Rapid transmission of the protozoan parasite Histomonas meleagrids in turkeys and specified pathogen free chickens following cloacal infection with a mono-eukaryotic culture. Michael Hess, Elvira Grabensteiner, Dieter Liebhart

To cite this version:

Michael Hess, Elvira Grabensteiner, Dieter Liebhart. Rapid transmission of the protozoan parasite Histomonas meleagrids in turkeys and specified pathogen free chickens following cloacal infection with a mono-eukaryotic culture.. Avian Pathology, Taylor & Francis, 2006, 35 (04), pp.280-285. ￿10.1080/03079450600815507￿. ￿hal-00540050￿

HAL Id: hal-00540050 https://hal.archives-ouvertes.fr/hal-00540050 Submitted on 26 Nov 2010

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Avian Pathology

For Peer Review Only

Rapid transmission of the protozoan parasite Histomonas meleagrids in turkeys and specified pathogen free chickens following cloacal infection with a mono-eukaryotic culture.

Journal: Avian Pathology

Manuscript ID: CAVP-2006-0017.R1

Manuscript Type: Original Research Paper

Date Submitted by the 24-Mar-2006 Author:

Complete List of Authors: Hess, Michael; Clinic for Avian, Reptile and Fish Medicine, Department for Farm Animals and Herd Management Grabensteiner, Elvira; Clinic for Avian, Reptile and Fish Medicine, Department for Farm Animals and Herd Management Liebhart, Dieter; Clinic for Avian, Reptile and Fish Medicine, Department for Farm Animals and Herd Management

Histomonas meleagridis, transmission, turkeys, specified pathogen Keywords: free chickens

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Cavp-2006-0017.R1

Edited by Dave 1 May 2006

Rapid transmission of the protozoan parasite Histomonas meleagrids in turkeys

and specified pathogen free chickens following cloacal infection with a mono-

eukaryotic culture.For Peer Review Only

M. Hess *, E. Grabensteiner & D. Liebhart

Clinic for Avian, Reptile and Fish Medicine, Department for Farm Animals and Herd Management, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria.

Short title: Transmission of Histomonas meleagridis in turkeys and specified pathogen free

chickens

All figures to be in Black and White in the printed version. Colour only in online version.

Figure 1 (the only figure) to be printed across two columns. EiC.

*To whom correspondence should be addressed. Tel: +43-1250775150. Fax: +43-250775192.

E-mail: [email protected]

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Cavp-2006-0017.R1

M. Hess *, E. Grabensteiner & D. Liebhart

Rapid transmission of the protozoan parasite Histomonas meleagrids in turkeys and specified pathogen free chickens following cloacal infection with a mono-eukaryotic culture. For Peer Review Only Abstract

In the present investigation the pathogenicity and transmission of a mono-eukaryotic culture of Histomonas meleagridis for commercial turkeys and specified pathogen free (SPF) chickens is described for the first time. Two separate trials with the same kind of experimental design, one with commercial turkeys and one with SPF chickens, were performed. In each experiment two different groups were included, which were housed in separate rooms. The first group contained 4 control birds whereas the second group consisted of 10 infected and 4 in-contact birds. The birds were infected via the cloaca at 14 days of age with 380 000 cells of a mono-eukaryotic culture of Histomonas meleagridis consisting of a cloned isolate

(Turkey/Austria/2922/04). Reisolation of the parasite from turkeys and chickens under experimental conditions was performed for the first time. The infected birds started to excrete the parasite as soon as two days post infection. Rapid spread of the parasite to in-contact turkeys and chickens was noticed, based on reisolation of live parasites. Reisolation of the pathogen was impossible from two of the 4 in-contact SPF-chickens at any time, whereas all of the infected turkeys were found positive. Intermittent shedding of the parasite was noticed in infected turkeys and SPF-chickens but the phenomenon was much more severe in the SPF- chickens as these birds survived the infection. All of the infected and in-contact turkeys died between day 11 and 14 post infection, whereas no death was recorded in the SPF-chickens, which were killed 6 weeks after the infection. Typical lesions were recorded in the caeca and

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livers of the infected turkeys. In addition, a heavy destruction of the bursa of Fabricius was

seen in all of the infected and one of the in-contact turkeys. Altogether, the present

investigations are of importance for an understanding of the pathogenicity and transmission of

Histomonas meleagridis in poultry.

For Peer Review Only

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Introduction

Histomonosis (histomoniasis), or blackhead disease, was first described in turkeys by

Cushmann (1894) but subsequently reports occurred of its presence in chickens (Chester &

Robin, 1901). The disease nearly disappeared in commercial poultry following the introduction ofFor chemotherapeutics Peer in the Review middle of the last century.Only In the EU the situation has changed recently due to the complete ban of available substances to be used for prophylaxis or therapeutic treatment of flocks to prevent histomonosis. The availability of substances is also very limited in other countries and histomonosis has to be regarded as a re- emerging disease in poultry. As a consequence, outbreaks are reported in turkeys, sometimes with the complete loss of turkey flocks due to the high mortality and the unavailability of any medication (Hess et al. , 2004). In general, mortality rates in chickens are somewhat lower in comparison with those reported for turkeys. However, recent reports underline the importance of the disease for chickens, especially for birds kept under free range conditions or on farms with low or inadequate disinfection procedures (Homer & Butcher, 1991; Hafez et al. 2001 ;

Esquenet et al. , 2003; Cortes et al. , 2004).

Soon after the description it was postulated that the occurrence of histomonosis is closely linked with the presence of the caecal worm Heterakis gallinarum (Graybill & Smith,

1920; Lund & Chute, 1973). Not surprisingly the earthworm was also described as a vector to transmit the protozoan parasite, explaining to some extent the increasing number of cases in free-range birds (Lund et al. , 1963; Lund et al. , 1966). However, other investigators reported outbreaks of histomonosis without the presence of the caecal worm or earthworm (Tyzzer &

Collier, 1925; Gerth et al. , 1985; Norton et al. , 1999; Cortes et al. , 2004). A possible way of infection without any vector was already reported by Tyzzer & Collier (1925). Recently, lateral transmission of Histomonas meleagridis between turkeys was demonstrated after cloacal infection with the in vitro grown parasite (Hu & McDougald, 2003). This

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phenomenon may explain the spread of the parasite once introduced into a poultry flock (Hu

et al. , 2004).

Despite all the infection experiments carried out so far no information is available

about the excretion and transmission frequency of Histomonas meleagridis . In addition, some

of the existing data depend on infection studies in which less well defined faecal material was used. Recently,For we successfully Peer established Review a mono-eukaroytic Only culture of Histomonas melegaridis by using a micromanipulation approach (Hess et al ., 2005).

The aim of the present investigation was therefore two-fold. First, to investigate the

transmission of Histomonas meleagridis in turkeys and SPF chickens, and second to

investigate the pathogenicity of the above mentioned cloned Histomonas meleagridis isolate

(Turkey/Austria/2922/04) for both species of poultry using the same kind of experimental

design.

Materials and methods

Animals. In the first experiment BUT-T9 turkeys and in the second experiment SPF (VALO,

Lohmann, Cuxhaven, Germany) chickens were used. Prior to the infection all birds were

individually marked using the swift tack system (Heartland Animal Health, Inc. Missouri,

USA). Un-medicated starter feed for turkeys or chickens together with water was provided ad

libitum . The experiments were performed in agreement with Austrian animal licence number

68.205/0017-BrGT/2005.

Housing . In each experiment four control birds (group 1) were kept together in a single room

in a pen of around 3 m2. The second group (group 2) consisted of 10 infected and four in-

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contact birds housed under the same conditions as birds in group 1. All birds were kept on wood shavings under negative pressure with a filtered air supply.

Culture of Histomonas meleagridis . A defined culture of Histomonas melegridis (Turkey/Austria/2922/04)For Peerestablished through Review micromanipulation Only was grown for the infection as described recently (Hess et al. , 2005). Briefly, Histomonas meleagridis was isolated from the caecal content of a diseased turkey. The culture was maintained by cultivation in medium

199 supplied with Earle´s salts, L-Glutamine, 25 mM HEPES and L – Amino acids (Gibco™,

Invitrogen), 1 mg of rice starch (Sigma Aldrich) and 15% FCS (Gibco™, Invitrogen). Out of this a single cell was selected using a micromanipulator (Narishige, Japan). Prior to infection the number of parasites was determined using a Neubauer cell counting chamber. The number of live parasites (given below) was adjusted such that the inoculum of 300 µl contained the appropriate number of parasites.

Infection. At 14 days of age 10 birds in group 2 were infected with 380.000 live histomonads per bird via the cloacal route. A conventional Eppendorf pipette was used to install 300 µl of the inoculation volume. Afterwards the birds were deprived of feed for 5 hours. Birds were monitored daily for any adverse effects and clinical signs.

Sampling and isolation of the protozoan parasite. Prior to infection all birds were swabbed

(day 0). Following infection further swabbing was performed at several intervals. At each sampling date a cotton swab was used and immediately placed in a microfuge tube containing

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300 µl of the medium described above. For isolation of live protozoa growth of Histomonas

meleagrids was monitored daily under a light microscope for up to 5 days.

Pathology. All birds were sectioned and typical morphological lesions were noticed in the turkeys. Due toFor the fact that Peer none of the Reviewchickens died in the secondOnly experiment all birds were killed 6 weeks post infection, at eight weeks of age.

Results

Clinical signs. Experiment 1, turkeys. No clinical signs were noticed in the control birds. First

clinical symptoms in the infected and in-contact birds were observed at 6 days post

inoculation (d.p.i.) when three of the infected birds and one in-contact bird showed signs of

depression. At day 8 p.i. the birds started huddling together and two birds (nos. 9 and 13) with

yellowish droppings were noticed. One day later six of the infected birds displayed diarrhoea

and all birds were located together under the heating lamp. At day 10 p.i all birds appeared

sick with ruffled feathers and displayed a severe apathy. At that time, 10 out of the 14 birds of

group 2 had sulphur-coloured diarrhoea. Seven birds (nos. 5, 6, 8, 9, 10, 12 and 13 and 17) out

of the 14 birds in group 2 died at day 11 p.i.. Two infected birds (nos. 7 and 14) died 12 d.p.i..

At day 13 p.i. one infected (no. 11) and one in-contact bird (no. 15) died, whereas the last two

in-contact birds (nos. 16 and 18) died 14 d.p.i.. As a consequence, the study was terminated

and all control birds which were housed as group 1 were killed.

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Experiment 2, specified pathogen free chickens. No adverse clinical signs were noticed throughout the whole experiment. All birds were killed 43 d.p.i. when the birds were about 8 weeks old.

Transmission For of live Histomomas Peer meleagrids Review between birds. Only Experiment 1, turkeys. No parasites could be isolated from any of the birds prior to infection (Tables 1a and 2). The control birds (group 1) stayed negative throughout the whole experiment. Excretion of

Histomonas meleagridis in infected turkeys started 2 d.p.i. when the parasite could be isolated from 7 of the 10 infected birds. At the same time two of the in-contact birds also started to excrete the parasite. Histomonas meleagridis could be isolated from all of the four in-contact birds at day 5 p.i., whereas only four of the infected birds were found positive. Most of the positive samples from the infected birds were noticed 7 and 9 d.p.i. (Table 2). Except for bird

7, all birds excreted the parasite intermittently.

Experiment 2: specified pathogen free chickens. No parasites could be isolated from the control birds during the whole experiment or from the infected and in-contact birds prior to the infection (Tables 1b and 2). The first positive samples from the infected birds were obtained at 2 d.p.i.. Afterwards the number of positives samples decreased to only three and two positive samples on 5 and 7 d.p.i., respectively. The maximum number of positive samples from the infected birds was recorded at day 23 p.i.. Subsequently the number of positive samples dropped again until the termination of the study. Live parasites were reisolated at least once from every infected bird and from two of the in-contact birds (nos. 15 and 17). Two in-contact birds (nos. 16 and 18) remained negative throughout the whole experiment. Two infected birds (nos. 13 and 15) were only positive at the beginning of the

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experiment. The majority of birds excreted the parasite intermittently. However, birds 8 and

10 excreted live parasite only at consecutive sampling dates.

Pathological lesions. Experiment 1, turkeys. None of the control birds showed any abnormal pathological signs,For whereas Peer necropsy ofReview all of the infected Only and in-contact birds revealed typical pathological lesions of histomonosis. All of these birds had grossly enlarged caeca

with thickened walls and their luminas were filled with fibrinous material, as well as swollen

livers with multiple rounded areas of necrosis (Figure 1). In most cases the whole liver was

involved. Pathological changes were also noticed in the bursa of Fabricius. In all cases the

bursa was enlarged, filled with caseous material and in some cases haemorrhages were also

noticed. These lesions were most severe in the infected birds (Figure 1a). However, they were

also present in bird no. 16, one of the in-contact birds (Figure 1b).

Experiment , specified pathogen free chickens. Since none of the SPF chickens died during

the experiment all of them were killed 43 d.p.i. at the termination of the experiment. Only

slight pathological changes like signs of a low grade inflammation of the mucosa were seen in

the caeca of some of the infected birds. Caeca were filled with caseous material and a

thickening of the caecal wall was only noticed in bird no. 17, one of the in-contact birds. No

liver lesions were recorded in any of the birds.

Discussion

The flagellated parasite Histomonas meleagridis is the aetiological agent of histomonosis,

sometimes also named blackhead disease (Tyzzer, 1920). Experimentally, Tyzzer & Collier

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(1925) showed that the cloacal installation of histomonads can be used to induce histomonosis in turkeys avoiding the presence of any other parasitic vector. Lateral transmission between turkeys kept under experimental conditions was demonstrated recently (Hu & McDougald,

2003). As a consequence, the same method of infection was used in the current experiments in order to investigate the transmission and pathogenicity of a defined cloned isolate (Turkey/Austria/2922/04)For Peerof Histomonas Review meleagridis in turkeys Only and SPF-chickens. Using the cloacal route of infection most the authors reported an installation volume of 0.5 ml up to 1 ml which was used sometimes in combination with the suspension of the birds´ feet

(Desowitz, 1951; Lund, 1955; Landman et al. , 2003; Hu et al. , 2004). In the present investigations the volume of 300 µl installed via the cloaca was found to be optimal in order to prevent any spoilage of the infectious material through active peristalsis of the colon as a consequence of manipulation at the cloaca. Prior to infection defecation was induced by stimulating the anal lips. No further manipulation was needed except that the birds were deprived of feed for 5 hours p.i..

The mono-eukaryotic isolate was found to be infectious and highly pathogenic as all of the infected and in-contact turkeys died by day 14 p.i.. Other investigators reported either a lower mortality and/or a delayed development of the disease. For example, Hu et al . (2004) noticed no mortality up to 12 d.p.i. using 100 000 cells/bird in two-week-old turkeys even though severe lesions in the caeca and livers were observed. A mortality of 80% was reported by Landman et al . (2003) after infection of 15-day-old turkeys with 200 000 histomonads/bird. In that experiment the birds died between two and 33 days of age. An even lower mortality of only 50% was reported recently after infecting three week-old female turkeys with 150.000 histomonads (Hauck et al. 2005).

Several factors have to be considered with regard to these discrepancies: the infectious dose, the age and genetic background of the birds and a possible variation amongst different isolates. The infectious dose of around 3.8 x 10 5 histomonads could be one reason for the

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fastidious development of the disease in the present investigation. However, the incubation

time and the development of the disease in the experimentally infected turkeys is in

agreement with earlier studies in which in vitro -grown parasites were used as well (Lund,

1955). The rapid transmission of the cloned isolate Turkey/Austria/2922/04 has certainly

contributed to obtaining such a high mortality. Infected and in-contact 4-week-old turkeys started to excreteFor live histomonads Peer by asReview early as 2 d.p.i.. Huber Only et al. (2005) infected turkeys with a somewhat higher dose of 3 x 10 6 and parasitic DNA was only detected from day 5 p.i.

onwards and some of the infected birds survived until day 19 p.i. when they were killed.

In the present study no clinical signs and mortality were recorded in the SPF chickens.

In comparison, there are many reports in literature which documented outbreaks with varying

mortality in young chickens (Milks, 1908; Eriksen, 1925; Tyzzer, 1934; Bayon, 1937;

Hungerford, 1937; Bishop, 1938; Goedbloed & Bool, 1962; Ivanics et al ., 1984; Gerth et al .,

1985; Reece et al ., 1986; Müller, 1990; Homer & Butcher, 1991; Ganapathy et al ., 2000;

Esquenet et al ., 2003). Furthermore, Desowitz (1951) reported the highest mortality during

the first two weeks after infection of commercial chickens and the infection status of the birds

varied by age. In addition, the susceptibility of different chicken breeds was demonstrated

some time ago (Lund, 1967; Chute et al. , 1976). According to the obtained results SPF

chickens behave as an asymptomatic carrier of the parasite, which favours these birds as a

good host to investigate the phenomenon of a latent infection caused by Histomonas

meleagridis .

McDougald & Galloway (1973) have highlighted already the advantage of in vitro

isolation for the diagnosis of histomonasis by growing parasites directly from the caeca. In

that study a high percentage of positive samples was noticed 7 d.p.i., similar to the current

experiments. However, birds were only sampled once as they had to be killed to obtain the

material. The intermittent shedding of the parasite noticed by both methods is of relevance for

future epidemiological studies.

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Beside the liver and caeca a heavy destruction of the bursa of Fabricius was observed in all of the infected turkeys. This may be due to the direct installation of the infectious material in the cloaca. In contrast to the present findings, Marx (1973) reported the presence of histomonads in only 1 of 200 bursa of Fabricius investigated by histology from turkeys and chickens experimentally infected via the cloaca. However, Cortes et al. (2004) were able to demonstrate HistomonasFor Peer meleagridis inReview the bursa of Fabricius Only in chickens during a field outbreak. The DNA of histomonads could also be detected in the bursa of Fabricius by PCR

(Hauck et al., 2005). The direct contact between the bursa of Fabricius and the intestinal content together with the cloacal sucking may provoke the infection of the bursa of Fabricius

(Schaffner et al. , 1974). The present investigation further proves the findings reported by Hu

& McDougald (2003) that turkeys are infected via the cloacal drinking mechanism in the field in the absence of any other vector for spreading of the flagellate. In the present investigation the same way of infection was demonstrated for SPF chickens. Monitoring excretion and transmission allows the precise differentiation between infected and non-infected birds, independent from clinical signs and pathological lesions.

In the present investigation a cloned isolate of Histomonas meleagridis , named

Turkey/Austria/2922/04, was used for the first time to infect turkeys and specified pathogen free chickens, and to investigate the transmission of the parasite under experimental conditions. Using this type of culture a very efficient model was established to demonstrate both (a) the high contagiousness of the disease in turkeys, and (b) the role of SPF chickens as asymptomatic carriers.

Acknowledgements

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The authors wish to thank the Austrian Ministry for Agriculture, Forestry, Environment and

Water Management and the Austrian Ministry for Health and Women’s Issues for financial

support to perform the present investigations.

References For Peer Review Only

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absence of Heterakis. Journal of Infectious Diseases, 37, 265-276.

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Figure 2:

a)

For Peer Review Only

b)

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Legend to figure

Figure 1. Caeaca, liver and bursa of Fabricius of two turkeys after sectioning.( a) bird no. 13

(infected) and (b) bird no. 16 (in-contact) which died at 11 and 14 d.p.i., respectively.

For Peer Review Only

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Table 1. Reisolation of Histomonas meleagridis from cloacal swabs of (a) turkeys and (b) SPF chickens

(a): turkeys Bird number Group 1 Group 2 control birds inoculated birds in-contact birds Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 p.i. a For Peer Review Only 0 ------

2 - - - - - +b + + + - + + - + + + - - 5 ------+ + - + + - - - + + + + 7 - - - - - + + - + + + + + + - - - + 9 - - - - + + + + + - - + + + - - + + 12 - - - - 11dpi c 11dpi 12dpi d 11dpi 11dpi 11dpi 13dpie 11dpi 11dpi 12dpi f 13dpi 14dpi g 11dpi 14dpi a Sample negative by reisolation b Sample positive by reisolation c Bird died 11 d.p.i. d Bird died 12 d.p.i. e Bird died 13 d.p.i.; reisolation at 12 d.p.i. positive f Bird died 12 d.p.i. with successful reisolation at this day g Bird died 14 d.p.i.; reisolation at 12 d.p.i. positive

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(b): specified pathogen free chickens Bird number Group 1 Group 2

control birds infected birds in-contact birds Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 p.i. 0 ------2 - - - - - + + - - - + + + + + - - - 5 - - - For - -Peer - - - Review - - + + - Only + - - - - 7 ------+ ------9 ------+ + ------12 ------+ + + - + + - + - - - - 14 ------+ + + - - + - + - - - - 16 ------+ + - + - + - - - - 19 - - - - - + + - + + + - - + - - + - 23 - - - - + + + - + + + + ------26 - - - - - + - - - + - - - + - - - - 30 - - - - - + - - + + + - - + - - - - 35 ------+ + - - + - - - - 43 ------+ ------+ - - - - a Sample negative by reisolation b Sample positive by reisolation

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Table 2. Reisolation of live Histomonas meleagridis trophozoites from cloacal swabs of turkeys and specified pathogen free chickens .

Day post infection

0 2 For 5 Peer 7 9 12Review 14 16 19 Only 23 26 30 35 43 Bird inf. a con. b inf. con. inf. con. inf. con. inf. con. inf. con. inf. con. inf. con. inf. con. inf. con. inf. con. inf. con. inf. con. inf. con. Turkey 0 0 7 2 4 4 8 1 8 2 not applicable Chicken 0 0 6 1 3 0 1 0 2 0 6 0 5 0 4 0 6 1 7 0 3 0 5 0 3 0 2 0

a Total number of positive birds (n=10 infected birds) b Total number of positive birds (n=4 in-contact birds)

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