Avian Pathology

ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: https://www.tandfonline.com/loi/cavp20

Rapid transmission of the protozoan parasite Histomonas meleagridis in turkeys and specific free following cloacal infection with a mono-eukaryotic culture

M. Hess , E. Grabensteiner & D. Liebhart

To cite this article: M. Hess , E. Grabensteiner & D. Liebhart (2006) Rapid transmission of the protozoan parasite Histomonas￿meleagridis in turkeys and specific pathogen free chickens following cloacal infection with a mono-eukaryotic culture, Avian Pathology, 35:4, 280-285, DOI: 10.1080/03079450600815507 To link to this article: https://doi.org/10.1080/03079450600815507

Published online: 18 Jan 2007.

Submit your article to this journal

Article views: 1647

View related articles

Citing articles: 10 View citing articles

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=cavp20 Avian Pathology (August 2006) 35(4), 280285

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

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

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

In the present investigation, the pathogenicity and transmission of a mono-eukaryotic culture of Histomonas meleagridis for commercial turkeys and specific pathogen free (SPF) chickens is described for the first time. Two separate trials with the same kind of experimental design were performed, one with commercial turkeys and one with SPF chickens. In each experiment, two different groups were included, which were housed in separate rooms. The first group contained four control , whereas the second group consisted of 10 infected and four 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 H. meleagridis consisting of a cloned isolate (/Austria/2922-66/04). 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 2 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 four 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 days 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 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 H. meleagridis in poultry.

Introduction

Histomonosis (), or blackhead disease, was Butcher, 1991; Hafez et al., 2001; Esquenet et al., 2003; first described in turkeys by Cushmann (1894), but Cortes et al., 2004). reports subsequently occurred of its presence in chickens Soon after the description it was postulated that the (Chester & Robin, 1901). The disease nearly disappeared occurrence of histomonosis is closely linked with the in commercial poultry following the introduction of presence of the caecal worm chemotherapeutics in the middle of the last century. In (Graybill & Smith, 1920; Lund & Chute, 1973). Not the European Union the situation has changed recently surprisingly, the was also described as a due to the complete ban of available substances to be vector to transmit the protozoan parasite, explaining to used for prophylaxis or therapeutic treatment of flocks some extent the increasing number of cases in free-range to prevent histomonosis. The availability of substances is birds (Lund et al., 1963, 1966). However, other investi- also very limited in other countries, and histomonosis gators reported outbreaks of histomonosis without the has to be regarded as a re-emerging disease in poultry. presence of the caecal worm or earthworm (Tyzzer & As a consequence, outbreaks are reported in turkeys, Collier, 1925; Gerth et al., 1985; Norton et al., 1999; sometimes with the complete loss of turkey flocks due to Cortes et al., 2004). A possible route of infection the high mortality and the unavailability of any medica- without any vector was already reported by Tyzzer & tion (Hess et al., 2004). In general, mortality rates in Collier (1925). Recently, lateral transmission of Histo- chickens are somewhat lower in comparison with those monas meleagridis between turkeys was demonstrated reported for turkeys. However, recent reports underline after cloacal infection with the in vitro grown parasite the importance of the disease for chickens, especially for (Hu & McDougald, 2003). This phenomenon may birds kept under free range conditions or on farms with explain the spread of the parasite once introduced into low or inadequate disinfection procedures (Homer & a poultry flock (Hu et al., 2004).

*To whom correspondence should be addressed. Tel: /43 1250775150. Fax: /43 250775192. E-mail: [email protected] ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/06/40280-06 # 2006 Houghton Trust Ltd DOI: 10.1080/03079450600815507 H. meleagridis transmission in turkeys and SPF chickens 281

Despite all the infection experiments carried out so symptoms in the infected and in-contact birds were far, no information is available about the excretion and observed 6 days post inoculation (d.p.i.) when three of transmission frequency of H. meleagridis. In addition, the infected birds and one in-contact showed signs some of the existing data depend on infection studies in of depression. At 8 d.p.i. the birds started huddling which less well-defined faecal material was used. Re- together, and two birds (numbers 9 and 13) with cently, we successfully established a mono-eukaroytic yellowish droppings were noticed. One day later six of culture of H. melegaridis using a micromanipulation the infected birds displayed diarrhoea and all birds were approach (Hess et al., 2006). located together under the heating lamp. At 10 d.p.i all The aim of the present investigation was therefore two- birds appeared sick with ruffled and displayed a fold. First, to investigate the transmission of Histomonas severe apathy. At that time, 10 out of the 14 birds of meleagridis in turkeys and specific pathogen free (SPF) group 2 had sulphur-coloured diarrhoea. Eight birds chickens, and second to investigate the pathogenicity of (numbers 5, 6, 8, 9, 10, 12, 13 and 17) out of the 14 birds the above mentioned cloned Histomonas meleagridis in group 2 died 11 d.p.i. Two infected birds (numbers 7 isolate (Turkey/Austria/2922-66/04) for both species of and 14) died 12 d.p.i. At 13 d.p.i. one infected bird poultry using the same kind of experimental design. (number 11) and one in-contact bird (number 15) died, whereas the last two in-contact birds (numbers 16 and 18) died 14 d.p.i. As a consequence, the study was Materials and Methods terminated and all control birds that were housed as Animals. In the first experiment BUT-T9 turkeys were used, and in the group 1 were killed. second experiment SPF (VALO, Lohmann, Cuxhaven, Germany) chickens were used. Prior to the infection all birds were individually Experiment 2, SPF chickens. No adverse clinical signs marked using the swift tack system (Heartland Animal Health, Inc., were noticed throughout the whole experiment. All birds Missouri, USA). Un-medicated starter feed for turkeys or chickens were killed 43 d.p.i. when the birds were about 7 weeks together with water was provided ad libitum . The experiments were old. performed in agreement with Austrian animal licence number 68.205/ 0017-BrGT/2005. Transmission of live H. meleagridis between birds. Housing. In each experiment four control birds (group 1) were kept Experiment 1, turkeys. No parasites could be isolated together in a single room in a pen of around 3 m2. The second group from any of the birds prior to infection (Tables 1 and 2). (group 2) consisted of 10 infected and four in-contact birds housed The control birds (group 1) stayed negative throughout under the same conditions as birds in group 1. All birds were kept on the whole experiment. Excretion of H. meleagridis in wood shavings under negative pressure with a filtered air supply. infected turkeys started 2 d.p.i. when the parasite could be isolated from seven of the 10 infected birds. At the Culture of H. meleagridis . A defined culture of H. melegridis (Turkey/ same time two of the in-contact birds also started to Austria/2922-66/04) established through micromanipulation was grown excrete the parasite. H. meleagridis could be isolated for the infection as described recently (Hess et al. , 2006). Briefly, from all of the four in-contact birds at 5 d.p.i., whereas H. meleagridis was isolated from the caecal content of a diseased turkey. only four of the infected birds were found positive. Most The culture was maintained by cultivation in medium 199 supplied with TM of the positive samples from the infected birds were Earle’s salts, l-Glutamine, 25 mM HEPES and l-amino acids (Gibco , noticed 7 and 9 d.p.i. (Table 2). Except for bird number Invitrogen), 11 mg rice starch (Sigma Aldrich) and 15% foetal calf serum (GibcoTM, Invitrogen). Out of this a single cell was selected using 7, all birds excreted the parasite intermittently. a micromanipulator (Narishige, Japan). Prior to infection, the number of parasites was determined using a Neubauer cell counting chamber. Experiment 2, SPF chickens. No parasites could be The number of live parasites (given below) was adjusted such that the isolated from the control birds during the whole experi- inoculum of 300 ml contained the appropriate number of parasites. ment or from the infected and in-contact birds prior to the infection (Tables 1 and 2). The first positive samples Infection. At 14 days of age, 10 birds in group 2 were infected with from the infected birds were obtained at 2 d.p.i. After- 380,000 live histomonads per bird via the cloacal route. A conventional wards the number of positives samples decreased to only Eppendorf pipette was used to install 300 ml of the inoculation volume. three and two positive samples on 5 and 7 d.p.i., Afterwards the birds were deprived of feed for 5 h. Birds were respectively. The maximum number of positive samples monitored daily for any adverse effects and clinical signs. from the infected birds was recorded at 23 d.p.i. Subsequently the number of positive samples dropped Sampling and isolation of the protozoan parasite. All birds were swabbed again until the termination of the study. Live parasites prior to infection (day 0). Following infection further swabbing was were reisolated at least once from every infected bird and performed at several intervals. At each sampling date a cotton swab was from two of the in-contact birds (numbers 15 and 17). used and immediately placed into a microfuge tube containing 300 ml medium (already described). For isolation of live , growth Two in-contact birds (numbers 16 and 18) remained of H. meleagridis was monitored daily under a light microscope for up negative throughout the whole experiment. Two infected to 5 days. birds (numbers 13 and 15) were only positive at the beginning of the experiment. The majority of birds Pathology. All birds were sectioned and typical morphological lesions excreted the parasite intermittently. However, bird num- were noticed in the turkeys. Due to the fact that none of the chickens bers 8 and 10 excreted live parasite only at consecutive died in the second experiment, all birds were killed 6 weeks post sampling dates. infection, at 8 weeks of age. Pathological lesions. Experiment 1, turkeys. None of the control birds showed any abnormal pathological signs, Results whereas necropsy of all of the infected and in-contact Clinical signs. Experiment 1, turkeys. No clinical signs birds revealed typical pathological lesions of histomo- were noticed in the control birds. The first clinical nosis. All of these birds had grossly enlarged caeca with 8 .Hess M. 282 tal et . Table 1. Reisolation of H. meleagridis from cloacal swabs of turkeys and SPF chickens

Time (d.p.i.) Group 1 Group 2

Control birds Inoculated birds In-contact birds

Bird 1 Bird 2 Bird 3 Bird 4 Bird 5 Bird 6 Bird 7 Bird 8 Bird 9 Bird 10 Bird 11 Bird 12 Bird 13 Bird 14 Bird 15 Bird 16 Bird 17 Bird 18

Turkeys

0 / / / / / / / / / / / / / / / / / / 2 / / / / / / / / / / / / / / / / / / 5 / / / / / / / / / / / / / / / / / / 7 / / / / / / / / / / / / / / / / / / 9 / / / / / / / / / / / / / / / / / / a b c d e 12 / / / / 11 d.p.i. 11 d.p.i. 12 d.p.i. 11 d.p.i. 11 d.p.i. 11 d.p.i. 13 d.p.i. 11 d.p.i. 11 d.p.i. 12 d.p.i. 13 d.p.i. 14 d.p.i. 11 d.p.i. 14 d.p.i. SPF chickens

0 / / / / / / / / / / / / / / / / / / 2 / / / / / / / / / / / / / / / / / / 5 / / / / / / / / / / / / / / / / / / 7 / / / / / / / / / / / / / / / / / / 9 / / / / / / / / / / / / / / / / / / 12 / / / / / / / / / / / / / / / / / / 14 / / / / / / / / / / / / / / / / / / 16 / / / / / / / / / / / / / / / / / / 19 / / / / / / / / / / / / / / / / / / 23 / / / / / / / / / / / / / / / / / / 26 / / / / / / / / / / / / / / / / / / 30 / / / / / / / / / / / / / / / / / / 35 / / / / / / / / / / / / / / / / / / 43 / / / / / / / / / / / / / / / / / /

a b c d /, sample negative by reisolation; /, sample positive by reisolation. Bird died 11 d.p.i. Bird died 12 d.p.i. Bird died 13 d.p.i.; reisolation at 12 d.p.i. was positive. Bird died 12 d.p.i., with successful reisolation on this day. eBird died 14 d.p.i.; reisolation at 12 d.p.i. was positive. H. meleagridis transmission in turkeys and SPF chickens 283

Table 2. Reisolation of live H. meleagridis trophozoites from grade inflammation of the mucosa were seen in the caeca cloacal swabs of turkeys and SPF chickens of some of the infected birds. Caeca were filled with caseous material and a thickening of the caecal wall was Turkeys Chickens only noticed in bird number 17, one of the in-contact 0 d.p.i. birds. No liver lesions were recorded in any of the birds. Infecteda 00 In-contactb 00 2 d.p.i. Discussion Infected 7 6 The flagellated parasite H. meleagridis is the aetiological In-contact 2 1 agent of histomonosis, sometimes also named blackhead 5 d.p.i. disease (Tyzzer, 1920). Experimentally, Tyzzer & Collier Infected 4 3 (1925) showed that the cloacal installation of histomo- In-contact 4 0 nads can be used to induce histomonosis in turkeys, 7 d.p.i. avoiding the presence of any other parasitic vector. Infected 8 1 Lateral transmission between turkeys kept under experi- In-contact 1 0 mental conditions was demonstrated recently (Hu & 9 d.p.i. McDougald, 2003). As a consequence, the same method Infected 8 2 of infection was used in the current experiments in order In-contact 2 0 to investigate the transmission and pathogenicity of a 12 d.p.i. defined cloned isolate (Turkey/Austria/2922-66/04) of H. Infected Not applicable 6 meleagridis in turkeys and SPF chickens. Using the In-contact Not applicable 0 cloacal route of infection, most of the authors reported 14 d.p.i. an installation volume of 0.5 to 1 ml, which was used Infected Not applicable 5 sometimes in combination with the suspension of the In-contact Not applicable 0 birds’ feet (Desowitz, 1951; Lund, 1955; Landman et al., 16 d.p.i. 2004; Hu et al., 2004). In the present investigations the Infected Not applicable 4 volume of 300 ml installed via the cloaca was found to be In-contact Not applicable 0 optimal in order to prevent any spoilage of the infectious material through active peristalsis of the colon as a 19 d.p.i. Infected Not applicable 6 consequence of manipulation at the cloaca. Prior to In-contact Not applicable 1 infection, defecation was induced by stimulating the anal lips. No further manipulation was needed except that the 23 d.p.i. birds were deprived of feed for 5 h post inoculation. Infected Not applicable 7 In-contact Not applicable 0 The mono-eukaryotic isolate was found to be infec- tious and highly pathogenic as all of the infected and in- 26 d.p.i. contact turkeys died by 14 d.p.i. Other investigators Infected Not applicable 3 reported either a lower mortality and/or a delayed In-contact Not applicable 0 development of the disease. For example, Hu et al. 30 d.p.i. (2004) noticed no mortality up to 12 d.p.i. using 100 000 Infected Not applicable 5 cells/bird in 2-week-old turkeys even though severe In-contact Not applicable 0 lesions in the caeca and livers were observed. A mortality 35 d.p.i. of 80% was reported by Landman et al. (2004) after Infected Not applicable 3 infection of 15-day-old turkeys with 200 000 histomo- In-contact Not applicable 0 nads/bird. In that experiment the birds died between 2 43 d.p.i. and 33 days of age. An even lower mortality of only 50% Infected Not applicable 2 was reported recently after infecting 3-week-old female In-contact Not applicable 0 turkeys with 150 000 histomonads (Hauck et al., 2005). Several factors have to be considered with regard to a b Total number of positive birds (n/10 infected birds). Total these discrepancies: the infectious dose, the age and number of positive birds (n/ 4 in-contact birds). genetic background of the birds, and a possible variation among different isolates. The infectious dose of around 5 thickened walls and their luminas were filled with 3.8/10 histomonads could be one reason for the fibrinous material, as well as swollen livers with multiple fastidious development of the disease in the present rounded areas of necrosis (Figure 1). In most cases the investigation. However, the incubation time and the whole liver was involved. Pathological changes were also development of the disease in the experimentally infected noticed in the bursa of Fabricius. In all cases the bursa turkeys is in agreement with earlier studies in which was enlarged, filled with caseous material; and in some in vitro grown parasites were used as well (Lund, 1955). cases haemorrhages were also noticed. These lesions The rapid transmission of the cloned isolate Turkey/ were most severe in the infected birds (Figure 1a). Austria/2922-66/04 has certainly contributed to obtain- However, they were also present in bird number 16, ing such a high mortality. Infected and in-contact one of the in-contact birds (Figure 1b). 4-week-old turkeys started to excrete live histomonads as early as 2 d.p.i. Huber et al. (2005) infected turkeys 6 Experiment 2, SPF chickens. Since none of the SPF with a somewhat higher dose of 3/10 histomonads, chickens died during the experiment all of them were and parasitic DNA was only detected from 5 d.p.i. killed 43 d.p.i., at the termination of the experiment. onwards*some of the infected birds survived until Only slight pathological changes such as signs of a low- 19 d.p.i., when they were killed. 284 M. Hess et al.

Figure 1. Caeaca, liver and bursa of Fabricius of two turkeys after sectioning. 1a: Bird number 13 (infected), which died at 11 d.p.i. 1b: Bird number 16 (in-contact), which died at 14 d.p.i.

In the present study, no clinical signs and mortality infectious material in the cloaca. In contrast to the were recorded in the SPF chickens. In comparison, there present findings, Marx (1973) reported the presence of are many reports in the literature that document out- histomonads in only one of 200 bursae of Fabricius breaks with varying mortality in young chickens (Milks, investigated by histology from turkeys and chickens 1908; Eriksen, 1925; Tyzzer, 1934; Bayon, 1937; Hun- experimentally infected via the cloaca. However, Cortes gerford, 1937; Bishop, 1938; Goedbloed & Bool, 1962; et al. (2004) were able to demonstrate H. meleagridis in Ivanics et al., 1984; Gerth et al., 1985; Reece et al., 1986; the bursa of Fabricius in chickens during a field out- Mu¨ller, 1990; Homer & Butcher, 1991; Ganapathy et al., break. The DNA of histomonads could also be detected 2000; Esquenet et al., 2003). Furthermore, Desowitz in the bursa of Fabricius by PCR (Hauck et al., 2005). (1951) reported the highest mortality during the first 2 The direct contact between the bursa of Fabricius and weeks after infection of commercial chickens, and the the intestinal content together with the cloacal sucking infection status of the birds varied by age. In addition, may provoke the infection of the bursa of Fabricius the susceptibility of different breeds was demon- (Schaffner et al., 1974). The present investigation further strated some time ago (Lund, 1967; Chute et al., 1976). proves the findings reported by Hu & McDougald (2003) According to the obtained results, SPF chickens behave that turkeys are infected via the cloacal drinking as an asymptomatic carrier of the parasite, which favours mechanism in the field in the absence of any other these birds as a good host to investigate the phenomenon vector for spreading of the flagellate. In the present of a latent infection caused by H. meleagridis. McDougald & Galloway (1973) have highlighted investigation, the same method of infection was demon- already the advantage of in vitro isolation for the strated for SPF chickens. Monitoring excretion and diagnosis of histomonosis by growing parasites directly transmission allows the precise differentiation between from the caeca. In that study a high percentage of infected and non-infected birds, independent of clinical positive samples was noticed 7 d.p.i., similar to the signs and pathological lesions. current experiments. However, birds were only sampled In the present investigation, a cloned isolate of once as they had to be killed to obtain the material. The H. meleagridis, Turkey/Austria/2922-66/04, was used intermittent shedding of the parasite noticed in the for the first time to infect turkeys and SPF chickens, current investigation is of relevance for future epidemio- and to investigate the transmission of the parasite under logical studies. experimental conditions. Using this type of culture a very Beside the liver and caeca, a heavy destruction of the efficient model was established to demonstrate both, the bursa of Fabricius was observed in all of the infected high contagiousness of the disease in turkeys and the role turkeys. This may be due to the direct installation of the of SPF chickens as asymptomatic carriers. H. meleagridis transmission in turkeys and SPF chickens 285

Acknowledgements a Blastocystis sp. established through micromanipulation. Parasitol- ogy, in press. Homer, B.L. & Butcher, G.D. (1991). Histomoniasis in Leghorn pullets The authors wish to thank the Austrian Ministry for on a Florida farm. Avian Diseases, 35 , 621 624. Agriculture, Forestry, Environment and Water Manage- Hu, J.H. & McDougald, L.R. (2003). Direct lateral transmission of ment and the Austrian Ministry for Health and Women’s Histomonas meleagridis in turkeys. Avian Diseases, 47 ,489492. Issues for financial support to perform the present Hu, J., Fuller, L. & McDougald, L.R. (2004). Infection of turkeys with investigations. Histomonas meleagridis by the cloacal drop method. Avian Diseases, 48 , 746 750. Huber, K., Chauvec, C. & Zenner, L. (2005). Detection of Histomo- niasis Meleagridis in turkeys cecal droppings by PCR amplification of References the small subunit ribosomal DNA sequence. Veterinary Parasitology, 131 , 311 316. Bayon, H.P. (1937). The pathology of Histomonas enterohepatitis in Hungerford, I.G. (1937). Blackhead in chickens. Agricultural Gazette, turkeys and other birds. Veterinary Record , 49 , 1010 1015. 48 , 647 651. Bishop, A. (1938). Histomonas meleagridis in domestic fowls (Gallus Ivanics, F., Glavits, R., Ratz, F. & Szelenyi, Z. (1984). Histomonadosis gallus ). Cultivation and experimental infection. Parasitology, 30 , el’fordulasa hazityukban [Histomonas in fowl]. Magyar Allatorvosok 181 194. Lapja , 39 , 562 566. Chester, F.D. & Robin, A. (1901). Enterohepatitis or blackhead of fowls. Landman, W.J.M., McDougald, L.R. & van der Heijden, H.M.J.F. Twelfth Annual Report of Delaware Agricultural Station Report (pp. (2004). Experimental infestation of turkeys and chickens with a 60 66). Dutch field isolate of Histomonas meleagridis. In H.M. Hafez (Ed.), Chute, A.M., Lund, E.E. & Wilkins, G.C. (1976). Comparative Proceedings of the 5th International Symposium on Turkey Diseases responses of White Leghorn and New Hampshire chickens to (pp. 258 268). Berlin, Germany. experimental infections with Histomonas meleagridis and Heterakis Lund, E.E. (1955). The progress of histomoniasis (blackhead) in turkeys gallinarium . Poultry Science, 55 , 710 713. as related to the size of the of the infective dose. Poultry Science, 34 , Cortes, P.L., Chin, R.P., Bland, M.C., Crespo, R. & Shivaprasad, H.L. 127 130. (2004). Histomoniasis in the bursa of fabricius of chickens. Avian Lund, E.E. (1967). Response of four breeds of chickens and one breed Diseases, 48 , 711 715. of turkeys to experimental Heterakis and Histomonas infections. Cushmann, S. (1894). The production of turkeys. Agricultural Experi- Avian Diseases, 11 , 491 502. ment Station of the Rhode Island State College of Agriculture and Lund, E.E. & Chute, A.M. (1973). Means of acquisition of Histomonas Mechanic Art Bulletin , 25 ,89123. meleagridis by eggs of Heterakis gallinarum. Parasitology, 66 , 335 Desowitz, R.S. (1951). Age as a factor influencing fatal infections of 342. histomoniasis in chickens. Journal of Comparative Pathology, 61 , Lund, E.E., Wehr, E.E. & Ellis, D.J. (1963). Role of in 231 236. transmission of Heterakis and Histomonas to turkeys and chickens. Eriksen, S. (1925). Blackhead in chicks. Poultry Science, 4 , 250 255. Journal of Parasitology, 49 , 50. Esquenet, C., De Herdt, P., De Bosschere, H., Ronsmans, S., Ducatelle, Lund, E.E., Wehr, E.E. & Elli, D.J. (1966). Earthworm transmission of R. & Van Erum, J. (2003). An outbreak of histomoniasis in free-range Heterakis and Histomonas to turkeys and chickens. Journal of layer hens. Avian Pathology, 32 , 305 308. Parasitology, 52 , 899 902. Ganapathy, K., Salamat, M.H., Lee, C.C.& & Johara, M.Y. (2000). Marx, D.J. (1973). A turkey bursa of Fabricius infected with Histomo- Concurrent occurrence of salmonellosis, colibaccillosis and histomo- nas meleagrids. Journal of Protozoology, 20 , 519. niasis in a broiler flock fed with antibiotic-free commercial feed. McDougald, L.R. & Galloway, R.B. (1973). Blackhead disease: in vitro Avian Pathology, 29 , 639 642. isolation of Histomonas meleagridis as a potentially useful diagnostic Gerth, C., Rudiger-Boesch, B., Schmidt, U., Mumme, J. & Friedhoff, aid. Avian Diseases, 17 , 847 850. K.T. (1985). Histomoniasis in pullet stock and its effect on later Milks, H.J. (1908). A preliminary report on some diseases of chickens. laying performance. Tiera¨ rztliche Praxis, 13 , 519 527. Louisiana Agricultural Experiment Station Bulletin , 108 ,311. Goedbloed, E. & Bool, P. H. (1962). The protozoan etiology of Mu¨ller, H. (1990). Enzootic typhlohepatitis in intensively kept pullets. blackhead. Avian Diseases, 6 , 302 315. Monatshefte fu¨ r Veterina¨ rmedizin , 45 , 464 467. Graybill, H.W. & Smith, T. (1920). Production of fatal blackhead in Norton, R.A., Clark, F.D. & Beasley, J.N. (1999). An outbreak of turkeys by feeding embryonated eggs of Heterakis papillosa . Journal histomoniasis in turkeys infected with a moderate level of Ascaridia of Experimental Medicine, 31 , 647 655. dissimilis but no Heterakis gallinarum . Avian Diseases, 43 , 342 348. Hafez, H.M., Mazaheri, A., Prusas, C., Bo¨hland, K., Po¨ppel, M. & Reece, R.L., Beddome, V.D. & Barr, D.A (1986). Diseases diagnosed in Schulze, D. (2001). Actual infectious diseases in layer flocks kept in replacement layer and breeder chicken flocks in Victoria, Australia, alternative rearing systems. Tiera¨ rtztliche Praxis Ausgabe Großtiere 1977 1985. Veterinary Record , 19 , 471 475. Nutztiere, 29 , 168 174. Schaffner, T., Mueller, J., Hess, M.W., Cottier, H., Sordat, B. & Ropke, Hauck, R., Lu¨schow, D. & Hafez, H.M. (2005). Pathogenesis of C. (1974). The bursa of Fabricius: a central organ providing for Histomoniasis: the spread of Histomonas meleagridis to different contact between the lymphoid system and intestinal content. Cellular organs after experimental infection. In H.M. Hafez (Ed.), Proceed- Immunology, 13 , 304 312. ings of the 3rd International Meeting of the Working Group 10 of the Tyzzer, E.E. (1920). The flagellate character and reclassification of the World Poultry Science Association (pp. 254 258), Berlin, Germany. parasite producing ‘‘blackhead’’ in turkeys-Histomonas (gen.nov.) Hess, M., Grabensteiner, E., Liebhart, D., Weissenbo¨ck, H. & Loupal, meleagridis (Smith). Journal of Parasitology, 6, 124 131. G. (2004). Diagnostic investigations on Histomonas meleagridis Tyzzer, E.E. (1934). Studies on histomoniasis or ‘‘blackhead’’ infection, following a severe outbreak in a turkey flock. In H.M. Hafez (Ed.), in the chicken and turkey. Proceedings of the American Academy of Proceedings of the 5th International Symposium on Turkey Diseases Arts and Sciences, 69 , 189 264. (pp. 254 257), Berlin, Germany. Tyzzer, E.E. & Collier, J. (1925). Induced and natural transmission of Hess, M., Kolbe, T., Grabensteiner, E. & Prosl, H. (2005). Cloned blackhead in the absence of Heterakis. Journal of Infectious Diseases, cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and 37 , 265 276. Avian Pathology (August 2006) 35(4), 1Á2 Non-English Abstracts Rapid transmission of the protozoan parasite Histomonas meleagridis in turkeys and specific pathogen free chickens following cloacal infection with a mono-eukaryotic culture

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

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

Transmission rapide du parasite protozoaire Histomonas meleagridis chez des dindes et des poulets SPF apre`s une infection par voie cloacale avec une culture mono eucaryote Dans cette e´tude, la pathoge´nicite´ et la transmission d’une culture mono eucryote d’ Histomonas meleagridis pour des dindes du commerce et des poulets exempts de microorganismes pathoge`nes spe´cifie´s (SPF) sont de´crites pour la premie`re fois. Deux essais diffe´rents avec le meˆme type de protocole expe´riemental, l’un avec des dindes du commerce et l’autre avec des poulets SPF, ont e´te´re´alise´s. Chaque essai, comprenait deux groupes diffe´rents qui e´taient he´berge´s dans des animaleries se´pare´es. Le premier groupe comportait 4 animaux te´moins, alors que le second groupe comportait 10 sujets infecte´s et 4 sujets contacts. Les animaux ont e´te´ infecte´s par voie cloacale a` l’aˆge de 14 jours avec 380 000 cellules d’une culture mono eucryote d’ Histomonas meleagridis qui correspondait a` un isolat clone´ (Turkey/Austria/2922/04). Le re´isolement du parasite a` partir des dindes et des poulets dans des conditions expe´rimentales a e´te´re´alise´ pour la premie`re fois. Les animaux infecte´s ont commence´a` excre´ter le parasite de`s le 2e`me jour apre`s l’infection. Une rapide diffusion du parasite aux dindes et poulets contacts a e´te´ observe´e, base´e sur le re´isolement des parasites vivants. L’agent pathoge`ne n’a pas pu eˆtre re´isole´a` partir de 2 des 4 poulets SPF contacts lors d’aucun pre´le`vement, alors que toutes les dindes infecte´es ont e´te´ positives. Une excre´tion intermittente du parasite a e´te´ note´e chez les dindes et les poulets SPF infecte´s, mais le phe´nome`ne a e´te´ beaucoup plus important chez les poulets SPF puisque ces animaux ont surve´cu a` l’infection. Toutes les dindes infecte´es et contacts sont mortes entre le 11e`me et le 14e`me jour apre`s l’infection, alors qu’aucune mortalite´ n’a e´te´ enregistre´e chez les poulets SPF qui ont e´te´ sacrifie´s 6 semaines apre`s l’infection. Des le´sions typiques ont e´te´ note´es au niveau des cæca et du foie chez les dindes infecte´es. De plus, une destruction importante de la bourse de Fabricius a e´te´ observe´e chez toutes les dindes infecte´es et chez une dinde contact. Dans l’ensemble ces recherches sont importantes pour la compre´hension de la pathoge´nicite´ et de la transmission d’ Histomonas meleagridis chez les volailles.

Schnelle U¨ bertragung des Protozoenparasiten Histomonas meleagridis in Puten und spezifiziert pathogen freien Hu¨hnern nach kloakaler Infektion mit einer mono-eukaryotischen Kultur Dies ist die Erstbeschreibung einer Untersuchung der Pathogenita¨t und U¨ bertragung einer mono- eukaryotischen Kultur von Histomonas meleagridis fu¨r kommerzielle Puten und spezifiziert pathogen freie (SPF) Hu¨hner. Es wurden zwei getrennte Versuche, einer mit kommerziellen Puten und einer mit SPF- Hu¨hnern, mit dem gleichen Versuchsplan durchgefu¨hrt. In jedem Versuch gab es zwei Gruppen, die in separaten Ra¨umen untergebracht waren. Die erste Gruppe bestand aus 4 Kontrolltieren, wa¨hrend in der zweiten Gruppe zehn infizierte und vier Kontakttiere waren. Die Tiere wurden am 14. Lebenstag via Kloake mit 380 000 Zellen einer mono-eukaryotischen Histomonas meleagridis-Kultur eines geklonten Isolats (Turkey/Austria/2922/04) infiziert. Die Reisolierung des Parasiten aus Puten und Hu¨hnern unter experimentellen Bedingungen wurde erstmalig durchgefu¨hrt. Bereits zwei Tage nach der Infektion begannen die infizierten Tiere den Parasiten auszuscheiden. Aufgrund der Reisolierung lebender Parasiten konnte die schnelle Ausbreitung der Parasiten auf die Kontakttiere nachgewiesen werden. Wa¨hrend bei zwei der vier Kontakt-SPF-Hu¨hner eine Reisolierung des nicht gelang, waren alle infizierten Puten positiv. Bei den infizierten Puten und Hu¨hnern wurde eine intermittierende Ausscheidung des Parasiten festgestellt, aber dieses Pha¨nomen war bei den Hu¨hnern, die auch die Infektion u¨berlebten, wesentlich deutlicher ausgepra¨gt. Alle infizierten und Kontakt-Puten starben zwischen dem 11. und 14. Tag nach der Infektion. Die SPF-

**To whom correspondence should be addressed. Tel: /43 1250775150. Fax: /43 250775192. E-mail: [email protected] ISSN 0307-9457 (print)/ISSN 1465-3338 (online)//40001-02 # 2006 Houghton Trust Ltd DOI: 10.1080/03079450600815507 2 M. Hess et al.

Hu¨hner u¨berlebten alle und wurden 6 Wochen nach der Infektion geto¨tet. In den Blindda¨rmen und Lebern der infizierten Puten wurden typische La¨sionen diagnostiziert. Außerdem wurde bei allen infizierten und einem Kontakttier eine schwere Zersto¨rung der Bursa Fabricii nachgewiesen. Die vorliegenden Untersu- chungsergebnisse sind fu¨r das Versta¨ndnis der Pathogenita¨t und der U¨ bertragung von Histomonas meleagridis beim Geflu¨gel wichtig.

Transmisio´n ra´pida del para´sito protozoario Histomonas meleagridis en pollos libres de pato´genos especı´ficos y pavos tras la infeccio´n cloacal con cultivo mono-eucariota En el presente estudio se describe por primera vez la patogenicidad y transmisio´n del cultivo mono-eucariota de Histomonas meleagridis en pavos comerciales y pollos libres de pato´genos especı´ficos (SPF). Se llevaron a cabo dos pruebas separadas con el mismo disen˜o experimental, una con pavos comerciales y la otra con pollos SPF. En cada prueba se incluyeron dos grupos diferentes alojados en corrales separados. El primer grupo contenı´a 4 aves control mientras que el segundo grupo incluı´a 10 aves infectadas y 4 aves centinelas. Las aves se infectaron vı´a cloacal a los 14 dı´as de vida con 380 000 ce´lulas de un cultivo mono-eucariota de Histomonas meleagridis procedente de la clonacio´n de un aislamiento (Turkey/Austria/2922/04). Por primera vez se llevo´ a cabo en condiciones experimentales el reaislamiento del para´sito de pavos y pollos. Las aves infectadas empezaron a excretar el para´sito tan pronto como 2 dı´as post infeccio´n. Se observo´ una diseminacio´n ra´pida del para´sito a los pavos y pollos centinelas, en base al reaislamiento del para´sito vivo. No se pudo reaislar el pato´geno de dos de los 4 pollos SPF centinelas en ningu´n momento, mientras que todos los pavos infectados fueron positivos. Se determino´ la excrecio´n intermitente del para´sito en pavos y pollos SPF infectados, pero el feno´meno fue mucho ma´s intenso en los pollos SPF, dado que estas aves sobrevivieron a la infeccio´n. Todos los pavos infectados y centinelas murieron entre los 11 y 14 dı´as post infeccio´n, mientras que ningu´n pollo SPF murio´, de modo que se sacrificaron a las 6 semanas post infeccio´n. Se observaron lesiones caracterı´sticas en los ciegos e hı´gados de los pavos infectados. Adema´s, se observo´ una destruccio´n intensa de la bolsa de Fabricio en todos los pavos infectados y en uno de los centinelas. En conjunto, estos estudios son importantes para entender la patogenicidad y transmisio´n de Histomonas meleagridis en aves.