Histomoniasis is an infectious entero-hepatitis caused by the protozoan parasite Histomonas meleagridis belonging to the order Trichomonadida, family Monocercomonadidae^1,2. It is ubiquitous in nature causing enterohepatitis which is often called as blackhead disease or enzootic typlohepatitits³. It is a disease of Galliformes causing high mortality in turkey poults. Chicken, guinea fowl, quails, pea-fowl, chukars, grouse, pheasants, patridges may also be worst affected^1,4,5,6 whereas ducks and ostriches act as carriers^7,8. Ring-necked pheasants are relatively resistant and are considered to be reservoir for histomonads⁹. Mode of disease transmission is still undetermined, as this protozoan requires no intermediate host for the completion of its life cycle, although vectors are thought to play a major role as carriers^3,10,11. Susceptible birds are infected after ingesting eggs of the caecal nematode, Heterakis gallinarum and or by earthworms¹². Many other insects including darkling beetles, houseflies, gross-hopper, sow-bugs have also been identified to carry protozoa¹³. Outbreaks of the disease are also recorded in the absence of vectors either by direct or lateral transmission which includes litter pecking, coprophagy, contaminated feed and water^14,15 and cloacal drinking^16,17. H. meleagridis causes reduced body weight gain, loss of productivity and high mortality in susceptible hosts producing fibrino-necro-haemorrhagic typhlitis and multifocal necrotic hepatitis¹⁸. No other organs are reported to develop lesions in natural outbreaks of histomoniasis¹⁹. Systemic infection of histomoniasis was reported to be rare in birds¹³. Among the Galliformes, it was reported in turkey¹³ and found that bursa of Fabricius harbour histomonads, microscopically^13,20. However, there have been no reports of multisystemic infection in desi chicken. This is the first report to describe multisystemic infection of histomoniasis with primary lesion in the bursa of Fabricius caused by Histomonas meleagridis in desi chickens.

A rural based private desi chicken farm near Orathanadu taluk of Thanjavur district, Tamil Nadu was reported to have high mortality in commercial chickens. Farm holds non-descript chicken of six months age with flock strength of 500 birds. Birds were reared in deep litter system for commercial purpose. Cumulative mortality of the farm reached 60% within 7-10 days and had the history of drowsiness, poor production and mild diarrhoea. Randomly selected dead birds (10 numbers) were collected and brought to the Department of Veterinary Pathology, Veterinary College and Research Institute, TANUVAS, Orathanadu for postmortem diagnosis. Owner reported that around 300 birds out of 500 total stock strength have died within a period of 10 days with similar symptoms. Detailed and systematic postmortem examinations were carried out and gross lesions in affected organs were recorded. Representative tissue pieces from affected organs were collected in 10% formalin for histopathological examination and processed as per standard paraffin embedding protocol, and haematoxylin and eosin staining²¹. Periodic-acid Schiff (PAS) special staining was done to demonstrate the protozoal bodies in tissue section as per the protocol²². Swab from liver surface and heart blood were collected for cultural examination. Cloacal swab was collected for parasitological studies.

Grossly, the birds were moderately emaciated. Gross lesions were noticed in the pharynx, lungs, heart, liver, spleen, small intestine, caecum and bursa of Fabricius. Pharynx revealed focal greyish-white, circumscribed (1 mm) diphtheritic area. Lungs were congested and slightly firm. Heart appeared pale. Liver was congested and covered with a patchy thin greyish membranous layer which peeled off easily (Fig. 1). Both the liver lobes showed multifocal greyish white to yellowish, circular (0.5 to 1 cm) to coalescing areas with sharp irregular borders. Many of these lesions were dark reddish-brown with raised centre surrounded by circular ring-like depressed zone bordered by elevated parenchyma resembling ‘press button’ (Fig. 2). Few of these lesions were similar to those observed in avian lymphoma characterised by greyish to white or yellowish foci, on incision, the lesions extended deep into the parenchyma (Lymphomatoid lesions). Kidneys were slightly enlarged, pale and revealed multifocal greyish white foci (1-5mm) which extended deep into the parenchyma (Fig. 3). Spleen was slightly enlarged and congested. Mucosa of duodenum and jejunum were slightly congested and thick. Caecum had a little dark brownish pasty content. Caecal mucosa showed moderate mucosal thickening. Bursa of Fabricius was distended and contained thick, creamy white firm content occluding the entire lumen having casting of the plicae (Fig. 4). The wall of the bursal sac was thick, glistening and had alternating prominent and less prominent mucosal plicae.

Microscopically, liver showed severe multifocal necrotic hepatitis. It was characterised by degeneration and necrosis of hepatocytes, syncytium formation, infiltration of heterophils, lymphocytes, plasma cells and macrophages (Fig. 5). Large number of vacuolar spaces containing pleomorphic, faintly stained eosinophilic bodies consistent with degenerative forms of Histomonas meleagridis were observed within the necrotic areas bearing a resemblance with the bull’s eye (oval or spherical bodies surrounded by clear halo) (Fig. 6). Hepatocytes surrounding the necrotic zone showed vacuolar degeneration, congestion of sinusoids with presence of mono- and or binucleated pleomorphic bodies measuring 5-20 µm with granular cytoplasm which were consistent with trophozoites of Histomonas meleagridis. Trophozoites were characterized by eosinophilic, oval or spherical bodies with clear nucleus, cytoplasmic vacuolations and without flagella. Spleen showed scattered histomonads in the red pulp. Bursa of Fabricius showed severe subacute to chronic cloacal bursitis characterised by widening of plicae with mild to moderate lymphoid depletion and necrosis. Follicles were infiltrated with heterophils, lymphocytes, plasma cells, macrophages and giant cells. Eosinophilic, pleomorphic bodies of H. meleagridis were observed within the necrotic lymphoid follicles. A few of the lymphoid follicles showed cystic degeneration with necrotic remnants of lymphoid tissue, inflammatory exudate and degenerative forms of H. meleagridis (Fig. 7). Small intestine showed severe subacute enteritis (Fig. 8) with scattered degenerative forms of histomonads in the mucosal epithelium. Caecum revealed diffuse severe subacute typhlitis characterised by thickening and widening of the mucosa with moderate to severe infiltrations of heterophils, lymphocytes, plasma cells, macrophages and syncytial cells. Epithelial crypts showed necrosis and cystic degenerative changes characterised by dilated crypt containing inflammatory exudate and degenerative forms of histomonads with rim of degenerative epithelium (Fig. 9). Crypts revealed random areas of hyperplasia with herniation into the muscularis layer (Fig. 10). Lungs revealed diffuse subacute pneumonia. Parabronchioles were rimmed by infiltration of heterophils, lymphocytes, plasma cells and macrophages (Fig. 11). Heart showed multifocal subacute myocarditis characterised by infiltrations of mononuclear cells separating the myocardial fibres (Fig. 12). Pancreas showed multifocal subacute pancreatitis with infiltrations of mononuclear cells around the islets of Langerhans. Kidney showed multifocal, severe chronic interstitial nephritis characterised by infiltration of lymphocytes, plasma cells, giant cells and macrophages in the interstitial space of the renal cortex (Fig. 13). Histologically, cerebral cortex revealed degeneration of neurons and satellitosis (Fig. 14).

Parasitological examination of cloacal swab revealed presence of flagellate bodies of Histomonas meleagridis. Escherichia coli was isolated from liver and heart blood by culture.

Special staining with PAS was carried out to demonstrate the protozoa in tissue sections. Among the tissues examined, liver and bursa of Fabricius revealed moderate to heavy PAS positive histomonads (Fig. 15 & 16) followed by caecum. Other organs were negative for PAS reaction. Degenerative forms and food vacuoles of trophozoite forms of Histomonas meleagridis stained intense magenta with PAS.

Blackhead disease was first described in Rhode Island during 1893 in turkey poults²³. Theobold Smith made an elaborative study of the protozoan and named as Amoeba meleagridis which was later renamed as Histomonas meleagridis by Tyzzer²⁴. It was initially thought to be the highly contagious disease with high mortality in turkey poults until first outbreak occurred during 1900 in chicken²⁵.

Histomoniasis was so named as enterohepatitis as it was reported to cause lesions which were restricted to the liver and caecum²³. More significantly there have been no reports regarding the histomonads induced lesions in bursa of Fabricius in turkeys. Deviation in the pathogenesis of histomoniasis was triggered after the entry of histomonads histologically in the bursa of Fabricius in turkeys¹³ and in chicken²⁰. The concept of enterohepatitis was later disproved by the occurrence of multisystemic histomoniasis in commercial turkeys causing high mortality with lesion severity in bursa of Fabricius, lungs and kidneys¹³. Until now systemic infections have not been reported in chicken. This was the first report which describes the systemic histomoniasis affecting multiple organ system in chicken and the lesions were very similar with the above findings in turkeys¹³.

Among galliform birds, turkeys were reported to be highly susceptible to histomoniasis followed by chicken, quail, pheasants, pea fowl and partridges which remained as important hosts^{4,6,13,27,28,29,30,31}. Histomoniasis caused mild clinical signs with lower mortality but could cause disease as severe as coccidiosis in chicken, the concept proposed by McDoughald³ was true with the high mortality of 60% in the present study. It was also supported by the earlier reports with severe clinical disease and high mortality in young chicken^{3,15,32,33,34}, layer hens³⁵ and in free range reared desi chicken³⁶ infected with histomoniasis.

Various predisposing factors were proposed by earlier workers for the occurrence of histomonas in chicken and they included 1) rearing of turkeys and chicken together as single flock, 2) rearing chicken near the turkey flock, 3) usage of contaminated water, feed, fomites and utensils interchangeably from turkey flock to chicken flock, 4) involvement of vectors, 5) engagement of workers to carryout routines in turkeys and chicken flocks together³. The above predisposing factors could be excluded in the present study as desi chicken were reared in deep litter system without any access to turkey flocks or population nearby. Ambiguity in the mode of transmission still persists and could be the possible reason for high mortality outbreaks in chicken and other Galliformes^15,31,34,36. Among the panel of questions raised by earlier researchers, few to be answered include, how did the histomonads reach the bursa of Fabricius ? what is the effect of histomonads on the bursa of Fabricius ? why does all stages of Histomonas not stain with PAS ? We attempt to justify the answer parallelly by discussing the present outbreak.

Oral infection could be a possible route of infection in earlier days due to the evidence that the protozoa were transported through vectors. Absence of caecal worm and necrotic core in caecum of the present outbreak justifies that the mode of transmission could possibly be either by earthworm or non-vector borne. Latter proves to the most possible route of transmission in the present case. Many researchers have claimed that histomoniasis occurred in the absence of possible vectors^15,34. As suggested by Hu and McDougald³⁷ and Tyzzer and Collier³⁸, direct or lateral transmission is possible in histomoniasis. It was further suggested that intracloacal infection of experimental histomoniasis was more effective in disease occurrence than oral inoculation^16,37,39. This was proved by applying the culture of histomonads in the cloaca which stimulated the cloacal drinking¹⁶. The elaborative studies on cloacal drinking carried out by Akester et al.⁴⁰ and Nechay et al.⁴¹ explained that retrograde movement of urine from the cloaca caused by reverse peristalsis could be important in the rapid spread of blackhead disease and other diseases in turkeys. Further, they explained that infection spread faster when inoculated birds comingle with uninoculated birds. Although cloacal drinking phenomenon was not well established in chicken, Sorvari et al.⁴² proved that chicken also showed similar phenomenon but with lesser transmission rate than turkeys. It was also suggested that differences in the behaviour and/or water content in droppings could account for difference in transmissibility⁴². Cloacal kissing is a similar phenomenon as cloacal drinking seen frequently in naturally reared desi birds during mating. This could also possibly transmit histomonads from cock to hen while mating¹⁷. The present study postulates that carrier cock might have transmitted the protozoa to the susceptible hens while cloacal kissing and transmission was exacerbated during comingling of infected hen with in the flock. Much research on cloacal transmission is warranted to understand alternative pathogenesis of histomoniasis in birds.

Involvement of bursa of Fabricius and the severity of bursal lesion in the present outbreak clarifies that bursa remains an important target organ. This deviates the regular pathogenesis of histomoniasis which starts colonization in the caecum after oral exposure, causing severe typhlitis with formation of caecal core. Parasites later infiltrate into circulation whereby reaches the liver via portal veins causing hepatic necrosis⁴³. Though finding of histomonads in the lymphoid follicles of bursa of Fabricius was surprising, earlier reports have also demonstrated the histomonads in bursa of Fabricius in chicken²⁰ and in turkeys¹³. It was considered as unusual in the pathogenesis of histomoniasis and raised infinite questions regarding the impact of bursal involvement. The bursa of Fabricius is a gut associated primary lymphoid organ in birds which plays a vital role in humoral immunity by development and differentiation of B cell for antibody production⁴⁴. Enormous etiological agents naming a few, infectious bursal disease virus, Marek’s disease virus, chicken infectious anaemia virus, reticuloendothelial virus, reoviruses⁴⁵ and Cryptosporidium baileyi⁴⁶ cause bursal atrophy, chronic bursitis and eventually immunosuppression. The suspicion of the earlier researcher regarding histomonads induced immunosuppression was refuted in the present outbreak where chronic bursitis with bursal core formation, severe lymphoid necrosis, cystic degeneration of follicles and the presence of degenerative form and trophozoite form of histomonads within the follicles and epithelial plicae unquestionably elucidate that histomonads could be the sole etiology for the cloacal bursitis induced immunosuppression. The present bursal severity in terms of lesion and immunosuppression was in obedience to the first recorded bursal lesions induced by histomoniasis²⁰.

Immunosuppression possibly was the reason for secondary bacterial infection occurred in the present outbreak. Lesions of multifocal myocariditis, pneumonia, pancreatitis not associated with histomonads recorded in the present study could be due to bacterial infection with E. coli which was isolated by culture from the affected carcasses. The above findings were in accordance with the Senties-Cue et al.¹³, who reported multifocal granulomatous pneumonia and pancreatitis without protozoan parasite. There have been no reports of lesions other than liver and caeca in natural outbreaks of histomoniasis in desi chicken. Multisystemic histomoniasis of the present study involving gross and microscopic lesions in unusual target organs like bursa of Fabricius, small intestine, kidneys, lungs, pancreas, in addition to the classical necrotic hepatitis and typhlitis was in-line with Senties-Cue et al.¹³. The discrepancy might be due to the virulence of histomonads. This was supported by earlier study suggesting the different genotypes of H. meleagridis differ in their virulence within the host, between the host and between the species of the birds⁴⁷. Few others suggested that concurrent infection with coccidiosis, helminths, viruses and bacteria plays a contributory role in exacerbating the lesions and increases the mortality of the birds⁴⁸. This explanation was in parallel with the present study where myocarditis, pancreatitis and pneumonia observed without association of histomonads and presence of thin membranous layer (perihepatitis) could be due to E. coli which was isolated in the present study.

Difference in the staining intensity of various forms of Histomonas meleagridis observed in this study was very similar with the observations made by Senties-Cue et al.¹³. Histomonas appears in two different forms in birds. A flagellated luminal form which resides inside the lumen of the intestine or caecum and non-flagellated transitional/amoeboid form (trophozoite) which resides in liver and other visceral organs. Present study revealed that majority were trophozoite form in liver, bursa of Fabricius and in caecum. In addition, degenerative form (tissue form) could also be detected inside the necrotic areas in affected organs. PAS stain was helpful in identifying these forms by staining the protozoa as deep purple magenta-coloured bodies. PAS stain could able to differentiate these forms. The degenerative forms within the necrotic areas took more intense purple-magenta than trophozoite outside the necrotic areas. A few of the trophozoites revealed PAS positivity especially in the food vacuoles appearing as dots inside the trophozoites. Tyzzer^24,49 described two different phases of H. meleagridis as invasive phase and resistant phase by its staining affinity with PAS stain. Lightly stained trophozoites were referred as invasive form and dark and intensely stained bodies as resistant phase of the protozoa. The explanation was equally justifiable with the present observations. Basically, PAS stains glycogen and mucosubstances in the cells²². Degenerative or resistant phase of the protozoa were small and had dense granular cytoplasm with tight aggregations of glycogen, glycoproteins, glycolipids and carbohydrates which stain deep coloured with PAS whereas trophozoites or invasive phase of the histomonads were comparatively larger with less dense mucosubstances except glycogen in food vacuoles and obviously food vacuoles were stained PAS positive rather entire trophozoites²⁰.

The present outbreak of systemic histomoniasis in desi chicken reared under deep litter system revealed the multiorgan infection, specifically the unusual lesions in bursa of Fabricius. Severe subacute to chronic cloacal bursitis with formation bursal core was observed in the affected carcasses which looks indistinguishable with caecal core with chronic transmural typhlitis, the classical lesion of histomoniasis in turkeys and chickens. The present outbreak emphasises the potential of histomonads causing high mortality in desi chicken as equal as turkeys with earlier reports. The present observations documented that the deviation of pathogenesis in causing lesions and also establishes that histomonads caused immunosuppression which was responsible for secondary bacterial infection and related lesions in various organs. The present study could not identify any other etiological agents as a primary cause for immunosuppression in bursa. This study records the first multisystemic histomoniasis in desi chicken with unusual lesions in the bursa of Fabricius, small intestine, heart, pancreas, lungs and brain. Neuronal degeneration and satellitosis in cerebral cortex of the affected birds observed in the present study are the first to be recorded and not reported in any other Galliformes affected with histomoniasis. Neuronal involvement of histomoniasis needs to be investigated. The severity of bursal lesions than other organs suggested that histomonads transmission might purely be by cloacal route rather than oral transmission. Intra-cloacal infection either as cloacal drinking or cloacal kissing paves ways for establishment of infection in the bursa of Fabricius and subsequent systemic spread could be plausible.

Many thousands of compounds have been tested against histomoniasis both invivo and invitro. Unfortunately, only preventive compounds are available today. Few of these compounds includes arsenicals (neoarsphenamine, tryparamide); nitrofurans; nitroimidazoles (dimetridazole); nitrothiazoles²⁹. Legislations were also laid down by Codex Alimentarius of the Food and Agricultural Organization of the United Nations and the World Health Organization regarding not to use nitroimidazole such as metronidazole or dimetridazole in food producing birds⁵⁰. To overcome the drawbacks in usage of allopathic compounds, herbal based compounds proved effective in controlling the disease transmission. Plant-derived oil from compounded product of cinnamon, lemon, rosemary, garlic and thyme were found effective in suppressing the growth of the histomonads, however the results were contradictory^51,52. Hence, strict hygiene and biosecurity measures are the best practices to be followed to control histomoniasis in chicken flocks including, 1) all sources infected or contaminated with Heterakis gallinarum must be kept away especially birds such as pheasants which host both Heterakis sp and histomonads, 2) strict control over the introduction of insects, rodents and other vermin which thought to play a possible role as vector, 3) litters and all other equipment and clothes must periodically be sanitized and dried before use. Free ranging and backyard birds are more likely to be exposed to the disease which had to be watched over to decrease mortality^1,29,53. Most importantly, it was reported that mortality tended to be high with poor hygiene and when acidification of drinking water was practised²⁹. Hence, the owner was advised to quarantine the affected birds and sanitize the deep litter flooring. The deep litter was replaced with the fresh dry litter for controlling the spread of the disease.

References