Histomonas meleagridis: Immunohistochemical localization of parasitic cells in formalin-fixed, paraffin-embedded tissue sections of experimentally infected turkeys demonstrates the wide spread of the parasite in its host Amarjit Singh a,1 , Herbert Weissenbo ¨ck b , Michael Hess a, * a 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 b Institute of Pathology and Forensic Veterinary Medicine, Department of Pathobiology, University of Veterinary Medicine, Veterina ¨ rplatz 1, A-1210 Vienna, Austria Received 27 August 2007; received in revised form 24 October 2007; accepted 1 November 2007 Available online 17 November 2007 Abstract In the present investigation, a polyclonal antibody-based immunohistochemical technique was developed to localize Histomonas mele- agridis in formalin-fixed, paraffin-embedded tissues of experimentally infected turkeys. The developed technique was highly specific for histomonads as no immunohistochemical reaction was observed with cultures of Tetratrichomonas gallinarum, Trichomonas gallinae and Blastocystis sp. In addition, tissues positive for various other protozoan parasites and fungi were also tested to evaluate the specificity of the technique. It was possible to detect immunohistochemically histomonad antigens in all the tested samples (n = 5) of caecum, liver, spleen and lung from infected turkeys, 3 out of 5 bursa of Fabricius, 1 out of 2 bone marrow, 2 out of 5 heart and 1 out of 5 each of proventriculus, pancreas and cerebellum. An immunohistochemical reaction indicative of presence of histomonads was also detected in blood vessels of various organs that indicated a possible hematogenous route of spread of the parasite in the host. A comparative study with routine diagnostic staining techniques indicated a high sensitivity and specificity of the newly developed immunohistochemical tech- nique. Altogether, the technique developed can be used to study the sequential pathogenesis of histomonosis in turkeys and to obtain new insights into the mechanisms of interaction with the host tissues. Ó 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Histomonas meleagridis; Protozoa; Mono-eukaryotic culture; Polyclonal hyperimmune serum; Immunohistochem- ical technique; Experimental infection; Detection; Diagnosis; Dissemination; Turkey; Spf, specific pathogen free; PCR, polymerase chain reaction 1. Introduction Histomonas meleagridis, a flagellated protozoan parasite of many gallinaceous birds, causes enterohepatitis com- monly known as histomonosis (syn. histomoniasis) or blackhead, which is an economically important disease of poultry that causes losses particularly in turkey flocks due to high mortality (McDougald, 2003; Hess et al., 2004). However, recent reports underline the importance of the disease for chickens, especially for birds kept under free-range conditions or on farms with low or inadequate disinfection procedures (Homer and Butcher, 1991; Esque- net et al., 2003; Cortes et al., 2004). The re-emergence of histomonosis in turkeys and chickens with serious and wide spread outbreaks, more recently in the European commu- nity, due to the increase of free-range housing of poultry 0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.11.004 * Corrersponding author. Fax: +43 1 250775192. E-mail address: [email protected](M. Hess). 1 Present address: Department of Veterinary Pathology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, India. www.elsevier.com/locate/yexpr Available online at www.sciencedirect.com Experimental Parasitology 118 (2008) 505–513
of experimentally infected turkeys demonstrates the widespread of the parasite in its host
Amarjit Singh a,1, Herbert Weissenbock b, Michael Hess a,*
a Clinic for Avian, Reptile and Fish Medicine, Department for Farm Animals and Herd Management, University of Veterinary Medicine,
Veterinarplatz 1, A-1210 Vienna, Austriab Institute of Pathology and Forensic Veterinary Medicine, Department of Pathobiology, University of Veterinary Medicine,
Veterinarplatz 1, A-1210 Vienna, Austria
Received 27 August 2007; received in revised form 24 October 2007; accepted 1 November 2007Available online 17 November 2007
Abstract
In the present investigation, a polyclonal antibody-based immunohistochemical technique was developed to localize Histomonas mele-
agridis in formalin-fixed, paraffin-embedded tissues of experimentally infected turkeys. The developed technique was highly specific forhistomonads as no immunohistochemical reaction was observed with cultures of Tetratrichomonas gallinarum, Trichomonas gallinae andBlastocystis sp. In addition, tissues positive for various other protozoan parasites and fungi were also tested to evaluate the specificity ofthe technique. It was possible to detect immunohistochemically histomonad antigens in all the tested samples (n = 5) of caecum, liver,spleen and lung from infected turkeys, 3 out of 5 bursa of Fabricius, 1 out of 2 bone marrow, 2 out of 5 heart and 1 out of 5 each ofproventriculus, pancreas and cerebellum. An immunohistochemical reaction indicative of presence of histomonads was also detected inblood vessels of various organs that indicated a possible hematogenous route of spread of the parasite in the host. A comparative studywith routine diagnostic staining techniques indicated a high sensitivity and specificity of the newly developed immunohistochemical tech-nique. Altogether, the technique developed can be used to study the sequential pathogenesis of histomonosis in turkeys and to obtain newinsights into the mechanisms of interaction with the host tissues.� 2007 Elsevier Inc. All rights reserved.
Index Descriptors and Abbreviations: Histomonas meleagridis; Protozoa; Mono-eukaryotic culture; Polyclonal hyperimmune serum; Immunohistochem-ical technique; Experimental infection; Detection; Diagnosis; Dissemination; Turkey; Spf, specific pathogen free; PCR, polymerase chain reaction
1. Introduction
Histomonas meleagridis, a flagellated protozoan parasiteof many gallinaceous birds, causes enterohepatitis com-monly known as histomonosis (syn. histomoniasis) or
0014-4894/$ - see front matter � 2007 Elsevier Inc. All rights reserved.
1 Present address: Department of Veterinary Pathology, College ofVeterinary Science, Guru Angad Dev Veterinary and Animal SciencesUniversity, Ludhiana 141004, India.
blackhead, which is an economically important disease ofpoultry that causes losses particularly in turkey flocksdue to high mortality (McDougald, 2003; Hess et al.,2004). However, recent reports underline the importanceof the disease for chickens, especially for birds kept underfree-range conditions or on farms with low or inadequatedisinfection procedures (Homer and Butcher, 1991; Esque-net et al., 2003; Cortes et al., 2004). The re-emergence ofhistomonosis in turkeys and chickens with serious and widespread outbreaks, more recently in the European commu-nity, due to the increase of free-range housing of poultry
506 A. Singh et al. / Experimental Parasitology 118 (2008) 505–513
and the ban of effective anti-histomonad drugs, has causedgreat concern regarding epidemiology and control of thedisease (McDougald, 2005).
In order to gain new insights into the epidemiology ofthe disease and to investigate the dissemination of H. mele-
agridis in the host, molecular diagnostic techniques likePCR (Hafez et al., 2005; Huber et al., 2005; Grabensteinerand Hess, 2006; Bleyen et al., 2007) and in-situ hybridiza-tion (Liebhart et al., 2006) have been developed recently,which indicated broad dissemination of nucleic acid ofthe parasite in different tissues of turkeys and spf chickens(Grabensteiner et al., 2006). However, these techniquesdetect only the nucleic acid of H. meleagridis that mightbe from circulating histomonads or remnants of dead his-tomonads present in the tissues. Principally, detection ofthe parasite in tissues is very important in order to establishits effect on the tissues, which is still lacking in histomono-sis. Consequently, the present investigation was set up inorder to close this gap and to gain new knowledge aboutthe impact of the parasite on the respective tissues. For thispurpose, an immunohistochemical technique was devel-oped using a rabbit polyclonal anti-H. meleagridis serumto detect parasitic cells in formalin-fixed, paraffin-embed-ded tissues following experimental inoculation of turkeyswith a mono-eukaryotic culture of H. meleagridis.
2. Materials and methods
2.1. Mono-eukaryotic culture of H. meleagridis
For the infection experiment a mono-eukaryotic cultureof H. melegridis (Histomonas meleagridis/Turkey/Austria/
2922-C6/04) was used. The culture was established throughmicromanipulation and was grown for the infection asdescribed recently (Hess et al., 2006a). Briefly, H. meleagri-
dis was isolated from caeca of a diseased turkey and main-tained by cultivation in medium 199 supplied with Earle’ssalts, 1-glutamine, 25 mM HEPES and 1-amino acids (Gib-co�, Invitrogen, Lofer, Austria), 11 mg rice starch (Sigma–Aldrich) and 15% foetal calf serum (Gibco�, Invitrogen,Lofer, Austria). Out of this a single cell was selected bymicromanipulation to grow up a whole stock. Prior tothe infection, the number of live parasites was determinedusing a Neubauer cell counting chamber and was adjustedso that the inoculum of 300 ll contained the appropriatenumber of parasites.
2.2. Samples
Tissue samples used in this study were from an infectiontrial conducted on 35-day-old BUT-9 turkeys that wereinfected with the mono-eukaryotic culture containing10,000 histomonads/300 ll of medium via the cloacal route.A conventional Eppendorf pipette was used to install 300 llof the inoculation volume as described earlier (Hess et al.,2006a). Afterwards the birds were deprived of feed for 5 h.Five birds from the infected group and three age matched
birds from the control group were analysed in this study.All infected birds died or had to be euthanized between 11and 13 days post infection. Samples included various tissueslike liver, caecum, spleen, lung, proventriculus, duodenum,pancreas, jejunum, kidney, bursa of Fabricius, thymus,bone marrow, cerebellum, cerebrum and medulla oblon-gata. All samples were placed in 7.5% buffered formalinand processed with conventional histological techniquesfor paraffin wax sectioning. Paraffin sections were stainedwith routine H&E and PAS staining techniques for histopa-thological analysis and detection of histomonads in thetissues following experimental infection. The stained slideswere analyzed at 10·, 20· and 60· magnifications usinglight microscope Nikon Optiphot-2.
2.3. Immunohistochemistry
2.3.1. Polyclonal sera and its purification
A polyclonal histomonad antiserum, produced in rab-bits using a mono-eukaryotic culture of H. meleagridis,
and purified through pre-absorption with acetone powderof bacterial flora and rice starch was used as primary anti-body to localize histomonad antigens in tissue sections.The purification was done according to the protocol ofZhao and Siu (1995) including some minor changes.Briefly, the bacterial supernatant harvested from the clonalculture of H. meleagridis was centrifuged at 4000g for30 min at 4 �C. The bacterial pellet was re-suspended in1 ml phosphate buffer saline (PBS) and the cells were col-lected by centrifugation at 16,000g. Cells were frozen inliquid Nitrogen and stored at �80 �C until further process-ing. Cells were thawed on ice and re-suspended in 300 ll,0.9% NaCl and split in two Eppendorf tubes. In one ali-quot, cells were opened with three subsequent freeze-thawcycles and the other aliquot was kept on ice. After thefreeze-thaw cycles, both samples were pooled together tohave a mixture of proteins and intact cells in one sample.The samples were mixed vigorously with 1.2 ml acetone(�20 �C) by vortexing and incubated on ice for 30 min.The cell and protein pellet was collected by centrifugationat 10,000g for 10 min at 4 �C. The pellet was re-suspendedin acetone (�20 �C) and further incubated for 10 min onice. Cells were collected by centrifugation, air dried andgrinded with a sterile spatula to make a fine powder. Thepowder was stored in Eppendorf tubes at �20 �C untilusage. For pre-absorption, a final concentration of 1%(w/v) acetone powder was used. To achieve this, the serumwas incubated by rocking, mixing 1 h at room temperatureor alternatively at 4 �C overnight. After incubation, theserum was centrifuged for 10 min at 10,000g, 4 �C andthe supernatant was collected to be used as a source forthe antibody.
2.3.1.1. Specificity of the polyclonal rabbit serum. To inves-tigate the specificity of the polyclonal anti-histomonadantibody, the clonal cultures of H. meleagridis, Tetratrich-
omonas gallinarum, Blastocystis sp. (Hess et al., 2006b) and
A. Singh et al. / Experimental Parasitology 118 (2008) 505–513 507
a culture of Trichomonas gallinae established from the cropof a pigeon (Liebhart et al., 2006) were stained using thedeveloped immunohistochemical technique. The fixationand paraffin wax embedding of the pellets of these cul-tures has been described previously (Liebhart et al.,2006). The pellets contained either 5 million cells of H.meleagridis, 50 million cells of Tetr. gallinarum, 10 millioncells of Tr. gallinae or 50 million cells of Blastocystis sp.The cultures of these protozoan parasites also containedrice starch and a variety of bacteria. In addition, archivedparaffin-embedded tissue samples from various animalspecies positive for protozoan parasites of the generaEimeria, Sarcocystis, Toxoplasma, Encephalitozoon, Cryp-
tosporidium or various fungi (Candida spp. and Aspergillusspp.), were also tested immunohistochemically using thepolyclonal serum.
2.3.2. Immunohistochemical technique
The avidin biotin complex (ABC) technique was used tolocalize cells of H. meleagridis in tissue sections. Paraffinwax sections (3 lm) were mounted on positively chargedglass slides (Superfrost plus; Menzel-Glaser, Braunschweig,Germany). Along with each batch of staining slides, paraf-fin-embedded pellets of the clonal cultures of H. meleagri-
dis and Tetr. gallinarum were included as positive andnegative controls, respectively. After dewaxing and rehy-dration, retrieval of antigen was performed by heatingthe slides in citrate buffer (pH 6.0) in a microwave oven.Endogenous peroxidase activity was blocked with 1.5%H2O2 in methanol for 30 min. The sections were then incu-bated with 1:10 dilution of a normal goat serum (VectorLaboratories, Burlingame, USA) mixed with 2% bovineserum albumin (Roche Diagnostics GmbH, Mannheim,Germany) for 45 min at room temperature in a humidified
Fig. 1. Examination of paraffin wax-embedded cultures of (A) Histomonas m
Trichomonas gallinae using polyclonal rabbit anti-H. meleagridis serum. Immu
chamber, followed by an overnight incubation with the pri-mary antibody (purified polyclonal anti-histomonadserum) at 4 �C at dilution 1:10,000. After extensive washingin PBS, the sections were incubated with a 1:400 dilution ofbiotinylated anti-rabbit IgG (Vector Laboratories, Burlin-game, USA) for 30 min, followed by Vectastain ABC Kit(Vector Laboratories, Burlingame, USA) for 60 min. Thereaction was visualized with a DAB Substrate Kit for per-oxidase (Vector Laboratories, Burlingame, USA). Aftercounterstaining with Mayer’s hemalum (Merck, Darms-tadt, Germany) and dehydrating, the sections weremounted under coverslips with Neomount (VWR, Vienna,Austria).
3. Results
3.1. Specificity of the polyclonal anti-histomonad serum
Initially a working dilution of the primary antibodywas established. Paraffin-embedded sections (3 lm) ofthe mono-eukaryotic culture pellet of H. meleagridis werestained with different dilutions of the primary antibody. Adilution of 1:10,000 was found to localize histomonads inthe embedded pellet without any background reactionwith bacteria and starch particles present in the culture(Fig. 1A). Applying the same testing conditions the pri-mary antibody showed no staining reaction with mono-eukaryotic cultures of other protozoa isolated from birds.Fig. 1B–D displays the immunohistochemical stainingreaction of the primary antibody with Tetr. gallinarum,Blastocystis sp. and Tr. gallinae. As shown in Table 1,none of the tissue samples in which other protozoan par-asites and fungi were localized gave a positive reactionwith the polyclonal anti-histomonad serum.
508 A. Singh et al. / Experimental Parasitology 118 (2008) 505–513
3.2. Localization of parasite in tissues
Histomonas meleagridis antigens were successfullylocalized using the polyclonal antibody-based immunohis-tochemical technique in several organs of experimentallyinfected turkeys. As shown in Table 2, histomonads weredetected in caecum (5/5), liver (5/5), lung (5/5), spleen (5/5), bursa of Fabricius (3/5), bone marrow (1/2), heart (2/5), proventriculus (1/5), pancreas (1/5) and cerebellum (1/5) of the infected birds. Fig. 2A–I shows the strongimmunohistochemical reaction specific to histomonadsin several organs of the infected turkeys. All the samplesfrom non-infected control birds were tested negative. Inaddition, a strong immunohistochemical reaction indica-tive of presence of histomonads was also detected inthe blood vessels of several organs as shown inFig. 3A–I.
In the caecum and liver of infected birds, invasion of his-tomonads was associated with extensive necrosis as it is evi-dent from Fig. 4A and B. In the caecum, histomonads were
Table 2Detection of H. meleagridis in organ samples of experimentally infected turke
a dpi, death of bird day post infection.b ND, not done.
observed in the lamina propria and muscularis mucosae(Fig. 4A) and invasion extended into muscular layer(Fig. 4B). In the necrotic zones, histomonads were seen sur-rounded by abundant mixed inflammatory cells (Fig. 4Band C). Histomonads were also localized within giant cells(Fig. 4D). In the liver, lesions were characterized by exten-sive hepatic necrosis with inflammatory exudate. Histomo-nads were seen in the periphery of the necrotic zone(Fig. 5A) and were also found surrounded by mixed inflam-matory cells (Figs. 2A, 5A and D). One or more parasiteswere also found within multinucleated giant cells(Fig. 5B) or within the hepatocytes or the sinusoids(Fig. 5C). Several histomonads were seen infiltrating thehepatic vessels (Fig. 3A) as well as the bile duct(Fig. 5D). In the bursa of Fabricius, infiltration of histomo-nads was observed in the follicles as well as interfollicular-ly, involving several plicae (Fig. 2D) with extensivelymphoid necrosis and depletion along with inflammatoryexudate. Mild to moderate pathological changes wereobserved in other organs, regardless of a positive immuno-
Fig. 2. Localization of histomonads in various formalin-fixed, paraffin wax-embedded tissues from turkeys infected cloacally with a mono-eukaryoticculture of H. meleagridis; (A) liver, (B) caecum, (C) lungs, (D) bursa of Fabricius, (E) bone marrow, (F) proventriculus, (G) spleen, (H) pancreas, (I)cerebellum. Immunohistochemistry. Bars, 20 lm.
Fig. 3. Immunohistochemical reaction specific to histomonads in blood vessels of tissues from experimentally infected turkeys; (A) liver, (B) lung, (C)proventriculus, (D) pancreas, (E) bursa of Fabricius, (F) thymus, (G) spleen, (H) bone marrow, (I) brain. Bars, 20 lm.
A. Singh et al. / Experimental Parasitology 118 (2008) 505–513 509
histochemical reaction specific to the parasite. However inthe brain, histomonads were detected without any patho-logical changes in the tissue except that a strong immuno-histochemical reaction was observed in the wall of severalblood vessels along with histomonad-like cells in the lumen(Figs. 2I and 3I).
Table 3 shows a comparative efficacy of the polyclonalantibody-based immunohistochemical, H&E and PASstaining techniques in detection of histomonads in theinfected tissues. A high percentage of tissues were foundpositive with immunohistochemistry (38.96%), followedby PAS (23.37%) and H&E (18.18%) techniques.
Fig. 4. Immunohistochemical detection of histomonads in caecum showing; (A) invasion of histomonads in lamina propria and muscularis mucosae withdestruction of mucosal epithelium, (B) invasion of histomonads deep into muscular layer with necrosis and inflammation, (C) various stages ofhistomonads in inflammatory exudate, (D) histomonads localized in multinucleated giant cells (arrows). Bars, 20 lm.
Fig. 5. Immunohistochemical detection of histomonads in the liver showing; (A) localization of histomonads in the periphery of necrotic lesions (arrow)and surrounded by inflammatory cells (arrow head), (B) in multinucleated giant cells (arrow), (C) in sinusoids (arrow) and hepatocytes (arrowhead), (D) inbile duct (arrow) and degenerated histomonads (arrowhead) surrounded by inflammatory cells. Bars, 20 lm.
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4. Discussion
The most significant aspect of this work was the attemptto localize immunohistochemically H. meleagridis antigenswithin the tissues of infected turkeys and to study interac-tions of the parasite with host tissues using a rabbit poly-clonal anti-histomonad serum.
Although disease diagnosis can be made on the basis ofcharacteristic gross and microscopic lesions in the caecumand liver of the infected birds, additional confirmation of
the disease is required to rule out concurrent protozoaninfections like coccidiosis, bacterial infections like salmo-nellosis, fungal infections like aspergillosis, upper digestivetract trichomonosis and leucosis tumors, all of which mayshow lesions in caecum and liver (Allen, 1941; McDougald,2003, 2005). Furthermore, the presence of other protozoanparasites like Blastocystis and Tetratrichomonas may arguefor increased specificity of the diagnosis (Grabensteinerand Hess, 2006). As histomonads in the tissue are non-flag-ellated (McDougald, 2003) and different morphological
Table 3Detection of H. meleagridis in formalin-fixed paraffin-embedded tissuesections of experimentally infected turkeys
Organ Detection of histomonads in tissue sections using
Total 30/77 (38.96%)b 18/77 (23.37%) 14/77 (18.18%)
a Number of positive samples/investigated samples.b Total number of positive samples/investigated samples (per cent
positive samples).
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forms were described in the tissue (Cortes et al., 2004), theclear differentiation from similar structures like macro-phages and yeast cells which may exhibit some morpholog-ical similarity is needed.
Until recently, the histopathological detection of histo-monads in liver and caecal samples was performed solelyby applying several staining techniques to diagnose and dif-ferentiate histomonosis (Kardevan and Vetesi, 1970; Kempand Reid, 1966; Esquenet et al., 2003). Histomonads canalso be isolated in vitro from the clinical samples of caecumand liver using a special culture medium (Bayon andBishop, 1937), however under field conditions, diagnosiscan be hampered due to mixed infections with other proto-zoan parasites.
The diagnosis of H. meleagridis by PCR has beendescribed in recent years (Hafez et al., 2005; Huber et al.,2005; Bleyen et al., 2007) by detecting nucleic acid of par-asite in infected tissues. Grabensteiner and Hess (2006)have developed a PCR for identification and differentiationof H. meleagridis, Tetr. gallinarum and Blastocystis sp.Hauck et al. (2006) used real-time PCR to demonstratethe dissemination of H. meleagridis DNA to differentorgans after natural and experimental infections of meatturkeys whereas Huber et al. (2006) used PCR to studythe dissemination kinetics of H. meleagridis DNA in exper-imentally infected turkey tissues. More recently, in-situhybridization technique has been developed by Liebhartet al. (2006) to specifically localize the nucleic acid of H.
meleagridis in the tissues of infected turkeys and chickens.Using this technique in combination with PCR, Grabenste-iner et al. (2006) studied the broad dissemination of nucleicacid of the histomonads in various tissues of experimen-
tally infected turkeys and chickens. However, all these tech-niques are based on detection of the nucleic acid of theprotozoan parasite that might be from viable histomonads,or from dead ones, or from parasites which were just pass-ing through the tissue. In principle, localization of a path-ogen within the tissue lesions is very important to establishits effect on host tissue, which in case of histomonosis is stilllacking for the parasite itself.
Considering the constraints and limitations describedabove the aim of the present investigation was to developa method suitable to detect whole cells of H. meleagridis
in tissue samples. A polyclonal anti-H. meleagridis serumraised in rabbits against a mono-eukaryotic culture of theparasite is the key element of the developed method. Usingthis approach specificity of the whole method could be suc-cessfully demonstrated similar as it was recently describedfor in-situ hybridization (Liebhart et al., 2006).
In order to obtain suitable tissue samples 35-day-oldBUT-9 turkeys were infected as described previously andthe disease was once again successfully reproduced withtypical lesions in the liver and caeca (Hess et al., 2006a).In the caeca it was possible to immunohistochemicallydemonstrate invasion of histomonads in lamina propriaand muscularis mucosa with extensive destruction of themucosa. The histomonad invasion was detected deep inthe muscular layer with extensive necrosis and severeinflammatory reaction which was composed of a mixedpopulation of heterophils, lymphocytes and macrophages.It was even possible to demonstrate histomonads withingiant cells. Following cloacal inoculation the parasite goesdirectly into the caeca by retrograde peristalsis, a cloacaldrinking phenomenon described already by Browne(1922). After multiplication in the caecal lumen, the para-site invades the caecal wall and migrates through the caecaltissue (Lee et al., 1969) with severe destruction of the hosttissue. In the present study, we observed invasion of histo-monads deep into the host tissue. As described previously(Hauck et al., 2006; Huber et al., 2006), the parasites gainaccess to the hepatic portal system and are carried to theliver, where they cause hepatic necrosis. Using PCR andin-situ hybridization the broad dissemination of the para-site in the host was described recently (Grabensteineret al., 2006). As immunohistochemical reactions specificto histomonads were detected in the blood vessels of sev-eral organs including liver and caeca this invasion occursmost likely via the bloodstream, which is in accordancewith earlier findings of Farmer et al. (1951). In the liver,histomonads were also seen focally in the bile ducts indicat-ing the likelihood of histomonads to ascend the intestine.However, Lund and Chute (1970) reported that bile greatlydiminishes the infectivity of histomonads and also they didnot detect histomonads in the gall bladder.
In the liver, a strong immunohistochemical reaction spe-cific to histomonads was observed in the periphery ofnecrotic zones and an immunohistochemical reaction indi-cating degenerated forms of histomonads was presentwithin the inflammatory zones. Histomonads were also
512 A. Singh et al. / Experimental Parasitology 118 (2008) 505–513
observed in the sinusoids in undamaged hepatic tissuewithout any inflammatory exudate. The mechanism bywhich histomonads interact with host tissue and producelesions in liver and other organs is still to be studied indetail but it seems from the present study that histomonadsinteract with hepatocytes in similar manner as reported forEntamoeba histolytica, a closely related parasite that causesamoebosis characterized by hepatic granulomas in humans(Ventura-Juarez et al., 2003). These authors revealed thatE. histolytica trophozoites express a protein with epitopesrecognized by anti-iNOS antibodies. Therefore the hepato-cellular damage in amoebosis relies on the release of prote-olytic enzymes and nitric oxide. In this study, few parasiteswere also found within multinucleated giant cells andwithin hepatocytes, a finding also described earlier by Tyz-zer (1934). However, this phenomenon requires furtherinvestigation.
Beside the liver and caeca as the main target organs, aheavy invasion of histomonads with tissue destruction wasobserved in the majority of bursal samples. This might bedue to the direct installation of the infectious material inthe cloaca. Cortes et al. (2004) were able to demonstrate H.meleagridis in the bursa of Fabricius in chickens during afield outbreak. The histomonad nucleic acid could also bedetected in the bursa of Fabricius by PCR and in-situ hybrid-ization (Hauck et al., 2006; Huber et al., 2006; Grabensteineret al., 2006). The direct contact between the bursa of Fabri-cius and the intestinal content together with the cloacal suck-ing may provoke the infection of the bursa of Fabricius(Schaffner et al., 1974). However, an immunohistochemicalreaction was also observed in the blood vessels of the bursaof Fabricius which indicates that a hematogenous spreadmight contribute to the invasion of this organ.
In addition to liver and caeca, parasite antigens couldalso be detected in all the tissues samples obtained fromlungs and spleens. Furthermore, 1 out of 2 samples fromthe bone marrow, and 2 out of 5 samples from the heart tis-sues were recorded positive. Finally, 1 out of 5 sampleseach from the proventriculus, pancreas and cerebellumwith mild to moderate pathological changes were noticedpositive. Therefore, histomonads are able to multiply andto induce pathological changes in several organs asobserved in the present study. Similarly, pathologicalchanges in organs other than liver and caeca were alsoobserved by earlier workers (Tyzzer and Collier, 1925;Farmer et al., 1951; Malewitz et al., 1958; McGuire andMorehouse, 1958; Kardevan and Vetesi, 1970). The pres-ence of histomonads in the blood stream confirms thehematogenous route of infection from caeca to liver, heart,lung, spleen and other organs as also reported earlier(Farmer et al., 1951; McGuire and Morehouse, 1958;Clarkson, 1962; Hauck et al., 2006). Although the immu-nohistochemical reaction specific to histomonads wasdetected in cerebellum of one bird without pathologicallesions, a strong immunohistochemical reaction indicatingpresence of histomonad-like cells or cells with histomonadantigens in vessels was observed in the brain.
The low incidence of parasites in the brain and someother organs might be attributed to the fact that higherinfective doses of the parasite are required to infect certaintissues (Lund, 1955) as most histomonads get filtered incapillary bed of lung after the liver (Hauck et al., 2006)and organs thereafter receive a low number of parasite.The low incidence of histomonads in the brain might alsobe due to strong resistance of blood–brain barrier (BBB).Silva et al. (2004) suggested that the parasitic entry of Try-
panosoma cruzi into the CNS through emigration of para-sitized mononuclear phagocytes may be possible but thisrequires peripheral infection, activation of leukocytes, acti-vation of CNS endothelium and leukocyte traffickingthrough BBB. Similarly, in the present study we observedimmunohistochemical reaction in the cells in blood vesselsof the brain indicating parasitized phagocytic cells thatmight carry histomonad antigens into the brain. Patholog-ical changes were not seen in the brain although parasiticcells were localized in brain tissue which might be due tothe fact that the parasite requires interaction with someinflammatory cells to produce pathological changes, acommon feature observed in all other organs positive forhistomonad antigens.
In the present study, a comparative evaluation of animmunohistochemical technique with routine H&E andPAS staining techniques was attempted to detect histomo-nads in tissues of infected turkeys. Overall, the immunohis-tochemical technique was found to be considerably moresensitive as compared with routine H&E and PAS stainingtechniques especially in tissues with a low amount ofparasites.
Altogether, the immunohistochemical technique devel-oped in the present investigation can be used to study thesequential pathogenesis of histomonosis in birds and toobtain new insights into the mechanisms of interactionwith the tissues they infect. Further investigations arerequired to study the mechanism by which histomonadsproduces lesions and their interactions with immune cellsof the host.
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