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Veterinärmedizinische Universität Wien (Vetmeduni Vienna)

28.05.2013 1

Histomonas meleagridis: from a single cell towards a new protection

University of Veterinary Medicine, Vienna

from a single cell towards a new protection strategy to prevent Histomonosis

Clinic for Avian, Reptile and Fish Medicine

Department for Farm Animals and Veterinary Public Health

Michael Hess

Clonal cultures as the core tool

V i ti t tVaccination as a new strategy

Conclusions and remarks

The disease: histomonosis

Zahoor et al. (2011), Avian Dis. 55, 29-34

Isolation and propagation of H. meleagridis

Establishment of clonal cultures

Histomonas meleagridis

Tetratrichomonas gallinarum

Blastocystis sp.

protozoa/species/country/diagnostic - clone number/year

Hess et al. (2006), Parasitol., 133, 547-554

New diagnostics: detection of histomonad DNAM Hm Tg Bl

bursa of Fabricius lungGarbensteiner & Hess (2006), Vet. Parasitol., 142, 223-230; Liebhart et al. (2006), J. Comp. Pathol., 135, 237-242


28.05.2013 2

New diagnostics: immunohistochemistry

bone marrowproventriculus

pancreas brain

Singh et al. (2006), Exp. Parasitol., 118, 505-513

New diagnostics: ELISA

Windisch et al. (2009), Vet.Parasitol., 161, 25-30

Grafl et al. (2009), Vet.Rec., 168, 160-164

H. meleagridis: morphology in culture

Zaragatzki et al. (2010), Parasitol.Res., 106, 1005-1007

Zaragatzki et al. (2010), Parasitol.Res., 106, 977-983

Mielewczik et al. (2008), Parasitol. Res. 103, 745-750

Interaction: H. meleagridis - bacteria

A DCB

Confocal laser micrographs of a monoxenic H. meleagridis culture grown with E. coliDH5α pGFPuv. Series of eight consecutive sections through a H. meleagridis cell labelled with polyclonal anti-histomonad serum (red) and E. coli DH5α pGFPuv(green). Nucleus of the parasite and bacterial DNA stained with DAPI (blue).

HGFE

Ganas et al. (2012), Int.J. Parasitol., 42, 893-901

Genetic investigations

Leberl et al. (2010), Mol.Biochem.Parasitol., 169, 101-107

Bilic et al. (2013), submitted for publication

Clonal cultures as a core tool




28.05.2013 3

Plant substances

conflicting results about the efficacy of plant substances Protophyt® , NatustatTM and Enteroguard®

Testing of 19 different plant substances altogether 45 plant samples

raw material

extracts (aqueous, ethanol, heptane)

results in vitro were not confirmed in vivo

Artemisia annua strong effect against Plasmodium spp.

antihistomonial effect in vitro

no effect in vivo

LogDose (mM)

No.

of

viab

le p

roto

zoa

(105 )

1 2 3 4 50.0

2.0

4.0

6.0

8.0

10.0

Thöfner et al. (2012), Avian Pathol., 41, 487-496

Grabensteiner et al. (2007), Parasitol.Res. 101, 193-199

Attenuation and VaccinationTyzzer (1934 and 1936), Proc. Am. Acad. Arts Sci. 69, 189-264 and J. Comp. Pathol. 49, 285-303

evidence that attenuation of H. meleagridis occurs by weekly passages but varies between isolates

vaccination with an in vitro cultivated isolate is possible but vaccinated birds displayed clinical signs and attenuation interferes with immunity

Clarkson J. (1963), Immunology, 6, 156-168

precipitating antibodies in fowls and turkeys succeeding infection performed in combination with treatment

protective immunity could not be transferred to other birds

Ruff and Hansen (1970), Avian Dis. 14, 646-653

gamma irradiation reduced the pathogenicity of H. meleagridis

no immunity of birds infected with non-treated H. meleagridis

Dwyer and Honigberg (1970), J. Parasitol. 56, 694-700

gradual attenuation of H. meleagridis by in vitro cultivation up to 9 weeks

Lund et al. (1959) J. Protozool. 6, 182-185

3 consecutive applications of a non-pathogenic isolate of H. meleagridis (H. wenrichi) are needed to infect 91% of turkeys; limited protection after challenge

Lund et al. (1966), Exp. Parasitol., 18, 403-407 in-vitro attenuation is not possible

Clonal histomonads: attenuation - vaccination

3 Years

vaccine: H. meleagridis/Turkey/Austria/2922-C6/04 P295

vaccination 2 weeks of age challenge 6 weeks of age

4 weeks later

Hess et al. (2008), Vaccine, 26, 4187-4193

Group vaccination on day 14 number of birds challenge on day 42 Mortality

Experimental vaccination of turkeys against histomonosis:

cloacal vaccination using 104 attenuated histomonads: Hm/Turkey/Austria/2922-C6/04 – passages 95, 215 and 295

cloacal challenge with 104 virulent histomonads: Hm/Turkey/Austria/2922-C6/04 – passage 21

Attenuation - Protection

I Passage (P) 95 10a/4b P 21 0%

II P 215 10a/4b P 21 0%

III P 295 10a/4b P 21 0%

IV not done (n.d.) 7c/3b P 21 100%

V N.d. 4 n.d. 0%

a vaccinated and challenged birds; b in-contact birds; c challenged birds;

Hess et al. (2008), Vaccine 26, 4187-4193

Group III(Infection at day 14: Passage 295 ----- Challenge at day 42: Passage 21)

group IV (room 1)

group III (room 4)

control birds infected birds in-contact birds

day p.i.1

(313)2

(314)3

(315)4

(316)1

(299)2

(300)3

(301)4

(302)5

(303)6

(304)7

(305)8

(306)9

(307)10

(308)11

(309)12

(310)13

(311)14

(312)

0 - - - - - - - - - - - - - - - - - -

2 - - - - - - x x - - - - - - - - - -

5 - - - - - - - x x - - x - - - - - -

7 - - - - - x - - x - - - - - - - - -

9 - - - - - - - - - - - - - - x - - -

12 - - - - - - - - - - - x - - - - - -

14 - - - - - x - x - - X - - - x - x x

16 - - - - - - - - - - - x - - - x x -

1919 - - - - - - - - x - - - - x - - - -

21 - - - - - x - - - - x - x - - - - -

23 - - - - x - - - - x - - x x - - - -

26 - - - - - x - - - x x - - - x - - -

28 - - - - - - - - - - x - - x - - x -

28 - challenge control birds challenged birds in-contact birds challenged birds

30 - - - - - x - - - x x x - - - - - -

33 - - - - - - x x - x x - x - - x - -

35 - - - - - - x - - x x x x - - - - x

37 - - - - - x - - - x x - x x - - - -

40 - - - - x x - - - x - x x x x - - -

42 - - - - - x x - x - - x x x x - x -

44 - - - - x x - - - x - x - x x - x -

49 - - - - - - x - - - - - - - - - - -

56- - - - - x - x x - x x - - x - - -

56a 56a 54b 56a 56a 56a 56a 56a 56a 56a 56a 56a 56a 56a 56a 56a 56a 56a

lesions in caeca (c)or liver (l)

no no no no no no no L:necro.C:fibrin no no no no no no no no L:necro.

C:ffibrin no

a killed at termination of the study; bbird died: no lesions for histomonosis

Vaccinated versus non-vaccinated

vaccinated birds: 15 d.p.c.

non-vaccinated birds: 12 d.p.c.


28.05.2013 4

Post mortem

vaccinated

non-vaccinated

Oral vaccination

groupa number of birdsb 1st day of life

14 days post vaccination (p.v.)

28 days p.v.

I 10c/4d vaccine (P295)e challenge (P21)f n.a.

II 10c/4d vaccine (P295) n.a. challenge (P21)

III 14c/5d vaccine (P295) n.a. n.a.

IV 5c/2d challenge (P21) n.a. n.a.

V 5c/2d n.a.g challenge (P21) n.a.

VI 5c/2d n.a. n.a. challenge (P21)

VII 10 n.a. n.a. n.a.

a group of birds kept in separated pens; b breed: B.U.T. 9; c infected birds / d in-contact birds e Hm/Turkey/Austria /2922-C6/04 passage 295 - 104 parasites orally; Hm/Turkey/ Austria /2922-C6/04 passage 21 - 104 parasites cloacallyg not applicable

Liebhart et al. (2010), Avian Pathol. 39, 399-403

Clinical signs

non-vaccinated(group VI)

orally vaccinated at day-old and challenge

4weeks later (group II)

day 50 post vaccination and day 22 post challenge


Progression of body weights

Vaccinated and challenged 14 days p.v.

Vaccinated and challenged 28 days p.v.

Vaccinated without challenge

Neither vaccinated nor challenged

The average live weight of all birds from each group is given.


Safety of the vaccine

B.U.T. Big 6 turkeys infected with different in vitro passages (P21 or P295) of Hm/Turkey/Austria/2922-C6/04 oral infection at 1st day of life with 104 histomonads

killing of 3 predetermined birds per groups I and II and 1 uninfected bird on days 4, 7, 10, 14 and 21 p.i.

Group I - P2115 birds infected with the

Group II - P29515 birds infected with the

Group III5 non-infected birds15 birds infected with the

virulent strain P2115 birds infected with the attenuated strain P295

5 non infected birds

Liebhart & Hess, (2011), Poult.Sci. 90, 966-1003

Lesion scores (LS): day 14 p.i.

LS4LS4LS4 LS4 LS4 LS3

Group I - P21- all birds infected with P21 died within 13 d.p.i.; no clincal signs in birds infected with P295

LS4LS4LS4 LS4 LS4 LS3

Group II – P295

livers and caeca: LS 0


28.05.2013 5

Immunohistochemistry: day 14 p.i.

Group I - P21

Caecum

Caecum

Liver Lung

Liver Lung

Group II - P295

Monoxenization - PathogenicityHM xenic P20 HM+DH5α P20 HM+DH5α P295 E. coli DH5α

Clinical signs of histomonosis yes yes no no

Pathological changes: caeca yes yesyes (light changes

in some of thecaeca)

no

Pathological changes: livers yes yes no no

90

100

HM xenic P20(14 dpi)

HM+DH5α P20(18 dpi)

HM+DH5α P295(35 dpi)

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5

cumulative

mortality per group [%]

week post‐infection

HM+DH5α P295

HM+DH5α P20

HM xenic P20

DH5α

Ganas et al. (2012), Int.J. Parasitol., 42, 893-901

50%

60%

70%

80%

90%

100%

rod

uct

ion

group C group V group V+C group NC

Vaccination of layers

** *

* significant difference between

control and challenged

group (P≤0.05).

# significant diff

#

-9,43%

-28,73%

0%

10%

20%

30%

40%

19 20 21 22 23 24 25 26 27 28 29

egg

pr

week of lifechallengevaccination*

1 2 3 4 5 6week p.c.*

1 2 3 4 5 week p.v.

difference between

vaccinated+ challenged and only

challenged group

(P≤0.05).*


*vaccination and challenge were performed with 104 histomonads given orally and cloacally

Necropsy

vaccinated

non-vaccinated

Clonal cultures as a core tool



Conclusions

histomonosis is a re-emerging disease in poultry

outbreaks in turkeys can co-incidence with high mortality and complete loss of the flock

micromanipulation to establish clonal cultures as basis for new diagnostic tools: PCR to discriminate between flagellates and other protozoag p ELISA to detect and quantify the antibody response Immunohistochemistry and in-situ hybridization to detect

H. meleagridis or its nucleic acid in tissues above mentioned diagnostics increase specificity and sensitivity

clonal cultures can also be used to: perform detailed genetic studies establish “single bacterial strain cultures”


28.05.2013 6

Conclusions

successful attenuation of clonal Histomonas meleagridis(Hm/Turkey/Austria/2922-C6/04) by long term in vitro cultivation

efficacy aspects of the prototype vaccine application of attenuated histomonads via the cloaca induced protection against a

severe challenge oral vaccination of day-old birds induced partial and complete protection at 2 and 4

weeks post vaccination, respectively vaccination of layers reduced a drop in egg production vaccination of layers reduced a drop in egg production

safety aspects of the prototype vaccine safety of the vaccine could be demonstrated based on clinical examination,

pathomorphological and histological findings vaccination had no influence on production parameters in turkeys and chickens vaccination never induced any adverse clinical signs

attenuated histomonads are confined to the caeca and do not invade other organs no reversion to virulence was noticed in turkeys and chickens 5x backpassaged attenuated histomonads induced only minor lesions in the caeca

Remarks

no accurate accessible records about disease outbreaks Histomonosis is not a listed disease = is it a problem?

funding situation Only very limited funds available – if at all!

registrationD i d b t i i th lt E li St t Do mixed bacteria in the culture, e.g. E. coli, Streptococcus sp. and Proteus sp., interfere with registration?

Are the existing data usable for registration?

vaccine technology time point: proven concept for oral vaccination at day old

(turkeys) and chickens prior to lay

application: so far: single bird vaccination orally or/and cloacally

Thanks for your attention!I. Bilic, P. Ganas, E. Grabensteiner, B. Grafl, M. Leberl,

University of Veterinary Medicine, Vienna

D. Liebhart, A. Singh, M. Windisch, A. Zahoor, T. Sulemanjovic

Heinrich Heine University, DüsseldorfH. Mehlhorn and E. Zaragatzki

All colleagues contributing with samples

Clinic for Avian, Reptile and Fish MedicineDepartment for Farm Animals and

Veterinary Public Health

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