hares during this work. A few of them had a negative effect on F. tularensis. These

contaminants are listed and described further. In these cases, Medium A, Medium B and

Medium C were not selective enough for the inhibition of the contaminants.

Bacterial contamination of media is a serious problem during diagnostic of tularemia in

Bacteriology. Insufficient inhibition of contaminants on selective media makes it often harder for

F. tularensis to grow. Therefore, contaminants were focused during this work and their ability to

influence F. tularensis by total suppression or delayed or weaker growth through them. The most

influencing contaminants were further isolated and characterised and through serial dilutions, the

counts of CFU were calculated. By doing this, an estimate to which extent the medium is

selective is possible.

3.5.1 Enterococcus faecalis

The predominant contaminant observed on Medium A was Enterococcus faecalis. The species

was confirmed by a negative catalase reaction and in smears, gram-positive cocci were seen. The

latex agglutination test (Streptococcal grouping kit, DR0585, OXOID) showed a positive reaction

with group D which was indicated by agglutination. Biochemical testing confirmed the species

Enterococcus faecalis, too (API 20 STREP V7.9, BioMérieux, Profile 5143711).

Enterococcus faecalis showed different effects on F. tularensis during this work. In low quantity,

Enterococcus faecalis was only able to inhibit partly and slowed down the growth of

F. tularensis (Figure 18, a, see arrow), whereas in quantity, the species was able to suppress the

growth of F. tularensis totally (Figure 18, b, see arrow).

48

a) b)

Figure 18: Effects of Enterococcus faecalis (see arrows) on Medium A by a) partly inhibition or b) total

inhibition the growth of F. tularensis.

Using ten-fold serial dilution, the ability of growth for Enterococcus faecalis on Medium A,

Medium B and Medium C was tested. For a better comparison, also a COS medium without

antibiotics was used (Figure 19).

After 10 ten-fold dilution steps, a concentration of 39 million CFU per ml was calculated.

Therefore, dilution step 1:100,000 was crucial. 39 CFU on COS and 24 CFU on Medium A were

counted, which was not a significant difference, and therefore, the desired selectivity of

Medium A was not reached.

Unfortunately, Medium A was in no way suitable for the inhibition of Enterococcus faecalis.

Growth on this medium was nearly as good as on COS.

More impressive was the selectivity of Medium B and Medium C. Growth of Enterococcus

faecalis was suppressed completely (Figure 19).

49

Figure 19: Comparison of growth of Enterococcus faecalis in stock solution on Medium A, Medium B,

Medium C and COS (from left to right)

3.5.2 Bacillus sp.

The most common contaminant observed on Medium B during this work was Bacillus sp.

In phase-contrast microscope large, spore-forming rods were seen. In smears prepared from

subcultures, large, gram-positive rods were visible. Bacillus sp. was catalase-positive and

oxidase-negative. Growth on Medium B occurred with haemolysis (Figure 20, a, b).

Bacillus sp. showed two different consequences on the growth of F. tularensis. On the one hand,

Bacillus sp. was able to delay the growth for F. tularensis (Figure 20, a, see arrow) and on the

other hand, F. tularensis was suppressed totally through the contaminant (Figure 20, b, see

arrow). a) b)

Figure 20: Effects of Bacillus sp. on Medium B by a) partly inhibition or b) total inhibition the growth of

F. tularensis.

50

Using ten-fold serial dilution, the ability of growth for Bacillus sp. on Medium A, Medium B and

Medium C was tested. For a better comparison, also a COS medium without antibiotics was used

(Figure 21, a).

After 10 ten-fold dilution steps, a concentration of 1.0 million CFU per ml stock solution was

calculated. Therefore, dilution step 1:1,000 was crucial. 50 CFU on Medium B and 100 CFU on

COS were counted which pointed out a selectivity in comparison to COS medium, but low

selectivity compared to Medium A and Medium C.

More impressive was the selectivity of Medium A and Medium C. Growth of colonies from this

contaminant was totally suppressed (Figure 21, a).

Figure 21: Comparison of growth of Bacillus sp. of stock solution on Medium A, Medium B, Medium C

and COS (from left to right)

Medium A and Medium C showed an impressive selectivity by total suppressing of Bacillus sp.

3.5.3 Yeast sp.

Yeast sp. was the predominant mycotic contaminant seen during this work. This pathogen

occurred equally on Medium A, Medium B and Medium C.

51

During this work, this contaminant never occurred in conjunction with F. tularensis (Figure 22).

This raised the question if Yeast sp. was able to completely suppress F. tularensis or no tularemia

was present.

Figure 22: Growth of Yeast sp. (see arrows) on Medium A

Using ten-fold serial dilution, the ability for growth of Yeast sp. on Medium A, Medium B and

Medium C was tested. A COS medium without antibiotics and a Sabouraud agar were inoculated

additionally.

After 10 ten-fold dilution steps, a concentration of 1.0 million CFU per ml stock solution was

calculated. Therefore, dilution step 1:1,000 was crucial (Figure 23). 100 CFU were counted on all

different media, which showed the bad selectivity for yeast sp.

52

Figure 23: Growth of Yeast sp. in dilution 1:1,000 on Sabouraud medium, COS medium, Medium C

(upper row), Medium B and Medium A (lower row) (from left to right)

Unfortunately, Medium A, Medium B and Medium C were not selective enough to inhibit

Yeast sp.

53

4 Discussion

F. tularensis is a fastidious organism making culture recovery a challenge. Culturing is the CDC

gold standard (VERSAGE, 2003). Due to the high degree of virulence of the organism, culture is

often avoided because laboratory staff is at high risk of acquiring clinical disease.

F. tularensis is extremely difficult to recover from field specimens, with recovery rates from

carcasses of prairie dogs at only 30 % (PETERSEN et al., 2004). Tissues from dead European

brown hares are often overgrown with contaminants. The isolation of F. tularensis from

contaminated samples requires the utilization of antibiotics to suppress growth of inhibitory

bacterial species.

In the present work, frozen (at – 80°C) and unfrozen tissue samples of European brown hares

from Austria and Germany were used for the direct isolation of F. tularensis. Medium A, a

CHAB medium supplemented with polymyxin B, amphotericin B, cefepime and vancomycin

(PETERSEN et al., 2009), Medium B, a cystine heart agar with 9 % sheep blood supplemented

with ampicillin, amphotericin B, colistin, lincomycin and trimethoprim (JOHANSSON et al.,

2000a) and Medium C, a cystine heart agar with 9 % sheep blood supplemented with ampicillin

and amphotericin B (OLSUFJEV et al., 1984) were used comparative.

Tissue samples of European brown hares were smeared on Medium A, Medium B and

Medium C, but growth of some bacterial species (Enterococcus faecalis, Bacillus sp.), as well as

yeasts were only inhibited partially.

Medium A showed unrestricted growth for Enterococcus faecalis and good growth for yeasts.

During the production of this medium, the media formulation was done without the addition of

cycloheximide in respect of its harmful side effects like inhibition of protein biosynthesis in

eukaryotic organism. Due to the known effects of suppressing yeasts, the antibiotic

cycloheximide should be added to Medium A onwards. Medium B enabled Bacillus sp. to grow.

54

An interesting phenomenon was observed in Medium A. Enterococcus faecalis showed a

delaying effect on the growth of F. tularensis. It seems that other bacteria can deplete nutrients

required for the growth of F. tularensis. Otherwise, it is possible that contaminants produce

inhibitors which prevent the growth of F. tularensis in the surroundings of the contaminant.

The terms of transport and storage conditions of European brown hares and the tissue samples

were an essential criterion concerning further diagnosis of tularemia. Tissue samples from 45 %

of German European brown hares were not evaluable because of overgrowing environmental

contaminants. There was a long time span between hunting the European brown hares, extraction

of the tissue samples, putting them into cryovials, and transportation to Austria. All these steps

were undertaken without cooling. At the FIWI, cryovials were frozen at – 80°C. Therefore, a

recovery of the culture of F. tularensis was more difficult because of the deterioration of the

samples and the loss of bacterial viability over time. It is very important to freeze tissue samples

(e.g. kidney, spleen, lung, liver, testes, and heart) as soon as possible after the extraction, if direct

cultivation of fresh samples is impossible. It is concluded that freezing tissues is more likely to

preserve predeath bacterial distributions and enable an improved recovery of F. tularensis.

Further, it is necessary to slice the tissues with sterile scissors and then the sliced portions must

be smeared directly on selective media, thereby more tissue material is transferred to the media.

In the case of chronic infections of tularemia with low bacterial counts of F. tularensis, the

chance to make a find is increased by using plane sections. But in this case, media have to be

very selective because sliced portions can contain high counts of contaminants, as well.

Therefore, it is recommendable to use in parallel a sterile loop to streak tissue material for colony

isolation. In an acute event, which means that the animal died of disease, using a sterile loop for

streaking the media would be sufficient.

Only few selective media are available commercially, which makes the use of cultures for the

routine diagnosis of tularemia very difficult. Without the necessary equipment and staff for self-

developed selective media, a direct isolation of F. tularensis is almost impossible. BD BCYE

55

Legionella agar was used for the isolation of F. tularensis in human samples (SCHOBER et al.,

2002). For wildlife samples, Legionella agar supplemented with antibiotics (eg. with

Vancomycin and Colistin) could be a valuable medium for the direct isolation of F. tularensis in

contaminated tissue samples, which was also shown during this work.

Detection of F. tularensis from frozen tissue samples has long been considered as impossible. In

this work, the direct isolation of F. tularensis in frozen (- 80°C) tissue samples from European

brown hares by using selective media was shown. Medium A, Medium B and Medium C were

suitable for direct culture. Per isolation attempt, it is recommended to use a CHAB medium with

antibiotics in parallel to a blood-based medium with antibiotics. In this way, the diagnostic of

tularemia could be enhanced.

As the use of cultures for the direct diagnosis of tularemia is highly promising, it should be spent

more time on the development of selective media and the best antibiotics combination.

Further evaluations with other wildlife samples, as well as additional potential contaminating

species will be important for a further assessment of the selectivity of media.

56

5 Zusammenfassung

Wildtiere stellen ein großes Reservoir für bakterielle Zoonoserreger wie Francisellen dar. Für die

direkte Isolierung von F. tularensis aus Proben von Wildtieren sind Nährmedien notwendig, die

durch den Zusatz geeigneter Antibiotika eine hemmende Wirkung auf Kontaminanten haben,

aber dennoch F. tularensis optimale Wachstumsbedingungen bieten.

Bisher wurden nur wenige selektive Nährmedien für die Humanmedizin, Veterinärmedizin oder

für Umweltproben beschrieben, die die direkte Isolierung von F. tularensis ermöglichen.

In dieser Arbeit wurden zwei selektive Nährmedien getestet, wobei eines ursprünglich für die

direkte Isolierung von F. tularensis aus Tupferproben vom Menschen verwendet wurde und das

andere Nährmedium für Umweltproben.

Ob diese Nährmedien auch für veterinärmedizinische Proben geeignet sind, wurde an gefrorenen

Organproben von 381 Feldhasen aus Österreich und Deutschland und an vier routinemäßig

eingesandten Feldhasen mittels Direktkultur im Vergleich zu einem in der Veterinärmedizin

verwendeten Medium getestet. Für den Kulturversuch wurde jede Organprobe mit einer sterilen

Schere durchgeschnitten und mit Hilfe einer sterilen Pinzette die erhaltene Schnittfläche direkt

auf den Nährmedien abgeklatscht. Medium A war ein Cystin-Herz-Agar mit 9 % Schafblut (auf

Kochblutbasis) und Zusatz von 13 mg Polymyxin B, 2,5 mg Amphotericin B, 4 mg Cefepime

und 4 mg Vancomycin, der für Umweltproben beschrieben wurde (PETERSEN et al., 2009).

Medium B war ein Cystine-Herz-Agar mit 9 % Schafblut und Zusatz von 10 mg Ampicillin,

2,5 mg Amphotericin B, 7,5 mg Colistin, 0,5 mg Lincomycin und 4 mg Trimethoprim, der für

Humanproben beschrieben wurde (JOHANSSON et al., 2000). Medium C war ein Cystin-Herz-

Agar mit 9 % Schafblut und Zusatz von 100 mg Ampicillin und 100 mg Polymyxin B

(OLSUFJEV et al., 1984).

Die Bebrütung der Nährmedien erfolgte bei 37°C für mindestens eine Woche. Kolonien, die

typisch für F. tularensis waren, wurden subkultiviert und anschließend mittels biochemischer und

molekularbiologischer Methoden, die Spezies, die Subspezies und das Biovar bestimmt.

57

Kontaminanten, die das Wachstum von F. tularensis teilweise gehemmt oder total unterdrückt

haben, wurden ebenfalls subkultiviert und die Wachstumshemmung durch die selektiven Medien

mittels Verdünnungsreihen untersucht.

F. tularensis subsp. holarctica biovar II wurde bei 7 (3,2 %) von 219 Feldhasen aus Österreich

gefunden. Die Qualität der Proben aus Österreich und Deutschland war sehr unterschiedlich.

Österreichische Proben waren zu 78,5 % vollständig auswertbar, Proben von deutschen

Feldhasen nur zu 29,7 %. Letztere wiesen durch einen ungekühlten Transport einen sehr hohen

Kontaminationsgrad auf. F. tularensis wurde aus keiner dieser Proben isoliert. Bei den vier

routinemäßig eingesandten Tularämie-infizierten Feldhasen konnte der Erreger mit allen

Selektivmedien isoliert werden.

Medium A und Medium B, die ursprünglich für Umwelt- bzw. Humanproben entwickelt wurden,

sind geeignet um F. tularensis aus gefroren (-80°C) und ungefrorenen Feldhasenproben direkt-

kulturell zu isolieren.

Infektionen mit F. tularensis können nur dann direkt-kulturell nachgewiesen werden, wenn

Nährmedien selektiv genug sind, um vorhandene Kontaminanten in den Proben ausreichend zu

hemmen.

Schlüsselwörter: Tularämie, Francisella tularensis, Isolierung, Selektivmedien

58

6 Summary

Wildlife constitutes a large reservoir for bacterial zoonotic pathogens including Francisellae. For

the direct isolation of the slow growing F. tularensis from wild animals, culture media have to be

supplemented with antibiotics to inhibit contaminants.

Only few selective media are described for the direct culture recovery of F. tularensis from

samples of humans, wildlife or environment. In this work, media developed for the direct

isolation of F. tularensis from human and environmental samples were tested for suitability in

comparison with the presently used selective medium for the direct cultural examination of tissue

samples from European brown hares, which may act as an important reservoir for F. tularensis

subsp. holarctica in the Central European natural focus of tularemia.

Cryo-conserved tissue samples from 381 European brown hares from Austria and Germany and

four routinely sent European brown hares were used for direct-culture to look for latent, chronic

or acute infection caused by F. tularensis, and to evaluate the selectivity of the media. Therefore,

tissues were sliced with sterile scissors. Using sterile forceps, the sliced portions were smeared

directly on selective media. Medium A was a cystine heart agar with 9 % chocolatized sheep

blood supplemented with 13 mg polymyxin B, 2.5 mg amphotericin B, 4 mg cefepime and 4 mg

vancomycin, which was described for environmental samples (PETERSEN et al., 2009).

Medium B was a cystine heart agar with 9 % sheep blood containing 10 mg ampicillin, 2.5 mg

amphotericin B, 7.5 mg colistin, 0.5 mg Lincomycin und 4 mg trimethoprim, which was

described for human samples (JOHANSSON et al., 2000). Medium C was a cystine heart agar

with 9 % sheep blood containing 100 mg ampicillin and 100 mg polymyxin B previously

described (OLSUFJEV et al., 1984). Cultures were incubated at 37°C at least one week. Colonies

characteristic for F. tularensis in morphology were subcultured and the identification of the

species, the subspecies and the characterisation of the biovar was done with biochemical and

molecular biological methods. Interfering contaminants were determined and dilution series on

Medium A, Medium B and Medium C were performed.

59

F. tularensis subsp. holarctica biovar II was isolated from 7 (3.2 %) out of 219 European brown

hares from Austria. Interestingly, frozen samples from Austria were to 78.5 % full evaluable,

whereas only 29.7 % of 158 frozen samples of European brown hares from Germany were full

evaluable, due to influencing contaminants. F. tularensis was not isolated in samples from

Germany. In all routinely sent European brown hares, infected with tularemia, F. tularensis was

isolated on Medium A, Medium B and Medium C.

Medium A and Medium B, developed for environmental samples and human samples, are

suitable for a direct-culture of frozen and unfrozen tissue samples from European brown hares to

isolate F. tularensis.

F. tularensis infections are only direct-cultural detectable if the growth of contaminants is

suppressed sufficiently through selective media.

Keywords: tularemia, Francisella tularensis, isolation, media, selectivity

60

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Use of selective media for direct isolation of Francisella ... · Francisella species, subspecies and biovar . Figures Figure 1: Segmentation of media Figure 2: Isolate 1 - Medium