Histomonosis in poultry: previous and current strategies for prevention and therapy^*

* Part of this review is based on the Avian Pathology Lecture entitled ‘Strategies to prevent histomonosis in poultry’, presented by Dieter Liebhart at the 19th Congress of the World Veterinary Poultry Association in Cape Town, South Africa, September 2015.

ABSTRACT

Histomonosis is a parasitic disease of poultry with worldwide prevalence. The disease can cause morbidity and mortality in chicken and turkey flocks entailing severe economic losses. In the first half of the last century, there was a high demand to control histomonosis as the turkey industry was severely affected by the disease. Consequently, numerous chemical compounds were tested for their efficacy against Histomonas meleagridis with varying outcomes, that are summarized and specified in this review. At the same time, preliminary attempts to protect birds with cultured histomonads indicated the possibility of vaccination. Several years ago antihistomonal drugs were banned in countries with tight regulations on pharmaceuticals in order to comply with the demand of consumer protection. As a consequence, outbreaks of histomonosis in poultry flocks increased and the disease became endemic again. New approaches to prevent and treat histomonosis are, therefore, needed and recently performed studies focused on various areas to combat the disease, from alternative chemotherapeutic substances to plant-derived compounds until vaccination, altogether reviewed here. Considering existing regulations and the overall outcome of experimental studies, it can be concluded that vaccination is very promising, despite the fact that various challenges need to be addressed until the first ever developed vaccine based upon live flagellates in human or bird medicine can be marketed.

KEYWORDS:

• Histomonosis
• Histomonas meleagridis
• prophylaxis
• therapy
• vaccination

Introduction

Emphasis of the review

The focus of the present review is to recapitulate reports investigating different approaches for preventive and therapeutic options against histomonosis since it was first reported (Cushman, 1893). Additionally, changes in management and husbandry that appeared in coincidence with the rise of the poultry industry, affecting the epizootiology of the disease, are described here. Basic knowledge about the parasite Histomonas meleagridis and the disease histomonosis is mentioned briefly, for a conclusive understanding regarding the prevention and therapy of the disease. For broader information on the biology of the parasite and general aspects of histomonosis, the authors would like to refer to previously published reviews (Hess et al., 2015; McDougald, 2005).

Histomonosis – from emergence to re-emergence

Histomonosis (syn.: blackhead disease or histomoniasis) was primarily observed as infectious enterohepatitis of turkeys (Smith, 1895). The start of the poultry industry at the end of the nineteenth century in the USA was in coherence with the emergence of the disease, causing high losses in turkey farms. Consequently, histomonosis became a severe threat for turkey production, which was evident by the decline from 6.5 million turkeys in 1900 to 3.6 million birds in 1920 (Animal and Plant Health Inspection Service, 1984). The need to control the disease at that time and in the following years is reflected by an increase in studies investigating not only the disease and the causative agent H. meleagridis itself, but also options for prevention and therapy. An important finding was the identification of Heterakis gallinarum as a vector for H. meleagridis, implicating practical suggestions on the prevention of histomonosis by aspects of biosecurity and worm control (Graybill & Smith, 1920). Later on, experimental studies demonstrated arsenicals and nitroheterocyclics to be effective against histomonosis (Tyzzer, 1923; Waletzky et al., 1950). Hence, those drugs became commercially available and by their use, outbreaks of the disease in turkeys and chickens could be effectively prevented or treated. However, health concerns were raised about the drugs of both chemical groups regarding food safety following administration in food-producing birds as recently reviewed (Baynes et al., 2016). Accordingly, antihistomonal drugs were banned in industrial countries, which caused a re-emergence of the parasitic disease in the last years (Hess et al., 2015).

Important features of the parasite and its interaction with the host

H. meleagridis belongs to the genus Histomonas, family Dientamoebidae, order Trichomonadida. The parasite is unicellular, 3–21 µm in size and is composed of an axosytle, a pelta, a costa, parabasal bodies and hydrogenosomes, organelles that are commonly found in trichomonads (Hess & McDougald, 2013). In general, two stages of H. meleagridis can be differentiated: (i) the flagellated caecal lumen form and (ii) the tissue form without flagella. The caecal lumen form propagates in the caecum and can be observed in liquid cultures following in vitro isolation. The shape is round or amoeboid, tentatively with the formation of pseudopodia. The tissue form is located intercellularly in the organs of the host. Non-flagellated stages can develop an additional membrane when transforming into a more resistant phase. Detailed light and transmission electron microscopy suggested the formation of such cyst-like stages of in vitro cultivated histomonads (Zaragatzki et al., 2010).

Turkeys suffering from histomonosis show ruffled feathers, drooped wings, apathy and sulphur-coloured diarrhoea. The mortality of turkeys can be up to 100% as demonstrated in various experimental settings, and summarized by Hauck and Hafez (2013). Histomonosis in chickens causes milder clinical signs and a lower mortality, but the disease was reported to be at least as severe as coccidiosis (McDougald, 2005). The pathogenesis of histomonosis starts with the colonization of the parasite in the caecum, leading to severe inflammation and necrosis. Following the destruction of intestinal tissue, the parasite may infiltrate into blood vessels and reach the liver via the portal veins. As a consequence, areas of inflammation and destruction can occur in the liver. In the final stage, the disease may become systemic when the parasite spreads to various organs of the host (Grabensteiner et al., 2006).

Lesions caused by H. meleagridis are noticed as thickening of the caecal wall, bleeding in the mucosa and fibrinous masses in the lumen of the caecum. Changes in the liver are found as round localized areas of necrosis that can be variable in size. Such lesions can be seen in turkeys and chickens, though they may vary in the severity of pathological changes. Microscopically, the inflammation is characterized by infiltrations of mixed populations of leukocytes, together with areas of necrosis and the presence of tissue stages of H. meleagridis as described above.

Drugs against histomonosis – benefits and limitations

In the past, efforts to prevent or treat the disease focused on the effect of chemical substances. The compounds that were used, along with further details on the findings, are given in this section and key facts on the drugs tested against histomonosis are specified (Table 1).

Table 1. Chemical agents investigated for their effect against H. meleagridis in vitro and/or in vivo.

Chemical group	Mode of action	Active component	In vitro effect	In vivo effect	Administration via	Reference
Arsenical	Interference of DNA repair processes and cellular energy metabolism	Arsenious acid	n.i.	Prophylactic, non-therapeutic	Feed	Tyzzer (1923) and Swales (1950)
		Neoarsphenamine	n.i.	Prophylactic and therapeutic	Intravenous application	Tyzzer (1923) and Blount (1938)
		Tryparsamide	n.i.	Prophylactic and therapeutic	Intravenous/subcutaneous application	Tyzzer (1923) and Sautter & Pomeroy (1950)
		Atoxyl	n.i.	Therapeutic	Intravenous/intramuscular application	Tyzzer (1923)
		Acetarsone	n.i.	Prophylactic and therapeutic	Feed	Blount (1938), Farmer (1950), Sautter & Pomeroy (1950) and Swales (1950)
		Mapharside (mapharsen)	n.i.	Prophylactic, non-therapeutic	Intramuscular application	Blount (1938), McCulloch & Nicholson (1941) and Jaquette & Marsden (1947)
		Nitarsone (4-nitro-phenyl-arsonic acid)	n.i.	Prophylactic	Feed and water	McGuire & Morehouse (1952) and Duffy et al. (2004)
		Carbarsone (p-ureidobenzenearsonic acid)	n.i.	Prophylactic	Feed	Welter & Clark (1961), Whitmore et al. (1968) and Bowen et al. (1971)
		Roxarsone (3-nitro-phenyl-arsonic acid)	Yes	No	Feed	Callait et al. (2002) and Hu & McDougald (2002)
		Sodium acetarsol	No	Prophylactic	Feed and water	Farmer (1950), Berks & Neal (1952), Sautter et al. (1952) and Swales (1952a)
Nitrofuran	Damage of DNA	Furazolidone	Yes	Therapeutic	Feed	Jowett (1911), McGregor (1953), Costello (1956), Horton-Smith & Long (1957), Jerstad (1957) and Vatne et al. (1969b)
		Furoxone	n.i.	Prophylactic	Feed	Costello & DeVolt (1956)
		Nihydrazone	n.i.	Prophylactic	Feed	Reid et al. (1960)
		Nifursol	n.i.	Prophylactic	Feed	Sullivan et al. (1972) and Vatne et al. (1969b)
		Nifurtimox	Yes	Prophylactic	Feed	Hauck et al. (2010a)
Nitroimidazole	Damage of DNA	Dimetridazole (1,2-dimethyl-5-nitroimidazole)	Yes	Prophylactic and therapeutic	Feed and water	McGuire et al. (1964), Flowers et al. (1965), Morehouse et al. (1968) and Hu & McDougald (2004)
		Metronidazole (1-(2′-hydroxyethyl)-2-methyl-5-nitroimidazole)	Yes	Prophylactic and therapeutic	Water	Lindquist (1962), Hegngi et al. (1999) and Hu & McDougald (2004)
		Ipronidazole	n.i.	Prophylactic and therapeutic	Feed	Mitrovic et al. (1968) and Mitrovic & Schildknecht (1970)
		Ronidazole (1-methyl-2-carbamoyloxymethyl-5-nitroimidazole)	n.i.	Prophylactic	Water	Whitmore et al. (1968), Peterson (1968), Shelton & McDougle (1970) and Sullivan et al. (1977)
		Ornidazole	Yes	Therapeutic	Feed	Hu & McDougald (2004)
		Tinidazole	Yes	Therapeutic	Feed
Benzimidazole	Binding on beta-tubulin, damage of cytoskeleton, interference of spindly apparatus, inhibition of cell division	Albendazole	No	Prophylactic	Direct oral application	Hegngi et al. (1999), Callait et al. (2002), Hu & McDougald (2004) and Hauck & Hafez (2009)
		Fenbendazole	No	Prophylactic	Direct oral application	Hegngi et al. (1999), Callait et al. (2002) and Hauck & Hafez (2009)
		Flubendazole	No	n.i.	n.i.	Hauck & Hafez (2009)
		Mebendazole	No	n.i.	n.i.	Hu & McDougald (2004) and Hauck & Hafez (2009)
		Nocodazole	No	n.i.	n.i.	Hauck & Hafez (2009)
Nitrothiazole		Enheptin-T (2-amino-5-nitrothiazole)	Yes	Prophylactic and therapeutic	Feed	Swales (1950), Waletzky et al. (1950), Berks & Neal (1952), McGregor (1952), Grumbles et al. (1952), Swales (1952b), Cooper & Skulski (1957), Horton-Smith & Long (1957), and Pino et al. (1955)
		Entramin A (2-acetylamino-5-nitrothiazole)	n.i.	Prophylactic and therapeutic	Feed	Grumbles et al. (1952), Brander & Wood (1955) and Price & Gingher (1956)
Benzothiazole	Inhibition of catalases and peroxidases	p-(6-nitro-2-benzothiazolylamino) benzoic acid	n.i.	Non-therapeutic	Feed	Swales (1952a)
		2-amino-6-nitrobenzothiazole	n.i.	Non-therapeutic	Feed
		p-(2-benzothiazolylamino) benzoic acid	n.i.	Non-therapeutic	Feed
		4-(4-nitrobenzylamino) benzoic acid	n.i.	Non-therapeutic	Feed
Nithiazide		Nithiazide (1-ethyl-3-(5-nitro-2-thiazolyl) urea)	n.i.	Prophylactic and therapeutic	Feed and water	Cuckler & Malanga (1956)
Hydroxyquinoline	Inhibition of transcription	Clioquinol (5-chloro-8-hydroxy-7-iodoquinoline)	Yes	Prophylactic	Feed	Wedgeforth & Wedgeforth (1921), De Volt & Holst (1948), Farmer (1950) and Berks & Neal (1952)
		5-chloro-8-hydroxyquinoline	n.i.	Prophylactic	Feed	De Volt & Holst (1948)
		5,7-diiodo-8-hydroxyquinoline	No	No	Feed	Swales (1950) and Hu & McDougald (2004)
Quinaldinic acid	n.i.	8-amino-5,7-diiodoquinaldinic acid	n.i.	Therapeutic	Feed	Swales (1950)
Dithiocarbamate	Inhibition of superoxide dismutases and metalloproteinases	Arsenic diethyldithiocarbamate	n.i.	Therapeutic	Feed	Swales (1950)
		Bismuth pentamethylene dithiocarbamate	n.i.	Therapeutic	Feed
		Sodium diethyl dithiocarbamate	n.i.	Therapeutic	Feed
Hydrazone	n.i.	Nidrafur (5-nitro-2-furaldehyde acetylhydrazone)	n.i.	Prophylactic	Feed	Reid et al. (1960) and Hall et al. (1965)
Phanquinone	n.i.	4,7-phenanthroline-5,6-quinone	n.i.	No	Feed	Hall et al. (1965)
Luminal amebicide	n.i.	Furamide (diloxanide furoate)	No	n.i.	n.i.	Hu & McDougald (2004)
Aminoglycoside antibiotic	Inhibition of protein synthesis	Paromomycin sulfate	Yes	Prophylactic	Feed	Lindquist (1962), Hu & McDougald (2004), Bleyen et al. (2009a), Hafez et al. (2010) and van der Heijden et al. (2011)
		Streptomycin	Yes	No	Intramuscular application	Swales (1950) and Berks & Neal (1952)
Pleuromutilin antibiotic	Inhibition of protein synthesis	Tiamulin	Variable	Prophylactic	Water	Burch et al. (2007), Hauck et al. (2010b) and van der Heijden et al. (2011)
Quinoxaline-di-N-oxide antibiotic	Intercalation and induction of mutations in DNA	Carbadox (methyl (2E)-2-[(1,4-dioxidoquinoxalin-2-yl) methylene]hydrazinecarboxylate)	Yes	No	Feed	Hu & McDougald (2004)

Abbreviation: n.i., not indicated.

Arsenicals. The first compounds which were used efficiently for the prevention and treatment of histomonosis were arsenicals. Interference with DNA repair processes and cellular energy metabolisms are mechanisms of these compounds and have a lethal effect on cells (Cobo & Castineira, 1997; Abernathy et al., 1999). Substances like neoarsphenamine and tryparsamide were found to be potent as therapeutics (Tyzzer, 1923), whereas other compounds such as mapharside and nitarsone were more suitable for prevention by using them as feed additives (Tyzzer, 1923; Jaquette & Marsden, 1947). Further investigations on nitarsone revealed a clinical relapse after withdrawal of medication (McCulloch & Nicholson, 1941; Morehouse & McGuire, 1950; McGuire & Morehouse, 1952) and continuous use of the drug could affect egg production and growth rates of birds (Moreng & Bryant, 1956). Recently, a reduced sensitivity of H. meleagridis against nitarsone was observed by in vitro assays following isolation from drug-fed turkeys (Abraham et al., 2014). The efficacy of the arsenical compound acetarsol against H. meleagridis was not consistently proven based on different experimental settings (Farmer, 1950; Berks & Neal, 1952; Swales, 1952a).

Nitrofurans. The mode of action of nitrofurans depends on the 5-nitro group of the furan ring. The substance damages the DNA of microbes by its reduction to multiple reactive intermediates inside the cells by the enzyme nitrofuran reductase (Zolla & Timperio, 2005; Jin et al., 2011). Different studies demonstrated a preventive effect of nifursol and nifurtimox against histomonosis (Sullivan, 1972; Vatne, 1969; Vatne, 1969; Hauck, 2010). Therapeutic intervention using furazolidone, another representative of the nitrofurans, was less effective against the disease (Jerstad, 1957; Vatne et al., 1969a, b).

Nitroimidazoles. The active part of the nitroimidazoles is the 5-nitro functional group of the imidazole ring which damages the DNA of the parasite (Lopez Nigro & Carballo, 2008; Boechat et al., 2015). The compounds serve as antibiotics as well as antiprotozoal medications. Dimetridazole especially was used for a long time for the therapy of histomonosis because of its good curative efficacy in chickens and turkeys (Flowers, 1965; Morehouse, 1968). However, other nitroimidazole compounds such as metronidazole, ipronidazole and ornidazole were also found to be effective against histomonosis (Lindquist, 1962; Mitrovic & Schildknecht, 1970; Hu & McDougald, 2004). Other studies showed the application of nitroimidazoles as food and/or water additives for prevention as well as treatment (Lindquist, 1962; Mitrovic & Schildknecht, 1970; Hu & McDougald, 2004).

Benzimidazoles. These bicyclic compounds consist of the fusion of benzene and imidazole. They interfere with the cells of intestinal worms by binding to tubulin proteins, causing damage to the cytoskeleton (Lacey & Gill, 1994; Jornet et al., 2016). Oral application of albendazole and fenbendazole to turkeys infected with H. gallinarum eggs carrying histomonads had a prophylactic effect (Hegngi et al., 1999). However, a direct effect of benzimidazoles against H. meleagridis can be excluded based on the results of in vitro studies (Callait et al., 2002; Hauck & Hafez, 2009). Therefore, it can be concluded that the anthelmintic effect of benzimidazoles reduces the risk of poultry infection with heterakis-harbouring H. meleagridis.

Nitrothiazoles. Nitrothiazoles are heterocyclic compounds that contain both sulphur and nitrogen. 2-amino-5-nitrothiazole and 2-acetylamino-5-nitrothioazole were successfully used for the prevention and treatment of histomonosis (McGregor, 1952; Swales, 1952b; Brander & Wood, 1955). Beside the direct impact of the compounds of this group on histomonads, investigations also described an antihelmintic effect of the substance against the larvae of H. gallinarum (Swales, 1952b).

Benzothiazoles. Members of these bicyclic compounds are thiazoles fused with benzene derivatives. Tests of different benzothiazoles such as 2-amino-6-nitrobenzothiazole as feed additives showed neither therapeutic nor prophylactic efficacy against histomonosis (Swales, 1952a).

Nithiazide. Nithiazide is a compound related to 2-amino-5-nitrothiazole. Mono-substituted nitrothiazolyl urea with 1-ethyl was found to be more effective and less toxic than other substituted nitrothiazolyl ureas and enheptin-T when used as feed additive to prevent histomonosis produced either by oral infection of turkeys with H. gallinarum eggs containing histomonads or by cloacal inoculation of an H. meleagridis culture (Cuckler & Malanga, 1956; Cuckler et al., 1956, 1957).

Quinolines. 8-hydroxyquinoline complexes exhibit antiseptic, disinfectant and pesticide properties based on functioning as a transcription inhibitor (Fraser & Creanor, 1975; Cameron et al., 2012). Their prophylactic activity was demonstrated against natural as well as induced infections of turkeys with histomonads (De Volt & Holst, 1948). In vitro studies demonstrated the inhibition of the growth of histomonads by the addition of clioquinol (Berks & Neal, 1952).

Carbamates. Dithiocarbamates are organic compounds derived from carbamic acid in which both oxygen atoms are replaced by sulphur atoms. Based on the effect of chelating metal ions (e.g. zinc), diethyldithiocarbamates inhibit enzymes such as superoxide dismutases and metalloproteinases (Hogarth, 2012). In vivo tests showed that these substances had the potential to treat histomonosis (Swales, 1950).

Hydrazones, phanquinones and luminal amebicides. Nidrafur was found to have a preventive potential when used as feed additive in the feed of chickens and turkeys (Reid et al., 1960; Hall et al., 1965), but therapeutic effects of the same substance and related compounds entobex and furamide were not observed (Hall et al., 1965; Hu & McDougald, 2004).

Antibiotics. In the past, antibiotics that were initially used against bacterial infections were also investigated for their antihistomonal effect. In this context, it should be mentioned that paromomycin sulphate was tested effectively against protozoan parasites, such as Entamoeba histolytica and other intestinal protozoa (Carter et al., 1962). Paromomycin has an antimicrobial effect by inhibiting the protein synthesis of target organisms (Eustice & Wilhelm, 1984; Mehta & Champney, 2003). Lindquist (1962) found that the drug was effective in preventing histomonosis, but application after infection of turkeys did not reduce the number of losses. Later on, further in vivo studies confirmed these outcomes despite a reduction of cultivated histomonads by in vitro experiments (Hu & McDougald, 2004; Bleyen et al., 2009a; Hafez et al., 2010; van der Heijden et al., 2011). Furthermore, tiamulin, a pleuromutilin antibiotic, was reported to be effective in a field observation (Burch et al., 2007) and was investigated for an anti-histomonad effect by in vitro assays (Hauck et al., 2010b; van der Heijden et al., 2011). However, the results of the last mentioned studies were not consistent, arguing for further investigations using in vivo experiments. Carbadox, which leads to intercalation and inclusion of mutations in the DNA of susceptible bacteria (Chen et al., 2008), was shown to reduce the growth of histomonads in vitro, but a reduction of liver and caecal lesions could not be observed in turkeys (Hu & McDougald, 2004). However, as the propagation of H. meleagridis strongly depends on the presence of bacteria (Bradley & Reid, 1966), it needs to be clarified whether an inhibitory effect targeted the protozoan or only the bacteria and with influence on histomonads.

Legislation on the use of drugs against histomonosis

The application of drugs for food-producing birds is regulated in many countries by national authorities in order to exclude or minimize side effects and to ensure consumer protection. Thus, there are major disparities between different countries on whether drugs can be applied to farm birds or not. International guidelines for food safety including veterinary drug residues in food are outlined in the Codex Alimentarius of the Food and Agriculture Organization of the United Nations and the World Health Organization. Regarding antihistomonals, it is clearly recommended not to use nitroimidazoles such as metronidazole or dimetridazole in food-producing birds (CAC, 2013). In the European Union (EU), decisions concerning food safety determined by European parliament are implemented by the member countries. The use of nitroimidazoles was forbidden by Commission Regulation No 1798/95 (CEC, 1995) and later on nifursol, the only available preventive drug against histomonosis, was banned by Council Regulation No 1756/2002 (CEC, 2002). Arsenicals used for prevention against histomonosis have never been registered in the EU according to the list in the Annex of European Medicines Agency on Maximum Residue Limits (MRL) of drugs (EMA, 2009). It is given by legislation that non-EU countries have to provide guarantees on the residue status of exported products into the EU for banned substances including nitroimidazoles and nitrofurans according to Directive 96/23/EC (CEC, 1996). These regulations implicate that food being imported fulfils the same specifications as domestic products. In the USA, the application of nitroheterocyclic compounds in food-producing birds is also not allowed (FDA, 2015a) and just recently, the approval for the use of the arsenical nitarsone in bird feed has been withdrawn based upon suspicions by the manufacturer (FDA, 2015b). Similar regulations that resulted in the loss of any preventive or therapeutic options against histomonosis were applied in other countries, such as Japan, Canada, Australia and New Zealand, that are directly or indirectly involved in the International Cooperation on Harmonisation of Technical Requirements for the Registration of Veterinary Medicinal Products (VICH). Furthermore, it was elsewhere described that many other countries of the Organisation for Economic Co-operation and Development have no approval for the application of nitroimidazoles in food-producing birds (Baynes et al., 2016). Despite this, the application of drugs against histomonosis is still permitted in most other countries. However, South Africa, China, Brazil, Argentina, Russia, India, the West African Economic and Monetary Union, the Veterinary Medicines Committee of the Americas and others attended one of the VICH Global Outreach Forums for the purpose of raising awareness of VICH guidelines among non-VICH countries (Smith, 2013). This indicates that laws, regulations, guidance and policies relevant to the control of veterinary medicines are of increasing relevance and, therefore, ban of antihistomonal drugs in food-producing birds may be implemented in future in additional countries. As a consequence of this, it can be hypothesized that the re-emergence of histomonosis may extend to a severe problem in different countries. The following case histories should demonstrate examples of the impact of histomonosis in different farms and restricted veterinary interventions according to European legislation.

Actual cases of histomonosis with restricted veterinary medical care

In an event of recent outbreaks of histomonosis in turkey flocks in Austria, measures had to be taken to prevent prolonged suffering of the birds or, if possible, the fatal outcome. In total, 10 outbreaks of histomonosis affecting seven different farms of commercial turkeys with mostly severe consequences have been recorded in a period from July 2014 until September 2015, of which three cases are briefly described below.

Case 1: Commercial turkeys were reared in three neighbouring houses owned by two different farmers located in an area of a few hundred square metres. The first outbreak occurred in the middle house populated with male turkeys at the age of 8 weeks, and 5000 of 7200 birds died within 9 days after the first appearance of clinical signs before the remaining birds were slaughtered. Four months later, birds in the neighbouring house belonging to the second farmer became affected. At this time, the house contained 2700 male and the same number of female turkeys separated by a simple wire fence. At the age of 5 weeks, typical clinical signs were noticed in male turkeys before a large number of them died. In order to avoid suffering of the remaining stags and to reduce the shedding of the parasite, all male turkeys were culled. However, one week later, the hens also had to be killed after they started to contract histomonosis. The third outbreak occurred in the so far unaffected third house containing different compartments with 3200 stags and 5000 hens at the age of 7 weeks, just 1 month after the second outbreak. This time all turkeys in this house were immediately slaughtered to prevent the rapid spread of the disease within the flock.

Following the first outbreak in the series of three, enhanced biosecurity measures were applied after depopulation, including sweeping and washing with water and detergents, formalin gassing and iodine-based disinfection in doubled concentrations along with insecticides and anticoccidial agents in the houses, silos and litter storages, together with spreading of lime around the premises. Additionally, herbal products Protophyt^® (Phytosynthese, Mozac, France) and/or Sangrovit^® (Phytobiotics, Eltville, Germany) were applied in the feed.

In a subsequent incidence of histomonosis in the middle house, 10 months after the last outbreak on this farm, 7500 female turkeys were affected at their fifth week of life. This time paromomycin sulphate (Parofor^®, Huvepharma, Sofia, Bulgaria) was administered via drinking water in a dosage of 12.5 mg/kg bodyweight for 10 days immediately after the birds showed clinical signs of histomonosis. Following treatment, only marginal losses due to histomonosis of overall eight birds were observed until the flock was slaughtered at the age of 16 weeks.

Case 2: The flock consisted of 11,500 turkey hens that showed adverse clinical signs and an increased mortality at week 7 of life. After histomonosis was confirmed by pathology and direct detection of the parasite, paromomycin sulphate (Parofor^®) was applied at week 9 of life with the same dose as mentioned above. However, the administration of the drug was stopped after 5 days of usage, because the daily mortality could not be reduced. Overall, 42% of the birds had died by the end of production 6 weeks later and 254 carcasses had to be condemned during slaughter.

Case 3: Histomonosis occurred in a house with 5100 male and 6900 female turkeys separated by a wire fence. Mortalities already started at 21 days of age in female turkeys, after which they were culled. Thereafter, the male birds started to show clinical signs and paromomycin sulphate (Parofor^®; 12.5 mg/kg bodyweight) was administered via the drinking water on the next day. The mortality was moderate in the first 4 days of treatment before a sudden increase in mortalities was noted and the birds had to be culled.

The short summary of field outbreaks of histomonosis given above did not provide encouraging results in regard to the actions taken to control histomonosis with currently available options. The importance of hygiene and biosecurity to prevent histomonosis is described specifically below; but as exemplified in the mentioned cases, it was not sufficient to prevent histomonosis. Paromomycin sulphate is allowed for “off-label” use in turkeys in Europe against histomonosis after the disease is diagnosed. This restriction in the application of the drug is supported by the development of resistant enteric bacteria in turkeys following administration as a continuous feed supplement (Kempf et al., 2013). However, as the drug has only a prophylactic and not a therapeutic effect, this procedure offers a certain dilemma. In any case, timely diagnosis and application are crucial to induce a metaphylactic effect against histomonosis.

Alternative and recent approaches to control the disease

Hygiene and biosecurity

The introduction of H. meleagridis into a poultry flock determines the outbreak of the disease; therefore, it is essential to reduce the risk of an initial infection of birds by measures of biosecurity. Since the parasite effectively multiplies in infected birds, transmission within a flock can occur rapidly, as was demonstrated experimentally (McDougald & Fuller, 2005; Hess et al., 2006; Landman et al., 2015). The knowledge how histomonads can be introduced into a flock is highly relevant. The unsheltered parasite has a low tenacity and survival time outside its hosts is relatively short. However, the parasite was shown to survive in contaminated water or faecal material for up to 9 hours (Lotfi et al., 2012), a time period in which transmission can occur also between houses or farms by indirect transmission. The most effective way to transmit the parasite is via the intermediate host H. gallinarum which can harbour H. meleagridis (Graybill & Smith, 1920). Eggs of the caecal worm are highly resistant against environmental impacts and can be infective for several years. Furthermore, earthworms are vectors for H. gallinarum and are accordingly a potential source of H. meleagridis (Lund et al., 1966b), which underlines the requirement for a proper biosecurity. Additionally, preventive measures to reduce the prevalence of H. gallinarum include the accurate control of the worm. Regular parasitic control is important to immediately start with deworming procedures in order to minimize H. gallinarum. Between bird replacements, cleaning and disinfection with substances effective against worm eggs are indicated. Disinfectants containing peracetic acid fulfil this requirement (van der Gulden & van Erp, 1972). All sources infected or contaminated with H. gallinarum must be kept away from poultry. This includes other birds, for instance pheasants that are known to transmit both parasites (Lund & Chute, 1972). Furthermore, insects, rodents and other vermin that can be considered to act as mechanical vectors must be strictly controlled. Litter contaminated with H. meleagridis and H. gallinarum was shown to induce the disease, and therefore, all the equipment and clothes that come in contact with it are potential vectors. The detection of histomonad-DNA in dust samples by polymerase chain reaction (PCR) was shown to be highly suitable for investigating the prevalence of the parasite in a poultry flock (Grafl et al., 2015). However, dust does not seem to be a potential source of infection because PCR-positive dust did not contain microscopically detectable cells of H. meleagridis and was not infective for turkeys in a preliminary experimental setting (Liebhart et al., 2013a).

Anyhow, the effect of biosecurity to prevent the introduction of histomonads depends on the housing system and is very much limited in free-range flocks or in houses that cannot be fully closed. This is consistent with the finding that the seroprevalence of H. meleagridis is higher in pullets and layers kept in free-range systems compared to chickens that are housed on deep litter (Grafl et al., 2011). Although biosecurity is important to reduce the risk of an outbreak in a chicken or turkey flock, it is not always possible to prevent the infection with H. meleagridis as described above and as reported previously from breeder flocks of turkeys and chickens, where a high level of biosecurity can be assumed (Callait-Cardinal et al., 2007; Dolka et al., 2015). Birds kept on free range are more likely to be exposed to parasites, although there are certain factors that influence the occurrence or severity of histomonosis. In a recent study, significant relationships of flock management and histomonosis were found (Callait-Cardinal et al., 2010). Most importantly, it was reported that mortality tended to be high when low hygiene and acidification of the drinking water were practised. However, more data are needed to identify possible entrance routes of the parasite into a flock and general measures to prevent or limit the infection.

Plant-derived compounds

Several compounds from plants were tested for their effect against H. meleagridis by applying in vitro and/or in vivo experiments (Table 2). Relevant parameters for in vitro studies were the growth rate and the inhibition of multiplication of the parasite. The impact of the used substances was often defined as the minimum lethal concentration. For infection trials, mostly turkeys were used to investigate clinical parameters and pathological changes in order to conclude a possible antihistomonal effect. In the following subsections, the different plant-derived compounds are categorized according to the type of extract or the chemical classes and the most relevant findings are briefly summarized.

Table 2. Plant-derived compounds investigated for their effect against H. meleagridis in vitro and/or in vivo.

Herbal form	Plant (commercial product)	Ingredient	In vitro effect	In vivo effect	Administration via	Reference
Oil	Cinnamomum aromaticum	Cinnamaldehyde	Yes	n.i.	n.i.	Zenner et al. (2003) and Grabensteiner et al. (2007)
	Citrus limon pericarps	Terpene	Yes	n.i.	n.i.	Zenner et al. (2003)
	Allium sativum	Diallyl tri- and disulphide	Yes	n.i.	n.i.
	Thymus vulgaris, Rosmarinus officinalis	Benzene, hydroxycinnamic acid, phenol, terpene	Yes	n.i.	n.i.	Grabensteiner et al. (2007)
	Unspecified	Carvacrol (terpene)	Yes	n.i.	n.i.
	Cinnamomum aromaticum, Allium sativum, Rosmarinus officinalis, Citrus limon (Protophyt®)	Cinnamaldehyde, diallyl tri- and disulfide, hydroxycinnamic acid, terpene	Yes	Inconsistent	Feed and water	Hafez & Hauck (2006), van der Heijden & Landman (2008a) and van der Heijden & Landman (2008b)
	Origanum vulgare, Cinnamomum aromaticum, Thymus vulgaris, Citrus limon, Capsicum annum (RepaXol®)	Alkaloid, benzene, cinnamaldehyde, phenol, terpene	Yes	n.i.	n.i.	Hauck & Hafez (2007)
	Cinnamomum aromaticum, Thymus vulgaris, Citrus limon, Capsicum annum	Alkaloid, cinnamaldehyde, benzene, phenol, terpene	Yes	n.i.	n.i.
Ethanol extract	Thymus vulgaris	Benzene, phenol, terpene	Yes	No	Water	Grabensteiner et al. (2008)
	Serenoa repens	Fatty acid, flavonoid, sterol	Yes	No	Water
	Vitis vinifera	Flavonoid, phenol, tannin	Yes	No	Water
	Cucurbita pepo	Amino acid, sterol, tocopherol	Yes	No	Water
	Cynara scolymus	Flavonoid, phenol, tannin	Yes	n.i.	n.i.
	Vaccinium myrtillus	Flavonoid, phenol, tannin	Yes	n.i.	n.i.
	Daucus carota	Polyacetylene	Yes	n.i.	n.i.
	Echinacea purpureae	Hydroxycinnamic acid	Yes	n.i.	n.i.
	Linum usitatissimum	Fatty acid, glycoside, phenols	Yes	n.i.	n.i.
	Mangifera indica	Benzole, flavonoid, phenolic, terpene	Yes	n.i.	n.i.
	Olea europaea	Fatty acid, flavonoid, phenolic acid	Yes	n.i.	n.i.
	Salix alba	Glycoside	Yes	n.i.	n.i.
	Helianthus annuus	Fatty acid	Yes	n.i.	n.i.
	Lycopersicum esculentum	Alkaloid, flavonoid, phenolic acid, terpene	Yes	n.i.	n.i.
	Crataegus oxyacantha	Flavonoid, oligomeric proanthocyanidin phenolic acid, tannin	Yes	n.i.	n.i.
	Primula veris, Gentiana lutea, Sambucus nigra, Rumex species, Verbena officinalis (Sinupret^®)	Fatty acid, glycoside, flavonoid, saponin, tannin, terpene	Yes	n.i.	n.i.
	Peganum harmala	Alkaloid	Yes	n.i.	n.i.	Arshad et al. (2008)
Water extract	Thymus vulgaris	Benzene, phenol, terpene	Yes	n.i.	n.i.	Grabensteiner et al. (2008)
	Serenoa repens	Fatty acid, flavonoid, sterol	Yes	n.i.	n.i.
	Vitis vinifera	Flavonoid, phenol, tannin	Yes	n.i.	n.i.
	Cucurbita pepo	Amino acid, sterol, tocopherol	Yes	n.i.	n.i.
	Cynara scolymus	Flavonoid, phenol, tannin	Yes	n.i.	n.i.
	Vaccinium myrtillus	Flavonoid, phenol, tannin	Yes	n.i.	n.i.
	Daucus carota	Polyacetylene	Yes	n.i.	n.i.
	Ginkgo biloba	Flavonoid, terpene	Yes	n.i.	n.i.
	Aesculus hippocastanum	Saponin	Yes	n.i.	n.i.
	Linum usitatissimum	Fatty acid, glycoside and phenol	No	n.i.	n.i.
	Mangifera indica	Benzole, flavonoid, phenolic, terpene	Yes	n.i.	n.i.
	Olea europaea	Fatty acid, flavonoid, phenolic acid	No	n.i.	n.i.
	Helianthus annuus	Fatty acid	Yes	n.i.	n.i.
	Lycopersicon esculentum	Alkaloid, flavonoid, phenolic acid, terpene	Yes	n.i.	n.i.
	Crataegus oxyacantha	Flavonoid, oligomeric phenolic acid, proanthocyanidin, tannin	Yes	n.i.	n.i.
Extract	unspecified (Natustat^™)	Unspecified active components of plants, yeast-derived mannanoligosaccharide, organic mineral nutrients	n.i.	Prophylactic	Feed	Duffy et al. (2004) and Duffy et al. (2005)
	Citrus x aurantium	Alkaloid, glycoside	Yes	n.i.	n.i.	Hauck & Hafez (2007)
Lyophilisate	Allium sativum, Cinnamomum aromaticum (Enteroguard^™)	Cinnemaldehyde, diallyl tri- and disulfide	Yes	No	Feed	van der Heijden & Landman (2008a) and van der Heijden & Landman (2008b)
	Tropical plants (Aromabiotic^™)	Unspecified active components of plants, fatty acid	No	n.i.	n.i.	van der Heijden & Landman (2008b)
β-Carboline alkaloid	Peganum harmala	Harmane	Yes	n.i.	n.i.	Arshad et al. (2008)
		harmalol hydrocholoride dehydrate	Yes	n.i.	n.i.
		Harmaline	Yes	n.i.	n.i.
		Harmine	Yes	n.i.	n.i.
Steroidal glycoalkaloid	Q. saponaria	Saponins	Yes	n.i.	n.i.	Grabensteiner et al. (2007)
Plant material	A. annua	Artemisinin	Yes	n.i.	n.i.	Thofner et al. (2012)
Sesquiterpene lacton			Yes	No	Feed
Hexane extract			Yes	n.i.	n.i.
Dichloromethane extract			Yes	No	Water
Methanol extract			No	n.i.	n.i.

Abbreviation: n.i., not indicated.

Plant-derived essential oils. Essential oils obtained from different plants such as cinnamon, lemon, rosemary, garlic and thyme were tested for their potential as medication against histomonosis. The oils were able to suppress the growth of the parasites when added to in vitro cultures of histomonads (Zenner et al., 2003; Grabensteiner et al., 2007; Hauck & Hafez, 2007; van der Heijden & Landman, 2008a). The commercial product Protophyt^® (Phytosynthese) contains the essential oils of cinnamon, garlic, rosemary and lemon and was previously surveyed for its antihistomonal effect in different studies (Hafez & Hauck, 2006; van der Heijden & Landman, 2008b). However, the results were very contradictory, ranging from a certain prophylactic activity to no efficacy.

Ethanol and water extracts. Tests of ethanol and water extracts derived from different plants such as Thymus vulgaris, Vitis vinifera, Olea europaea, Peganum harmala, Ginkgo biloba and Aesculus hippocastanum showed an influence on the growth behaviour of histomonads in in vitro cultures, whereas extracts used as water additives for turkeys experimentally infected with H. meleagridis did not have any appreciable efficacy regarding prevention of histomonosis (Arshad et al., 2008; Grabensteiner et al., 2008).

Further extracts and lyophilisates. The product Natustat^™ which contains unspecified plant extracts was described to have an effect against histomonosis in turkeys and chickens kept on infected litter (Duffy et al., 2004, 2005). However, investigations on this plant-derived feed additive using a well-defined infection model are not available. Other commercial products such as the lyophilic products Enteroguard^™ and Aromabiotic^™ could not prevent experimental histomonosis in turkeys, although an in vitro effect of Enteroguard^™ was observed (van der Heijden & Landman, 2008a, 2008b).

Alkaloids and sesquiterpene lactones. In vitro investigations using the β-carboline alkaloids harmane, harmalol, harmaline and harmine, as well as the steroidal glycoalkaloid saponin derived from the plant Quillaja saponaria, demonstrated the effectiveness of these substances on the growth rate of histomonads (Grabensteiner et al., 2007; Arshad et al., 2008). In the same way, the sesquiterpene lactone artemisinin and material of Artemisia annua, as well as extracts derived from this plant, showed the potential to suppress the growth of histomonads in culture (Thofner et al., 2012). On the other hand, the addition of artemisinin and a dichloromethane A. annua extract to feed or water, respectively, was not suitable to prevent chickens or turkeys from histomonosis (Thofner et al., 2012).

Vaccination

Investigations on vaccination against histomonosis include several studies, starting already at the beginning of the last century with experiments that assessed differences in the virulence of H. meleagridis, until the recent establishment of a well-defined experimental vaccination model for turkeys and chickens (Table 3). During the first attempts to isolate H. meleagridis in vitro followed by in vivo application, it was found that the parasite lost its virulence by long-term cultivation (Tyzzer, 1934). In a subsequent manuscript, Tyzzer (1936) summarized all of his immunization experiments describing a certain protective effect of in vitro attenuated histomonads in chickens and turkeys, but found no uniformity of attenuation in regard to the duration of cultivation between different isolates. He also observed in this study that immunizing properties decreased during extended time of cultivation in vitro. Furthermore, it was found that the pathogenicity of long-term in vitro attenuated histomonads could not be restored by serial passages in chickens or turkeys (Tyzzer, 1936; Dwyer & Honigberg, 1970). Nevertheless, the investigations at that time did not result in the development of an efficient vaccine.

Table 3. Vaccination studies performed to prevent histomonosis.

Bird species	Age of birds	Vaccination with	Vaccination route	Vaccine dose	Side effects of the vaccine	Challenge	Challenge dose	Challenge time post-vaccination	Protection^a	Reference
Turkey	2–55 days	In vitro attenuated H. meleagridis	Cloacal	n.i.	Death of several birds	Virulent isolate or kept on contaminated soil	n.i.	0–125 days	Partial	Tyzzer (1936)
Chicken	1–47 days	In vitro attenuated H. meleagridis	Cloacal	n.i.	n.i.	Virulent isolate	n.i.	10–46 days	Partial
Turkey	6 weeks	Non-pathogenic strain directly or via eggs of H. gallinarum	Cloacal: isolate of H. meleagridis; oral: eggs of H. gallinarum containing H. meleagridis	1 × 10^4–4 × 10^4 H. meleagridis repeatedly or 160–162 eggs of H. gallinarum	n.i.	Virulent isolate directly or via eggs of H. gallinarum or kept on contaminated soil	1 × 10^4–6 × 10^4 H. meleagridis repeatedly or 162–200 eggs of H. gallinarum or not defined	3–10 weeks	Partial	Lund (1959)
Chicken and turkey	3 weeks	Antiserum	Intra-peritoneal	2–3 ml of antiserum	n.i.	Virulent isolate	n.i.	1 day	None	Clarkson (1963)
Turkey	5 weeks	In vitro attenuated H. meleagridis	Cloacal	5.6 × 10^3–2 × 10^5 repeatedly	n.i.	Virulent isolate	1.25 × 10^4–2 × 10^5 H. meleagridis	3 weeks	Partial
Turkey		In vitro attenuated H. meleagridis	Cloacal	3 × 10^5–5 × 10^5 H. meleagridis repeatedly	n.i.	Virulent isolate	1.25 × 10^4–2 × 10^5 H. meleagridis	3 weeks	Partial	Lund et al. (1967)
Turkey	2 weeks	In vitro attenuated H. meleagridis	Cloacal	1 × 10^4 H. meleagridis	No clinical signs	Virulent isolate	1 × 10^4 H. meleagridis	4 weeks	Full	Hess et al. (2008)
Turkey	1 and 2 weeks	Inactivated H. meleagridis	Intramuscular	∼2.3 × 10^6 inactivated H. meleagridis	No clinical signs	Virulent isolate	1 × 10^4 H. meleagridis	3–4 weeks	None
Turkey	3 and 6 weeks	Inactivated H. meleagridis	Intramuscular	250 µg of protein	No clinical signs	Virulent isolate	1 × 10^5 H. meleagridis	4 hours	None	Bleyen et al. (2009b)
Turkey	5 weeks	Antiserum	Intra-peritoneal	4 ml of antiserum	No clinical signs	Virulent isolate	3 × 10^5 H. meleagridis	4 weeks	None
Turkey	1 day	In vitro attenuated H. meleagridis	Oral	1 × 10^4 H. meleagridis	No clinical signs	Virulent isolate	1 × 10^4 H. meleagridis	2–4 weeks	Partial	Liebhart et al. (2010)
Chicken	18 weeks	In vitro attenuated H. meleagridis	Cloacal and oral	2 × 10^4 H. meleagridis	No lesions	Virulent isolate	2 × 10^4 H. meleagridis	5 weeks	Partial	Liebhart et al. (2013b)
Turkey	12 days	In vivo attenuated H. meleagridis	Cloacal	1 × 10^3–1 × 10^5 H. meleagridis	No clinical signs	Virulent isolate	1 × 10^5 H. meleagridis	3 weeks	Full	Nguyen Pham et al. (2013)
Turkey	1 day and 2 weeks	In vivo attenuated H. meleagridis	Cloacal and oral	1 × 10^4–2 × 10^4 H. meleagridis repeatedly	No clinical sings	Virulent isolate	2 × 10^4 H. meleagridis	4 weeks	Full	Sulejmanovic et al. (2016)

Abbreviation: n.i., not indicated.

^aIndicated by mortalities in turkeys or postmortem lesions in chickens.

Later on, Lund (1959, 1963) showed that virulent histomonads did not cause the disease when the birds were initially inoculated with a non-pathogenic strain specified as Histomonas wenrichi. In further experiments, the same author and colleagues (Lund et al., 1966a) observed immunizing properties of H. meleagridis following in vitro cultivation, but they hypothesized that the loss of virulence during cultivation was due to a selection process between different virulent strains of the parasite present in the material used for cultivation. This hypothesis was later disproven by establishing clonal cultures of H. meleagridis by micromanipulation and attenuation by long-term cultivation (up to 295 in vitro passages) (Hess et al., 2008). However, prolonged in vitro attenuation of more than 1000 passages was shown to obliterate the immunogenicity of H. meleagridis (Lund et al., 1967).

Around the middle of the last century, chemotherapy was proven to be effective against histomonosis as described above. At that time, it was also found that birds which recovered from an infection by antihistomonal treatment can develop resistance against histomonosis, supporting the idea of vaccination (Brackett & Bliznick, 1949; Sautter & Pomeroy, 1950; Kendall, 1957). Although Clarkson (1963) confirmed this result, he demonstrated that precipitating antibodies did not protect naive turkeys against a severe challenge. He concluded that protective immunity against histomonosis must be produced by a serum factor, leukocytes or local immunity of the caecal mucosa. In agreement with his observations, Hess et al. (2008) and Bleyen et al. (2009b) were unable to protect turkeys with killed histomonads. In contrast to this, we could achieve full protection from fatal histomonosis by vaccination using a well-defined clonal culture of in vitro attenuated histomonads in turkeys (Hess et al., 2008). In continuation, successful oral administration of attenuated histomonads to day-old turkeys was demonstrated successfully for the first time (Liebhart et al., 2010). The established vaccine candidate was further investigated for its safety, with the outcome that (i) no clinical signs or pathological lesions were found in chickens and turkeys that were vaccinated (Liebhart et al., 2011) and (ii) the strain used for vaccination did not revert to virulence following consecutive passages in both host species, and only minimal lesions were noted (Sulejmanovic et al., 2013). In another study, the vaccine was applied as a monoxenic inoculum which had no effect on the attenuation and efficacy (Ganas et al., 2012). Further experiments in adult layers have shown that vaccinated birds can be protected from a severe drop in egg production caused by histomonosis (Liebhart et al., 2013b). Most recently, cross-protection by the mentioned attenuated clonal strain against genetically different isolates of H. meleagridis was demonstrated in turkeys (Sulejmanovic et al., 2016).

Finally, Nguyen et al. (2013) reported the implementation of serial in vivo passages in turkeys to obtain a low-virulent isolate which protected turkeys from a severe challenge, supporting the concept of a live vaccine.

Outlook on the control of histomonosis

Requirements of new approaches

This review summarized studies on drugs, plant-derived compounds and vaccination that were performed in order to elucidate options for the prevention or treatment of histomonosis. The urgent need for an effective prophylaxis and therapy is evident since several countries banned effective and widely used drugs due to safety concerns. Consequently, new substances as alternatives to be used against the disease have to fulfil comprehensive regulations when used in food-producing birds, as outlined above. The main requirement of chemical agents or its derivates applied against histomonosis is, beside a significant effect against the parasite, the safety of the product. Most important are criteria regulating the prevention of residues or at least the compliance of MRL in eggs or meat of poultry. Furthermore, it should also be excluded that a certain drug induces resistant microorganisms, either the parasite itself or the microbiota in general. Standards and recommendations are given by the Codex Alimentarius. Considering the high demand to fulfil all regulations for a substance to be used in food-producing birds, it seems very unlikely that such a drug will enter the market in the near future.

Botanicals may be applied as feed additive, and therefore, the application is specifically regulated by law, exemplarily given from the EU (EC, 2003). As mentioned above, several plant substances were tested for their effect to prevent histomonosis without substantial impact on the progression and outcome of the disease. It needs to be determined in controlled experiments whether new plant-derived substances have an antihistomonal effect. For instance, approaches to alleviate coccidiosis in poultry with different botanicals were recently summarized (Bozkurt et al., 2013). Anyhow, the characteristics of the parasite H. meleagridis and the pathogenicity of histomonosis require proper investigations to confirm an antihistomonal effect, and well-defined in vitro and in vivo assays are essential for this. In vitro studies are a prerequisite in assessing a potential effect against H. meleagridis and are needed to minimize bird experiments. However, it should be pointed out that in vivo experiments are mandatory to confirm an impact against the disease, considering that various recent investigations noted considerable differences between in vitro and in vivo studies (Table 2).

As already pointed out, vaccination based upon an attenuated clonal strain of H. meleagridis was shown to be highly effective and a live vaccine seems at present the most suitable tool to successfully prevent the disease. However, further efforts are needed to standardize the production and to optimize the administration of the vaccine in the field. It remains questionable whether the introduction of advanced molecular and genomic techniques which were just recently applied to study H. meleagridis will lead to new achievements with regard to vaccine development.

In any case, a better understanding of the host–parasite interaction is needed in order to elucidate the mechanisms of protection. In a recent study, it was shown that that chickens raised an earlier immune response than turkeys in the caecum based on cytokine expression profiles, which could possibly explain the milder course of the disease in chickens (Powell et al., 2009). Considering the severe impact on the gut epithelium, a comparison with Eimeria spp. seems very obvious, although both parasites display a very different biology. Similarities in the immune reactions against coccidia and H. meleagridis include severe inflammation and destruction of intestinal host tissue and the accumulation of cytokines (Rose, 1996; Powell et al., 2009). However, the most relevant distinction is the intracellular presence of Eimeria spp. compared to the intercellular infiltration of H. meleagridis in tissues. This should fundamentally affect the immune cascade during infection. For Eimeria spp., it is known that activation and involvement of T cells occur and they are essential to establish immunity against reinfection (Smith et al., 2014). It is well known that the mammalian immune system can be divided into type 1 and type 2 pathways, depending on the causative pathogen. Birds show a similar range of immune response, as reported by Degen et al. (2005) who demonstrated Th1/Th2 polarization by intracellular (viral) and extracellular (helminth) infection in chickens. However, an experimental helminth infection model is not very reliable because of the different development stages of the nematode (egg – larvae – adult worm). H. meleagridis, an extracellular protozoan parasite that propagates only by binary cell division, should be a good candidate to elucidate the development of the type 2 pathway in chickens, highlighting a broader view to study this pathogen.

Conclusion

Histomonosis is a re-emerging disease in various countries without permission to use effective chemotherapeutics. Different strategies to prevent the disease including the use of chemicals, plant-derived compounds or vaccination were comprehensively investigated in the last years. Chemotherapeutics against histomonosis are not licensed anymore in many countries and compounds from plants did not show promising effects in vivo. Experimental vaccination using in vitro attenuated H. meleagridis effectively protects chickens and turkeys from histomonosis and is safe in use, arguing for the most promising approach for the future prevention of histomonosis in poultry.

Acknowledgements

The authors thank all colleagues from the Clinic for Poultry and Fish Medicine and the Christian Doppler Laboratory for Innovative Poultry Vaccines (IPOV) at the Vetmeduni Vienna for their continuous support. The first and corresponding author would like to extend his sincerest thanks to the Houghton Trust for having the opportunity to present the Avian Pathology Lecture at the XIXth World Veterinary Poultry Association (WVPA) Congress on the reviewed subject.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Substantial funding to support the research on H. meleagridis and histomonosis at the Clinic for Poultry and Fish Medicine was obtained by the establishing the Christian Doppler Laboratory for Innovative Poultry Vaccines (IPOV) and from the Austrian Science Fund (FWF).

Notes

* Part of this review is based on the Avian Pathology Lecture entitled ‘Strategies to prevent histomonosis in poultry’, presented by Dieter Liebhart at the 19th Congress of the World Veterinary Poultry Association in Cape Town, South Africa, September 2015.