Signalment:  

Six week-old, Welsh Harlequin Ducks (Anas platyrhynchos domestics). A group of eleven Welsh Harlequin ducklings hatched in Northern Ontario in late June were housed indoors until approximately 4-5 weeks of age, and then were allowed access to an outdoor enclosure during the day. Other than green watery diarrhea, the ducks were clinically normal. However in mid-August, at six weeks of age, one of the ducks became inappetent and lethargic, exhibited labored breathing, and died overnight. In the following two days, three more ducks died, the last two exhibiting similar clinical signs as the first. In total, the owner lost 5 of the 11 ducks; two recovered following intensive supportive care. Chickens were also maintained on the same property but housed in a separate enclosure and suffered no morbidity or mortality. Pens were cleaned every 1-2 days; feed and water were always available and refreshed daily.


Gross Description:  

Two ducks were received for postmortem examination. Both ducks had similar findings and were in good body condition with good muscle mass, external and internal fat stores but the tissues were generally pale. There was mild generalized subcutaneous edema and focal hemorrhage in the caudal pectoral muscle. The lungs were very edematous, pink/purple, and pleural spaces of the lateral margins of the lungs contained small amounts of clear fluid. There was focal hemorrhage on the epicardium. The spleen was very enlarged and soft. The liver was enlarged, tan/red and soft. The esophagus and proventriculus were empty; the gizzard contained grit and the intestine contained only a small amount of bile. Kidneys and bone marrow were pale. 


Histopathologic Description:

BRAIN (Slide A): Variable numbers of large protozoal megaloschizonts containing enlarged host nuclei and numerous cytomeres and infrequently degenerate/ruptured megaloschizonts are present in the brain, occasionally within distended endothelial cells filling capillary lumens, infrequently rimmed by a small amount of hemorrhage with a few mononuclear cells and granulocytes in the surrounding parenchyma. The ruptured megaloschizonts are often surrounded by larger numbers of inflammatory cells, including multinucleated cells and released merozoites. Numerous small vessels are infiltrated and surrounded by narrow cuffs of mononuclear cells and granulocytes. Hypertrophied astrocytes and reactive microglia are present within the parenchyma surrounding affected vessels, and also in close proximity to infrequent small foci of acute parenchymal necrosis.

LIVER (Tissue not provided): Within the liver, there is widespread multifocal to coalescent acute hepatic necrosis with mild to marked hemorrhage and hepatic dissociation. Sinusoids and vessels contain numerous red blood cells with eccentric compressed nuclei and round cytoplasmic structures morphologically compatible with protozoal gametocytes (Figure 2). Kupffer cells are enlarged and contain pale orange pigment, cellular debris and occasionally phagocytized erythrocytes.


Morphologic Diagnosis:  


Brain: Mild encephalitis with vasculitis and perivasculitis, mild multifocal necrosis with numerous intraparenchymal megaloschizonts
Liver: Marked acute multifocal to coalescent hepatic necrosis with numerous intraerythrocytic gametocytes and mild erythrophagocytosis


Lab Results:  

No bacterial pathogens were isolated from Duck A spleen and Duck B heart blood. RT-PCR testing of lung/trachea and cecal tonsil pools for Avian influenza virus (AIV) and avian paramyxovirus-1(APMV-1), and pooled brain/kidney for West Nile virus (WNV) and Eastern equine encephalitis virus (EEEV) were negative.


Condition:  

Leukocytozoon simondi, duck


Contributor Comment:  

The megaloschizonts with the enlarged host nuclei found only in the brain in these ducks are characteristic of Leukocytozoon spp.; Leukocytozoon simondii is the species that infects ducks and geese. The vector for L. simondi is the blackfly (Simuliidae spp.) and infective sporozoites carried in the salivary gland are introduced into the circulation of the bird at the time of the insect bite.(1,3) The sporozoites travel to the liver, enter hepatocytes and develop into first-generation meronts. These enlarge, go through multiple rounds of nuclear division, and form sections called cytomeres which are multinucleated. These further develop into uninucleate merozoites and multinucleate syncytia.(1,3) Some of the merozoites re-enter hepatocytes for a second round of merogony, while some merozoites and syncytia enter the circulation. The merozoites penetrate erythrocytes and develop into round gametocytes, and the intravascular syncytia travel to various organs where they are phagocytized by reticuloendothelial cells, including macrophages, and develop into second generation meronts called megalomeronts or megaloschizonts because of their large size (100 to 200 μm).(1,3) A megaloschizont characteristically contains a markedly enlarged centrally located host nucleus and numerous cytomeres.(1) L. simondi megaloschizonts can be found many tissues including brain, lung, spleen and heart.(1) When the megaloschizonts mature, they release large numbers of tiny merozoites that enter erythrocytes or leukocytes and develop into round or fusiform gametocytes.(1,3) The infective gametocytes are ingested by a biting fly, undergo sexual reproduction, produce a zygote that becomes a motile ookinete. Upon entering the small intestinal epithelium, the ookinete transforms into an oocyst, undergoes sporogony to produce sporozoites which then relocate to the salivary gland of the biting fly to complete the life cycle.(1,3)

The principal clinical effect of Leukocytozoon spp. infection is intravascular hemolytic anemia, thought to relate to the release of an antierythrocyte factor produced either by the meronts or their host cells rather than the primary mechanical destruction of the red blood cells by the protozoa, since the lowest hematocrit values occur before the parasitemic spike.(4)

In discussions with the referring veterinarian, the black fly population in the area appeared to be very low in early August, but in susceptible ducks, very limited exposure to infected blackflies is sufficient for the intravascular introduction of sufficient sporozoites to cause mortality. The prepatent period for L. simondi infection is approximately two weeks, so this fits well with the length of time from the initial release of the ducks into the outside enclosure and the onset of mortality. The producer has been advised that leukocytozoonosis is one disease that will have to managed if they wish to continue to raise susceptible ducks in Northern Ontario.


JPC Diagnosis:  

Brain: Intraendothelial hemoprotozoal megaloschzonts, multifocal, moderate, with intraerythrocytic gametocytes and minimal perivascular inflammation.


Conference Comment:  

The histologic features discussed during the conference were very similar to the contributors histologic description. Slide variability was noted with some slides containing sections of pineal gland. Participants also discussed the ecology of avian hemo-parasites and how it relates to host pathology. Infection with hemoparasites in the reservoir host is very common and often an incidental finding, resulting in minimal pathology. However, when in aberrant host is infected, the megaloschizonts can cause marked tissue damage, often in the liver and spleen, resulting in clinical signs and death in severe cases. 

Avian blood parasites include three Haemosporidia genera which are closely related: Plasmodium, Haemoproteus and Leucocytozoon. All are transmitted by a variety of biting insect vectors, which vary depending on the specific parasite, geographic location and distribution. The majority of bird species can become infected, but some species are more susceptible to infection than others (i.e. waterfowl, wild turkeys and penguins are commonly infected, but infection is less common in migratory shorebirds). 

In general, Plasmodium and Leucocytozoon species are capable of causing more severe disease, and species of Haemoproteus are considered less pathogenic. Hepatomegaly and splenomegaly may be seen in infection with all three genera, but Plasmodium and Haemoproteus infection results in production of hemozoin pigment from digestion of hemoglobin, which can be seen in the liver, spleen and other organs, and may impart a black or brown appearance to the organs grossly. Leucocytozoon infection does not result in production of hemozoin pigment, so organs will not have the dark discoloration. A presumptive diagnosis can be made based on microscopic examination of a blood film, and the appearance of the parasite in red blood cells (RBC).  Leucocytozoon results in the most dramatic change in RBC structure, with enlargement and elongation of RBCs and formation of hornlike extensions at each end of the cells; splitting of the nucleus may also be seen. With Haemoproteus and Plasmodium, less dramatic changes are seen and include slight enlargement of the cells, lateral displacement of the nucleus, hemozoin pigment, and the presence of the small schizonts or gametocyte nuclei.(2) Because the different hemoprotozoa can cause similar lesions, both grossly and histologically, PCR is often necessary to reach a precise etiologic diagnosis.(5)

There are many species of Leucocytozoon, but not all are pathogenic. The species of Leucocytozoon which are pathogenic to wild birds include L. simondi (waterfowl), L. marchouxi (doves and pigeons) and L. toddi (raptors). The species which may cause disease in domestic birds include L. simondi (waterfowl; U.S., Canada, Europe), L. smithi (turkeys; U.S., Canada), L. macleani (chickens; S.E. Asia), L. struthionis (ostriches; S. Africa), L. schoutendeni (chickens; sub-Saharan Africa, S.E. Asia) and L. caulleryi (chickens; south and S.E. Asia).(1)

In Leucocytozoon infection, gross lesions seen in infected birds include splenomegaly, hepatomegaly, tissue pallor and thinning of the blood. Histologically, capillaries may be distended by gametocytes with absence of host tissue reaction. The large megaloschizonts, which are often associated with small vessels, typically elicit a mononuclear inflammatory reaction but the meronts may or may not be associated with an inflammatory response. After the megaloschizonts rupture they may be filled with eosinophilic debris and mononuclear cells. Severe centrilobular hepatic necrosis may be seen along with periportal hepatitis and pigment laden macrophages / Kupffer cells. The enlarged spleen is congested, with loss of normal architecture, and contains large macrophages distended with pigment and cellular debris. Lymphocytic and histiocytic infiltration may be seen in other organs such as the lung and myocardium.(1)


References:

1. Forrester DJ, Greiner EC. Leucocytozoonosis. In: Atkinson CT, Thomas NJ, Hundter DB, eds. Parasitic Diseases of Wild Birds. Ames, Iowa: Blackwell Publishing; 2008:54-107.

2. Friend M, Franson JC, Ciganovich EA. Field manual of wildlife diseases, general field procedures and diseases of birds. Washington DC: U.S. Department of the Interior, U.S. Geological survey; 1999:193-200.

3. Gardiner CH, Fayer R, Dubey JP. Leucocytozoon. In: An Atlas of Protozoan Parasites in Animal Tissue. USDA Agricultural Research Service. Agriculture Handbook # 651; 1988:72.

4. Kocan R. Anemia and Mechanism of Erythrocyte Destruction in Ducks with Acute Leukocytozoon Infections. J Protozol. 1968;15(3):455-462.

5. Schmidt RE, Reavill DR, Phalen DN. Pathology of pet and aviary birds. Ames, IA: Wiley Blackwell; 2015:109-110.



Click the slide to view.



4-1. Spleen, liver


4-2. Cerebellum


4-3. Cerebellum


4-4. Cerebellum



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