Haemoproteus sp infection in a white gerfalcon (Falco rusticolus)

S. Knöpfler1, D. Brandstetter2, C. Hochleithner2, M. Hochleithner2, E. Leidinger1 1In Vitro Labor, Vienna, Austria 2Tierklinik Strebersdorf Hochleithner GmbH, Vienna, Austria

Signalment: White gerfalcon (Falco rusticolus), 6 months old, sex unknown

Hx: weakness, declines in food consumption and activity levels, reduced laying performance in several private owned falcons

Blood smear: A smear was prepared and sent to a private diagnostic lab for a routine haematology screening. A leukocyte estimate from the blood smear and differential blood count were performed.

Selected haematology analytes are presented in Table 1.

Table 1: Laboratory data (Reference intervals according to ISIS, 2002) Analyte Result Reference interval WBC adequate < 10 G/l Differential Heterophils 69% 40-60% Lymphocytes 29% 30-50% Monocytes 2% 0-5% Eosinophils 0% 0-4% Basophils 0% 0-4% Platelets adequate 100-500

By microscopic examination, most RBCs revealed an unremarkable morphology with a small number of polychromatic erythrocytes (physiological appearance). At a higher magnification, RBCs numerous, pleomorphic intra-erythrocytic organisms could be detected. Some of those were irregular round to elongated or „U“-shaped, with a round amphopilic central nucleus, and a moderate amount of basophilic cytoplasm that frequently contained yellow-brownish pigment granules. This pigment was most likely consistent with haemazoin (“malaria pigment”). Other structures were elongated and curved, basophilic and with dark pigment. The intra-erythrocytic structures often partly surrounded the host erythrocytes’ nucleus without dislocating it.

WBC numbers can be estimated from the stained blood smear by multiplying the average number of leukocytes per HPF by a given factor (e.g., 2000 for the 40x high-dry objectives). Due to an uneven distribution of the WBC and the platelets, it was not possible to apply this formula, but the WBC number appeared to be WRI. The granules of the heterophils were not always visible. There were a mild increase in the relative heterophil count and a very mild decrease in the relative lymphocyte numbers in differential blood count. Platelet estimate and morphology were also inconspicuous. Partly, they formed small aggregates.

Figure 1: Blood smear, Romanowsky-type stain, 400x magnification

Figure 2: Blood smear, Romanowsky-type stain 1000x magnification PCR The referring veterinarian subsequently requested a PCR-analysis for sp, , Haemoproteus sp, and Leukocytozoon sp. Results: - Plasmodium/Haemoproteus specific mitochondrial cytochrome B DNA positive - Plasmodium specific 18S rDNA negative - Leucozytozoon negative

Summary of the results The haematology examination revealed intra-erythrocytic organisms of different sizes, frequently containing pigment. The morphologic findings were consistent with Haemoproteus sp or Plasmodium sp.

The PCR revealed the presence of Haemoproteus sp and was negative for Plasmodium sp. Diagnosis Haemoproteus sp infection, morphologically, a Plasmodium sp infection could not be excluded.

Discussion The spezies of the apicomplexans Haemoproteus, Plasmodium and comprise a diverse group of vector-transmitted parasites that infect red blood cells (in the case of Leucocytozoon sp also white blood cells) and other organs within their vertebrate hosts1. Species of these parasite genera share several characters with human malaria parasites and all three, but most frequently Plasmodium sp only are referred to as avian malaria2. Apicomplexans have a very complex life circle, with the complete life cycles of Plasmodium sp being investigated best of all2. Haemoproteus is the most common blood parasite especially in nondomestic . More than 120 species have been reported. Haemoproteus sp have been described in free-ranging ducks, quails and turkeys but are rare to absent in commercial flocks, probably because of limited vector exposure or very specific feeding habits of sp and Hippoboscid sp flies, the invertebrate vectors. The protozoa has mainly a continental distribution and is absent from the majority of oceanic regions, what can also be explained by the vector exposure2. However, the complete life cycle, specifically transmission dynamics between avian and vector hosts, have poorly studied3. Typically, gametogony of Haemoproteus sp occurs within erythrocytes whereas schizogony occurs within endothelial cells. Consequently only gametocytes are observed within erythrocytes (in contrast to Plasmodium sp)3. Multiple gametocytes within a single erythrocyte are commonly observed with some species. Classically, the gametocytes of Haemoproteus sp are halteridial; i.e. elongated and curved, often partly surrounding the host erythrocyte´s nucleus. In this case we suspected macro- and microgametocytes in the erythrocytes. The predominant form, however, of the gametocytes varies between species. In this case we could identify macro- and microgametocytes in the erythrocytes. In general, the gametocytes have a distinct peripheral outline, the cytoplasm that contains variable amounts of a yellow to black-brown granular pigment and punctate purple granules, and a centrally located nucleus. Macrogametocytes, in comparison to microgametocytes, typically have amphophilic to basophilic cytoplasm. In contrast, microgametocytes typically have cytoplasm that stains a pale blue to pinkish colour with few granules, that are often polar in distribution and a nucleus that is more diffuse and stains an amphophilic to eosinophilic colour4. Haemoproteus is considered nonpathogenic in most avian species. Infected birds are often seen as asymptomatic carriers, showing mainly chronic parasitaemia2. In contrast, Plasmodium infection in birds is often associated with progressive weakness, declines in food consumption and activity levels. Loss of up to 30% body weight were described1.

Species of the Plasmodium have a wide distribution and can infect nearly all avian taxa5. The use of molecular methods has uncovered a greater genetic diversity and phylogenetic complexity of parasites and has resulted in the suggestion that there could be as many species of avian malaria parasites as there are host species6.

The development of Plasmodium sp in birds may be divided into exoerythrocytic and erythrocytic merogony and formation of gametocytes7. Sporozoites injected by the vector into a give rise to the first generation of primary exoerythrocytic meronts (cryptozoites). They develop predominantly in the reticular cells of many organs (, , , ). The merozoites induce the second generation of primary exoerythrocytic meront (metacryptozoites). They are able to infect RBCs 7.

Plasmodium sp infections are characterized by the concomitant presence of several stages of the organism within the erythrocytes of the host. Notably, gametocytes and schizonts (containing merozoites) may be recognized. The morphology of the organism varies between species of Plasmodium. In general, the gametocytes may be irregularly round, elongate, „U“- shaped, with a round amphophilic central nucleus, and moderately basophilic cytoplasm that contains several brown-black pigment granules2.

The diagnosis of Haemoproteus or Plasmodium infection in birds is usually based on morphological evaluation of the blood smear. However, the large gametocytes of these species look very similar by microscopic evaluation and haemazoin cannot be used for a reliable diagnosis. PCR analysis can differentiate the species and can also be used when parasites are too few to be identified in blood smear2. Despite PCR analysis for Plasmodium sp was negative in this case; a co-infection could not be excluded. Low parasitaemia as well as the primer used for PCR, especially in cases of co-infections, can lead to false negative results3. Unfortunately, the PCR protocol was not exactly known in this case.

In clinical cases different therapies, especially for poultry flocks were described. No antimalarial drug is commercially available or approved to treat birds. Mixtures of trimetoprim and sulfaquinoxaline or chloroquine and primaquine have been proposed3.

The prognosis depends on the infectious species. According to the fact, that birds are usually asymptotic carriers the prognosis of Haemoproteus sp infection in birds is good and a treatment is not needed3. Moreover, combinations of parasites may have different effects on host fitness because they may, positively or negatively, interact with each other8. According to the clinical signs, a co-infection with Plasmodium sp cannot be ruled out in this case.

References: 1. Atkinson CT, Dusek RJ, Woods KL, et al. Pathogenicity of avian malaria in experimentally-infected Hawaii Amakihi. Journal of wildlife diseases 2000;36:197-204.

2. Clark NJ, Clegg SM, Lima MR. A review of global diversity in avian haemosporidians (Plasmodium and Haemoproteus: ): new insights from molecular data. International journal for parasitology 2014;44:329-338.

3. Valkiunas G, Iezhova TA, Loiseau C, et al. Nested cytochrome B polymerase chain reaction diagnostics detect sporozoites of hemosporidian parasites in peripheral blood of naturally infected birds. The Journal of parasitology 2009;95:1512-1515.

4. Valkuinas G. Genus Haemoproteus. In: Valkuinas, G (Ed): Avian Malaria Parasites and other Haemosporidia 2005;1:259-566.

5. Valkiunas G, Anwar AM, Atkinson CT, et al. What distinguishes malaria parasites from other pigmented haemosporidians? Trends in parasitology 2005;21:357-358.

6. Waldenstrom J, Bensch S, Hasselquist D, et al. A new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood. The Journal of parasitology 2004;90:191-194.

7. Valkuinas G. Genus Plasmodium. In: Valkuinas, G (Ed): Avian Malaria Parasites and other Haemosporidia 2005;1:589-727.

8. Hellgren O, Waldenstrom J, Bensch S. A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. The Journal of parasitology 2004;90:797-802.