Leucocytozoon caulleryi is an etiological agent for leucocytozoonosis which was first discovered and reported in domestic chicken¹. It is a haemoprotozoan parasite, assigned to order of Haemospororina of the phylum Apicomplexa. It affects red blood cells and endothelial cells of internal organs of birds². Natural infection of L. caulleryi is transmitted by culicoides^2-3.

Leucocytozoon caulleryi has three growth stages such as schizogony and gametogony in chicken and sporogony in culicoides. Leucocytozoon infection starts when an infected culicoides takes a blood meal from a chicken, simultaneously injecting sporozoites into the blood stream of chicken. Such sporozoites invade capillary endothelial cells in the spleen, lung, liver and bursa of Fabricius and develop into first generation schizonts. Mature first generation schizonts are variable in size and range from 40-60 µm in diameter. The schizonts mature and release first generation merozoites on day-5 post-infection. These merozoites in turn invade the capillary endothelial cells of various tissues and organs and become large second generation schizonts (megaloschizonts) that release second generation merozoites on day-14 or 15 post-infection. Mature second generation schizonts are found extracellularly in various organs and reach sizes upto 400 µm in diameter. The second generation merozoites are oval in shape which invade erythrocytes and grow into gametocytes. Mature gametocytes freed from host cells appear in the peripheral smear of the chicken on day 18-24 post-infection. Culicodes that take a blood meal therefore ingest mature gametocytes and sporogony occurs to form sporozoites which migrate to the salivary gland. The sporozoites enter the chicken during biting by culicoides³. The clinical features and the required method of diagnosis of this leucocytozoonosis in chicken flocks mainly depend on its life cycle, especially whether the parasite is in the stage of schizogony or gametogony. The second generation schizont, observable as megaloschizonts, can be detected with histopathology. Subsequently, megaloschizonts release second generation merozoites which are oval in shape, invade and develop into gametocytes in erythrocytes, thus marking the gametogony stage. At this point, leucocytozoonosis can be diagnosed with a blood smear. As the disease progresses, chickens suffer from internal haemorrhage and anaemia, resulting in increased mortality and decreased egg production^5-7. Present communication reports the occurrence of leucocytozoonosis in commercial white Leghorn layer chickens.

During the first week of September 2021, seven white Leghorn layer chickens from a commercial layer flock containing 1300 birds were received for necropsy and gross lesions were recorded. During necropsy, smears made from the fluid in the cysts of the heart, intestine and liver were collected and examined under light microscope. The tissue samples of comb, skeletal muscle, heart, trachea, lungs, proventriculus, gizzard, intestine, liver, pancreas, kidney, ovary, oviduct, thymus, spleen, bursa of Fabricious and brain were collected and fixed in 10% formalin for histopathological examination. The tissues samples were routinely processed, sectioned (4 µm thickness) and stained with Hematoxylin and Eosin (H&E) stain.

On necropsy, externally the birds showed pale comb, wattle, skin, beak and conjunctival mucous membrane. In one bird, comb showed multifocal reddish cysts of 0.5 to 1 mm size (Fig. 1). On internal examination, tracheal lumen and peritoneal cavity contained blood mixed fluid. Subcutaneous tissues of neck, pectoral muscle (Fig. 2), proventricular mucosa, gizzard mucosa, intestine mucosa, liver, pancreas, thymus, and bursa of Fabricious showed multiple reddish cysts of 0.5 to 1 mm diameter. In addition, epicardium of heart (Fig. 3), crop mucosa, proventriculus and gizzard mucosa (Fig. 4) intestinal serosa (Fig. 5) as well as mucosa, liver (Fig. 6), pancreas (Fig. 5), oviduct serosa and broad ligament (Fig. 7), spleen, skull and meninges (Fig. 8) revealed multiple raised white thin cysts of 1 to 2 mm diameter. Liver was enlarged, yellowish and friable in two cases. Both kidneys were enlarged, haemorrhagic with adhered blood clots (haematoma). Two birds showed perirenal haemorrhages masking the kidneys (Fig. 9). In two birds, ovarian follicles were mishappened, shrunken or haemorrhagic and showed a few scattered raised red or white thin cysts of 1 to 2 mm diameter. On microscopic examination, contents in the cyst on the heart, intestine and liver revealed various sizes of round schizonts of leucocytozoon (Fig. 10).

Histopathologically, comb revealed ruptured megaloschizont (Fig. 11) with haemorrhage. Pectoral muscle showed ruptured megaloschizont (Fig. 12) with haemorrhage along with infiltration of macrophages, lymphocytes and giant cells. Heart, lung, proventriculus, gizzard, intestine, liver, pancreas kidney, ovary, oviduct, thymus, spleen and bursa of Fabricious were revealed the megaloschizonts of Leucocytozoon. The megaloschizonts appeared either as singular or aggregates with round unilocular structure having well defined thin eosinophilic capsular wall and contained numerous basophilic merozoites. Most of the megaloschizonts were intact or had irregular capsular wall and a few showed ruptured wall with haemorrhage around it. Heart showed scattered clumps of megaloschizonts (Fig. 13). Myocardial fibres near the megaloschizont showed atrophy. The megaloschizonts observed near the epicardium caused its bulging (Fig. 14). Tracheal mucosa showed desquamation, congestion with infiltration of lymphocytes and macrophages. Lungs showed moderate congestion, megaloschizonts with irregular wall (Fig. 15 & 16) and lymphocytes and macrophages infiltration. Proventriculus showed megaloshizonts in the mucosa causing compression of mucosal epithelium and glandular acini (Fig. 17). Gizzard mucosa as well as the muscular layer showed clumps of megaloschizont of various sizes (Fig. 18) wherein the nearby muscles showed atrophy (Fig. 19). Intestinal mucosa showed lymphocytes and macrophages infiltration along with megaloschizonts in the mucosa as well as the serosa (Fig. 20). Liver showed periportal hepatitis with scattered megaloschizonts (Fig. 21). Around megaloschizonts, the hepatocyte showed atrophic changes. Pancreas showed aggregates of megaloschizonts (Fig. 22) with atrophy of surrounding acinar cells. Kidneys showed either single or clumps of megaloschizonts (Fig. 23). In the kidney, lesions consist of vacuolar changes in the tubular epithelial cells and ruptured megaloschizont with haemorrhages were also noticed. Ovary showed multiple clumps of megaloschizonts with compression of stroma seen adjacent to the normal ovarian follicles which lined by cuboidal follicular epithelial cells (Fig. 24). Oviduct mucosa as well as muscularis layer showed scattered multiple clumps of megaloschizonts with compression of nearby structures (Fig. 25). Thymus showed megaloschizont (Fig. 26) and one lobe of thymus revealed ruptured megaloschizont with infiltration of erythrocytes, lymphocytes, macrophages and giant cells. Spleen showed intact megaloschizonts (Fig. 27) as well as ruptured megaloschizonts with infiltration of mononuclear cells. The bursa of Fabricious revealed multiple aggregates of megaloschizonts (Fig. 28) with round intact as well as irregular wall and follicle showed lymphoid depletion. The organs which showed intact megaloschizonts did not show any inflammatory reaction in the tissues. But the organs which had ruptured megaloschizonts showed inflammatory reaction with infiltration of lymphocytes, macrophages and giant cells.

An outbreak of Leucocytozoon caulleryi infection in the farming area might be related to the Culicoides spp. vector population. Constant high humidity is important for the development and fecundity of arthropod vectors⁹. External features of pale comb, wattle, skin, beak and conjunctival mucous membrane were in accordance with earlier report⁴. Grossly, megaloschizonts apperead as red or white thin cyst are found in numerous tissues including the heart, lungs, proventriculus, ventriculus, intestines, liver, pancreas, kidneys, spleen and brain⁸. Multiple reddish cysts observed in various organs like subcutaneous tissues of neck, pectoral muscle, proventriculus, gizzard, intestine mucosa, liver, pancreas, thymus and bursa of Fabricious were in agreement with earlier report⁹ who observed similar lesion in subcutaneous region, thigh, pectoral muscle, thymus, heart, pancreas and kidneys. Multiple raised white thin cysts observed in various visceral organs, skull and meninges were in accordance with recent reports⁴. Haemorrhage with adhered blood clots masking the kidneys was in agreement with earlier ovary and oviduct might result in malfunction of ovum production and oviductal secretion¹⁰.

Microscopic examinations provide definitive diagnostic tool for diagnosis of leucocytozoonosis. Histopathologically, L. caulleyri presents its characteristic megaloschizont as singular or aggregates and contained numerous basophilic merozoites in various organs, which are distinctive from other avian hemosprodians including Leucocytozoon spp. infective for other avian hosts, such as L. simondi for duck leucocytozoonosis¹⁰. Megaloschizonts specific for Leucocytozoon caulleryi were observed in various organs like comb, pectoral muscle, heart, lung, proventriculus, gizzard, intestine, liver, pancreas kidney, ovary, oviduct, thymus, spleen and bursa of Fabricious which were similar to earlier reports^4,9-11. Most of the megaloschizonts in tissues were intact or had irregular capsular wall. The organs with intact megaloschizont did not show any inflammatory reaction of the tissues. Some organs showed ruptured wall with haemorrhage around it. Leucocytozoonosis infections in chicken cause a hemorrhage in soft tissues due to the release of second generation schizogony³. In the organs with ruptured megaloschizont, inflammatory reaction with infiltration of lymphocytes, macrophages and giant cells were also seen. Similar lesions were observed in various organs in a earlier report¹⁰. Leucocytozoon caulleryi might cause severe hemolytic anemia due to destruction of erythrocytes by gametocytes and subsequent hemorrhagic anemia due to vascular endothelial damage³.

The present study reports the occurrence of leucocytozoon infection in white Leghorn layer chicken. The affected chickens suffer from internal hemorrhages and anemia, resulting in increased mortality and decreased egg productions with severe economic loss to the farmers. Hence, the farmers and veterinarians need to be aware of leucocytozoonosis infection in chicken.

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