Avian haemosporidian parasites: An updated review

: Avian hosts are vulnerable to many infectious agents including parasitic diseases. Haemosporidians (Sporozoa: Haemosporida) is a group of internal parasites of blood that infect domestic and wild birds causing loss of productivity and sometimes death. More than 200 species of haemosporidian parasites were identified in the blood and tissues of avian hosts. However, Leucocytozoon , Plasmodium , and Haemoproteus spp . are the most common and widely distributed haemosporidian parasites. Insect vectors including Culex mosquitoes, Simulium spp . , midges or hippoboscid flies, and Argas persicus are the main route of infection and transmission of haemosporidian parasites. Climatic conditions such as temperature, humidity, and vector activities can also play an important role in the distribution of infections. Affected birds may show no clinical signs during mild infection, however, signs of loss of weight, drop in egg production, anemia, anorexia, pale comb, green droppings, dyspnea, and variable mortalities may be observed especially in heavy infection. Different lesions in the internal organs such as the liver, spleen, and kidneys can be detected. Diagnosis of haemosporidian parasites is mainly based on microscopic examination of stained blood smears and/or molecular identification of the different stages of the parasite in the blood or tissues. Control of infection depends on the eradication of insects and the treatment of the affected birds using specific drugs. This review article was designed to take a look at avian haemosporidian parasites regarding types, distribution, diagnosis, and control .


Introduction
Avian species are susceptible to numerous types of bacterial, viral, fungal, and parasitic infections.Among the various parasitic diseases of poultry, haemoparasitic infections are considered very significant (Dunn and Outlaw, 2019).Haemosporidians (Sporozoa: Haemosporida) are a group of endoparasites of blood that inhabit a broad range of avian species (Sehgal, 2015).The significant economic importance of blood parasites include increasing mortalities (Valkiūnas, 2005), retardation of growth, and reproductive failure (Marzal et al., 2005).A reduced immune response is a sequence of events due to the destruction of immunological cells such as white blood cells (La Puente et al., 2010).
There are more than 200 species of identified avian blood protozoon parasites (Atkinson et al ., 2009).The common avian haemoprotozoans are Leucocytozoon, Plasmodium, Haemoproteus, Aegyptinella, Eperythrozoon, Fallisia, Haemobartonella, and Trypanosomes spp.(Mohammed et al., 2019).Nematode Microfilariae are also common in the blood of birds (Silveira et al., 2010).Besides, sporozoan Hepatozoon, Babesia, and Atoxoplasma genera are other vector-borne avian blood parasites.Blood parasites show some stages of development in both tissues and circulating red blood cells (RBCs) of the infected hosts.Wild and domestic birds are vulnerable to that intracellular haemosporidian (Buranapim et al., 2019;Lawal et al., 2021;Sadaf et al., 2021;Aiyedun et al., 2022).However, Haemoproteus and Leucocytozoon spp.are somewhat host-specific and restricted to species of birds in the same family.Plasmodium spp.show a much broader host specific and may infest several families of birds by changing their morphology and genetic characters (Atkinson, 1986).Free-ranging birds act as a link between commercial poultry and wild bird populations.Accordingly, the free-range breeding systems show high incidences and susceptibility to this type of parasitic infestation (Opara et al., 2014).
The interaction between the blood parasites and the avian host is complex (Martinez and Merino, 2011).Factors that influence the prevalence's variations of haemosporidian in avian communities may include environmental conditions, species, age, sex, and the reproductive and behavioral status of the host, as well as the presence of vectors (Laurance et al ., 2013;Rodrigues et al., 2021).Climatic changes such as warm and dry climates reduce the prevalence of blood parasites (Mirzaei et al ., 2020;Castaño Vázquez and Merino, 2022).When the vector populations are low, infected birds may show no clinical manifestations but parasites can survive during dry seasons.Haemoparasites might induce depression, anorexia, reduced productivity, growth retardation, green feces, dyspnea, and mortality, and having a negative influence on the birds' behavior (Sørci and Møller, 1997;Dunn et al., 2011).
The diagnosis of blood parasites has relied on the conventional microscopic examination of stained blood smears and/or the molecular techniques using polymerase chain reaction (PCR) assays (Tostes et al., 2015;Bernotienė et al., 2016;Chawengkirttikul et al., 2021).Insufficient preventive or treatment measures could increase infection rates and birds mortality (Pérez-Tris and Bensch, 2005).Therefore, the main objective of the present review article was to describe the avian haemosporidian parasites regarding their genus/species, world distribution, ways of diagnosis, and methods of controL.

Leucocytozoon spp.
Leucocytozoonosis is a protozoan disease that affects the blood and the internal organs of different avian species (Forrester and Greiner, 2008).Chickens, turkeys, pigeons, raptors, waterfowl, ostriches, and wild birds are susceptible to such infection (Bennett et al., 1993).Leucocytozoon (L.) spp.are named according to the species of birds in which they were detected.For instance, L. smithi in turkeys, L. sabrazesi in fowl, L. simondi and L. caulleryi in anseriformes, L. marchouxi in columbiformes, L. toddi in falconiformes, and L. ziemanni in owls (Soulsby, 1982).Leucocytozoon spp.are generally transmitted by insect vectors such as Culicoides midges and Simuliidae (black fly) spp.(Desser and Bennett, 1993).Leucocytozoon spp.have a complex life cycle; a schizogony (merogony) stage in tissues, a sexual (gametogony) stage in RBCs or leukocytes of vertebrates, and a sporogony stage in simuliid fly or culicids midges (Valkiūnas 2005).Biting of the host with the mosquito vectors results in the inoculation of sporozoites in the blood and then attacks the endothelial cells of the lung, liver, and spleen (Zhao et al., 2015).In the liver cells and vascular endothelium of other tissues, the sporozoites of Leucocytozoon spp.transform to small schizonts and megaloschizonts, and then form gametocytes in the erythroblasts and mononuclear leucocytes (Zhao et al., 2015).Yin et al. (2002) found that intravenous inoculation of L. caulleryi sporozoites in 26-day-old chickens induced early production of schizonts in the lung, spleen, and thymus at the 6 th -day post-inoculation.Moreover, L. simondi was detected as a comma shape parasite in the hepatocytes, alveolar epithelial cells of lungs, and renal tubules of ducks' tissues (Shutler et al., 1999;Dey et al., 2008).
Heavy infections with Leucocytozoon could lead to a high mortality rate (Hunter et al., 1997).Naturally infected chickens with L. sabrazesi may show acute mortality of more than 50%, or chronic drop in egg production, depression, anorexia, anemia, pale comb, and green droppings (Zhao et al., 2015).In South Korea, an outbreak of L. caulleryi infection has been histopathologically and molecularly detected in a broiler breeder chicken flock with a history of depression, sudden death, and subcutaneous hemorrhages in the wings and legs, muscles, thymus, epicardium, pancreas, and kidneys (Lee et al ., 2014).A further study by Lee et al. (2016) showed mortality and decreased egg production in 59 and 82-week-old layer chickens due to L. caulleryi infection.Dead birds displayed enlarged, fragile, yellowish, and hemorrhagic livers, and the protozoon megaloschizonts were microscopically and molecularly detected in the liver and ovaries tissues (Lee et al ., 2016).Moreover, naturally infected Pekin ducklings with L. simondi showed anemia, gametocytaemia, and osmotic fragility of erythrocytes (Maley and Desser, 1977).

Plasmodium spp.
Avian malaria is a parasitic disease caused by protozoan parasites of the Plasmodium genus (Jennings et al., 2006).This genus has a close linkage to Haemoproteus and Leucocytozoon genera.Over 65 Plasmodium spp.have been detected in more than 1.000 different avian hosts.However, P. gallinaceum, P. juxtanucleare, and P. durae are the most pathogenic to avian species and cause a 90% mortality rate (Springer, 1991).Additionally, P. gallinaceum, in particular, is known to cause severe illness and mortality rates up to 30% to 80% (Soulsby, 1982).The transmission of Plasmodium spp. to birds via mosquito was first detected in India by Ross in 1898 (Carlton, 1938).Avian orders including Columbiformes, Galliformes, and Passeriformes are highly susceptible to Plasmodium spp., while Struthioniformes, Coliiformes, and Trogoniformes are resistant (Valkiūnas et al., 2005;Martinsen et al., 2008).Plasmodium sporozoites are transmitted to the avian hosts through the salivary glands of Culicinae, Coquillettidia, and Anophelinae mosquitoes (Njabo et al., 2011).Schizonts present in the macrophages and fibroblasts develop into merozoites in the hepatocytes, and then finally to gametes in the erythrocytes (Campbell, 1995).Both Plasmodium (P.) gallinaceum and P. juxtanucleare (Bisseru and Lim, 1971) and P. vaughani (Ludin et al., 1994) were isolated from crows, P. formosanum from White-breasted Waterhens (Yap et al., 1986), and an unidentified Plasmodium spp.from the Mountain Fulvetta (Paperna et al., 2008) in Malaysia.Therefore, wild waterfowl and wetland birds showed high rates of Plasmodium spp.infections because of their proximity to the insect vectors along waterways and tall reed beds.
The pathogenicity of Plasmodium in avian hosts usually varies during acute and chronic infections (Atkinson et al., 1995;Palinauskas et al., 2011).Affected birds may show no apparent clinical signs, while others show hepatomegaly with necrosis, fatty liver, splenomegaly, pericardial effusion and hydropericardium, nephritis, edematous lungs, occlusion of the brain capillaries, and hemorrhagic conjunctivitis (Omar and Lim, 1962;Ishtiaq et al., 2012;Dimitrov et al., 2015).Moreover, Plasmodium causing malaria induces pale watery blood, numerous immature erythrocytes, hemolytic and acute anemia, lymphocytosis, leukocytosis, hypoalbuminemia, diffuse areas of extramedullary erythropoiesis in the liver and kidney, and hemoglobinuria (Atkinson et al., 2000;William, 2005;Ferrell et al., 2007).Experimental infection of native Hawaiian forest birds with P. relictum revealed a reduction of feed intake and body weight as well as deaths due to anemia-associatederythrocyte parasitemia (Atkinson et al., 1995).For human health considerations, Plasmodium spp.inducing avian malaria are closely related to the malarial parasites of humans, but they are not able to infect people.
Infections with Haemoproteus spp.have been associated with muscle affection, hepatomegaly, and splenomegaly (Atkinson and Forrester, 1987;Cardona et al., 2002).Moreover, hepatic hemorrhage, hemocoelom, and sudden death could also be observed in passerine birds (Donovan et al., 2008).Under natural or experimental conditions, H. meleagridis infection could be accompanied by muscle pathology in turkeys (Atkinson et al., 1988).The scarcity of studies on Haemoproteus infections in commercial poultry may be attributed to the difficulties in differentiating this parasite from Plasmodium during microscopic examination (Atkinson et al., 2009).Moreover, the pathogenesis of Haemoproteus infections in birds is still well not understood (Atkinson et al., 2009).

Incidence and distribution
Avian haemosporidian affections are genetically diverse (Ishtiaq et al., 2012;Padilla et al., 2017;Nourani et al., 2022).Infections with blood protozoon parasites have been reported in many countries all over the world, particularly those of hightemperature climates and vector activities.The distribution of blood parasites relies on host-parasite (Knowles et al ., 2011) and vector-parasite (Carlson et al., 2015) compatibilities, mosquito feeding behavior (Medeiros et al., 2015), and climatic conditions (Garamszegi, 2011).The temperature, relative humidity, and rainfall reflect the numbers and activities of their insect vectors (Zamora-Vilchis et al., 2012).The broad host susceptibility to haemoprotozoan may increase the transmission rate and the prevalence of the parasites in numerous avian species (Bensch et al ., 2009).Table 1 shows the incidence of avian haemosporidian parasites in different countries, from 1991 to 2023.

George et al. (2004)
The infection rate of Plasmodium spp. in pigeons was 3.0%.

Bui et al. (2005)
The prevalence rate of Plasmodium spp. in 575 chickens and Guinea fowls was 9.4 %.

Opara et al. (2014)
A total of 1820 chicken blood samples were examined for haemoparasites and the results showed a prevalence of 19.6% for 3 genera of haemoparasites in a single or mixed infections.Plasmodium spp.were the most prevalent (13.9%) followed by Haemoproteus (2.6%) and Leucocytozoon (0.4%) in single infection, but the prevalence of mixed infection with Plasmodium and Haemoproteus was 2.6%.Moreover, the prevalence rate was higher in male chickens (28.5%) than females (8.9%), and in adults (23.0%) than growers (11.0%).The prevalence rates were 39.3%, 12.5%, and 7.7% in the rainy, cold dry, and hot dry seasons, respectively.

Lawal et al. (2019)
Out of 108 local chickens (49 males and 59 females) were examined.Females showed higher infection rate with blood parasites (53.1%) than males (46.9%).Plasmodium spp.were mostly found with a prevalence of 54.6%, in both males and females' chickens.

Malawi
The prevalence rate of blood parasites was 71%.Moreover, the indigenous scavenging chickens revealed the higher prevalence of infection (29.5 %) compared to the commercial and intensively managed chickens (0 %).

Sabuni et al. (2011) Uganda
Haemoproteus and Plasmodium spp.were detected in the blood samples of 43 pigeons.The prevalence of Haemoproteus was 76.5%.

Dranzoa et al. (1999)
Microscopic and PCR approaches were done on blood samples of 304 free-ranging chickens, 70 ducks, 14 turkeys, and 19 Guinea fowl.The results showed presence of Haemoproteus (17.25%, n=69), Plasmodium (22%, n=88), and Leucocytozoon (1.75%, n=7) in the sampled birds.The sequences from these genera were nested within their respective clades in a phylogenetic tree constructed using sequences from the MalAvi database.

Nakayima et al. (2019)
Uganda and Cameroon Using of PCR and microscopy, out of 148 samples, 18.3% and 4.1% had L. schoutedeni and T. gallinarum, respectively.The phylogenetic analysis of the cytochrome b gene of L. schoutedeni identified 2 distinct lineages that were found at all 3 sampling locations in Uganda.The sequence divergence between these 2 lineages Sehgal et al. (2006) http://dx.doi.org/10.5380/avs.v28i2.91244

Country
Incidence in avian spp.Reference is 1.5%.One of these lineages was also detected in Cameroon.

Earlé et al. (1991)
Aegyptianella botuliformis and Aegyptianella pullorum were detected in the blood of Guinea fowls.Argas spp.were the vectors of the detected blood parasite.

Elahi et al. (2014)
A total of 213 pigeons blood samples were collected from Chittagong and Khulna districts.The results showed that mature and immature stages of Haemoproteus gametocytes were seen in 43.63% and 58.25% in Chittagong and Khulna district, respectively.The prevalence was almost similar in the areas of Khulna district (55.55 -62.86%), whereas, a fluctuation observed in Chittagong district from 33.33 to 59.52%.

Momin et al. (2014)
The prevalence rate of Leucocytozoon was 12% in chickens.

Mirzaei et al. (2020)
Molecular diagnosis of 152 avian hosts belonging to 17 species revealed presence of haemosporidian in an overall prevalence of 22.36%.Haemoproteus spp.were detected in pigeons, while Plasmodium spp.were found in Hooded crows and Carrion crow.
Costa Rica Haemoproteus spp.and Microfilariae were found in the examined 248 blood samples and 114 birds, respectively.The prevalence of Haemoproteus spp. was 0.8% and 4.4% in Hitoy Cerere and Barbilla, respectively, However, the prevalence Microfilariae was 8.1% and 3.5%, respectively.

Benedikt et al. (2009)
Colombia A total of 315 birds including 75 species (23 families) showed that 50 birds (15.9%) had blood parasites.Microfilariae were the most common blood parasites, followed by Haemoproteus, Plasmodium, and Trypanosoma spp.

Germany
The prevalence rate of blood parasites in 1149 birds was 11% (adult birds 18%, immature birds 16%, and nestlings 4%).Among the Falconiformes 11% of 976 birds were infected, and 13% of 173 Strigiformes.Out of 17 falconiform spp., 9 were positive whereas the Eurasian buzzard showed the highest prevalence for haematozoa; i.e.L. toddi (31%), the highest prevalence (25%) for Haemoproteus spp. was found in the hobby.The tawny owl showed a highest prevalence with H. syrnii (22%).In the one pygmy owl, T. avium and P. fallax were found.The white-tailed sea eagle was a host of L. toddy.

Laboratory diagnosis
The different developmental stages of the haemoparasites could be microscopically detected in blood or tissue smears after staining with Giemsa (Valkiūnas, 2005).The gametocytes are seen in stained blood films, while schizonts are detected in tissue sections.However, the microscopic examination is insufficiently sensitive to detect the slight or early parasitism with a low number of sporozoites or gametophytes as in the case of Leucocytozoon spp.infections.Thus, the possibility of a falsenegative diagnosis by this method is common (Valkiūnas et al., 2005;Garamszegi 2010).The centrifugation of the blood samples facilitates the concentration and detection of the parasites in the buffy coat and this method is significantly better than the direct blood smear (Bennett, 1962).Further, the plasma layer over the buffy coat is rich in Trypanosoma and Microfilariae spp., while the stained buffy coat film is rich in Haemoproteus and Leucocytozoon spp.Besides, the diagnosis of blood parasites in dead birds could be achieved via the histopathological examinations of the affected organs or through the prepared tissue smears before formalin fixation.
The molecular diagnostic techniques of amplification and sequencing of DNA or RNA are also useful, potential, and sensitive to detect the different stages of parasites in the blood or tissues even when the blood smears are negative (Palinauskas et al ., 2013;Valkiūnas et al., 2014).However, some reports have shown that microscopic and molecular identifications have the same sensitivities for detecting avian haemoparasites (Valkiūnas et al., 2008(Valkiūnas et al., , 2009)).The PCR assay is regarded as an accurate and easy as well a rapid approach to investigate the presence of some blood parasites such as Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp.(Hellgren et al., 2004).Gq et al. (2003) considered PCR as a special and sensitive test that shows no cross-reaction between L. caulleryi and other protozoon parasites, besides it can detect the lowest DNA concentration of the parasite in the tissues.The molecular-based detection of avian blood parasites usually depends on the mitochondrial genes (mtDNA) and the genomic markers that have been developed in the phylogeny of Plasmodium and related haemoprotozoon (Martinsen et al., 2008;Braga et al., 2011).Moreover, the molecular detection of a mitochondrial cytochrome b (cytb) gene could be a reliable, highly sensitive, and specific method of diagnosis, particularly in cases with false-negative blood smear results (Ortego and Cordero, 2009;Ramey et al., 2012;Suprihati and Yuniarti, 2017).By the amplification of a 478 bp fragment from the cytb gene, 6 Haemoproteus and two Plasmodium lineages were identified in the biting midges and the blood of 123 bird's spp.(Ferraguti et al., 2013).

Prevention and control
The adoption of modern intensive poultry production programs could reduce the incidences of parasitic infections.Moreover, the implementation of specific eradication measures for arthropod vectors could alleviate the possibility of parasite transmission (Piersma, 1997).Despite the employment of intensive control and management measures, few interventions have been directed toward managing infections in free-ranging domestic birds (Wong et al., 1998).
Primaquine is a member of the 8-aminoquinoline compounds that are effectively used to inhibit the sexual stages of some parasites such as Plasmodium spp.(Ashley et al., 2014) and Leucocytozoon spp.(Merino et al., 2000;Zhao et al., 2016).
Pyrimethamine is used to control the asexual stages of the parasite in the blood (Baird and Hoffman, 2004;Baird, 2005).In addition, ketotifen, desipramine hydrochloride, and clomipramine hydrochloride are tricyclic antihistamines/antidepressants that have transmission-blocking activities against Plasmodium spp.inducing malaria.They inhibit the fertilization of gametocytes and the early development of oocysts in mosquitoes (Eastman et al., 2013).The gametocytocidal and transmission-blocking activities of artesunate in a P. gallinaceum-avian model were successfully evaluated (Kumnuan et al., 2013).Aminoguanidine, an inhibitor of inducible nitric oxide synthase, could treat P. gallinaceum-infected chickens in terms of increasing the survival rate, thrombocytopenia, and reducing anemia and hemozoin levels in the spleen and liver (de Macchi et al., 2013).However, L. simondi was not affected by paludrine, atebrin, and sulphamerazine treatments in ducklings (Fallis, 1948).Zhao et al. (2014) demonstrated that gametocytaemia of L. sabrazesi in blood samples of infested chickens showed 'relapse' or persistence of low-level for 4-5 months, which could be useful as an in-vivo model for the evaluation of the drug against liver stages of the parasite.

Conclusion
The present study brings an update on the incidence, distribution, and diversity of avian haemoparasites worldwide.The constant monitoring of the occurrence of avian haemoparasites in domestic poultry and wild migratory birds is required to reduce the risks of potential outbreaks.This could affect the international poultry industry as well as disrupt fragile wild bird populations.
Senlik et al. http://dx.doi.org/10.5380/avs.v28i2.91244Out of 313 blood samples of chickens, the PCR assay showed cytb gene of L. sabrazesi in 80.51% (252/313).Chawengkirttikul et al. (2021) Malaysia Giemsa-stained blood smears of 728 Galliformes, Anseriformes, Phoenicopteriformes, Pelecaniformes and Gruiformes revealed presence of Plasmodium in a prevalence of 8.0%.The Anseriformes and Gruiformes showed infection rates (31.8-50.0%).The prevalence of Plasmodium was 2.7% in domestic poultry.Moreover, L. sabrazesi and L. caulleryi were limited to the Galliformes with infection rates of 0.7% and 0.5%, respectively.Haemoproteus was detected in domestic poultry and Red Jungle fowls with an average prevalence of 0.8%.Trypanosomes and microfilaria were only present in the village chickens and Red Jungle fowls, with a high microfi laraemia rate (19.0%) in Red Jungle fowls.Indonesia The microscopic examination and molecular sequencing of cytb gene revealed presence of Plasmodium spp.and L. caulleryi in broiler chickens.The genetic distance between L. caulleryi taxa from various endemic areas is very close (<5%).Blood smears of 136 individuals belonged to 10 different families of songbirds were Nourani et al. http://dx.doi.org/10.5380/avs.v28i2.91244 Blood smears of 100 diseased pigeon's revealed presence of H. columbae in an infection rate of 30%.It was more pronounced in males (35.71%) than in females (16.66%) and more in adults (57.14%) than in young (15.38%).The sequence of H. columbae showed 100% identity with other related Haemoproteus spp. in Brazil and United Kingdom.

Table 1 -
The incidence of avian haemosporidian parasites in different countries.