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A new Sarcocystis species has recently been reported in the domestic pigeon (Columba livia f. domestica) as intermediate host, causing severe central nervous signs similar to Paramyxovirus-1 or Salmonella typhimurium var. cop. infection. Transmission of the parasite via the Northern goshawk (Accipiter gentilis) as definitive host has been established. Experimental infection of domestic pigeons with sporocysts excreted by experimentally infected Northern goshawks reproduced the natural infection in the pigeon, proving the causative role of the parasite in the disease. Here, we describe the course of a fulminant biphasic disease depending on the infectious dose. Pigeons infected with 103 and 104 sporocysts showed clinical signs of polyuria and apathy as a first phase 10 to 11 dpi and sudden neurological signs 51 to 57 dpi as a second phase. Pigeons infected with higher doses died within 7 to 12 dpi also showing polyuria and apathy. At necropsy, liver and spleen had multifocal necroses and infestations with schizonts. Pigeons infected with 102 sporocysts kept symptomless until 58 to 65 dpi, when sudden central nervous signs occurred. Major histopathologic findings of pigeons with neurological signs were encephalitis and myositis of the skeletal muscles with high infestations of sarcocysts. By contrast, in the same experiment, chickens failed to develop any clinical signs or pathologic lesions. In conclusion we
Worldwide, a large number of Sarcocystis species (Protozoa: Apicomplexa) have previously been reported to infect avian species but have only occasionally been described in Columbiformes (Olias et al., 2009c). Some avian Sarcocystis species such as Sarcocystis neurona and Sarcocystis falcatula are known for their variety of natural and aberrant host species. S. neurona causes neurological disease in a broad range of animals, including domestic horses, dogs, cats, raccoons, skunks, minks and Rhesus monkeys (Klumpp et al., 1994; Dubey et al., 2001; Dubey et al., 2003; Dubey et al., 2006). S. falcatula, which is transmitted by the North American opossum (Didelphis virginiana), has been shown to be infectious for domestic pigeons but only in experimental infectious studies (Box & Smith, 1982). Among the very few species naturally infecting domestic poultry are Sarcocystis horvathi and Sarcoystis wenzeli which cyle between chickens (Gallus gallus) as intermediate host and dogs (Canis lupus) as definitive host, whereas S. wenzeli also uses cats (Felis catus) as alternative definitive host (Wenzel et al., 1982). Sarcocystis rileyi uses ducks as intermediate host and skunks (Mephitis mephitis) as definitive host (Riley, 1931).
We recently reported a novel neurologic disease with lethal outcome in domestic pigeons (Columba livia f. domestica) in Berlin, Germany, caused by a so far unknown Sarcocystis species (Olias et al., 2009a). Clinical signs, very similar to those caused by Paramyxovirus-1 or Salmonella typhimurium var. cop., included apathy, polyuria, torticollis, opisthotonus, paralysis, trembling and death. Pigeons had parasitic cysts in their muscles and obviously serve as intermediate hosts in a two-host life cycle characteristic for Sarcocystis, in which intermediate hosts (prey) are infected by ingestion of sporocysts shed with the feces by the definitive host (predator) (Mehlhorn & Heydorn, 1978; Dubey, 1989). The definitive host of this new Sarcocystis species was therefore hypothesized to be a typical pigeon predator, having regular contact to domestic pigeons. Experimental infectious studies of dogs (Canis familaris, beagle), ferrets (Mustela putorius furo), rats (Rattus norvegicus f. domestica), mice (Mus musculus domesticus), Northern goshawks (Accipiter gentilis) and Gyr-saker (Falco rusticolus Ã- Falco cherrug) hybrid falcons identified the Northern goshawk as definitive host, shedding high numbers of sporocysts in the feces (Olias et al., 2009b). Subsequent experimental infections of domestic pigeons with goshawk-derived and purified sporocysts clearly reproduced the natural disease and determined the causative role of the parasite, then named Sarcocystis calchasi, in this novel disease in pigeons (Olias et al., 2009c).
Here, we further describe the clinical course of the disease in the pigeons after experimental infection with S. calchasi. Further experimental infectious studies were conducted to test chickens (Gallus gallus) as alternative intermediate hosts of the parasite.
Material and Methods
As intermediate and potential intermediate hosts 17 adult domestic pigeons (Columba livia f. domestica) and ten 5-week-old SPF-chicken (Gallus gallus f. domestica; Valo, Lohmann, Germany) were used in this study. Biopsies of pectoral muscle of all birds had no parasitic structures prior to the experiment. The animals were examined 14 days prior to infection and were clinically healthy. Fecal examination confirmed absence of Salmonella species and endoparasites.
Source of Sarcocystis Sporocysts
The Sarcocystis species used for experimental infection was derived from the small intestine of one Northern goshawk (Olias et al., 2009b) experimentally infected with naturally cyst-infested pectoral muscle of a racing pigeon from a recent outbreak in Berlin, Germany in 2008 (Olias et al., 2009a).
Sporocysts were purified and counted as described elsewhere (Rommel et al., 1995). Sixteen pigeons were separated in eight groups of two birds each; one additional pigeon was housed in group 1 as sentinel animal. Infectious doses of sporocysts for the pigeons were 3 Ã- 106 sporocysts (group 1); 3 Ã- 105 (group 2); 105 (group 3); 104 on eight consecutive days (104 Ã- 8; group 4); 104 (group 5); 103 (group 6) and 102 (group 7). Pigeons of group 8 served as control animals. Sporocysts suspended in 2 ml sterile water were administered directly into the crop. The sentinel pigeon and the birds of the control group received only 2 ml of sterile water (table 1). The chickens were housed together in a standardized isolator. Two control chickens received 2 ml of sterile water and two chickens each a dose of 102, 104, 3 Ã- 105 or 3 Ã- 106 sporocysts, respectively. All pigeons and chickens were observed four times daily during the entire course of the experiment. Animals with neurological signs were euthanized. Surviving pigeons were euthanized 59 and 120 dpi, respectively. Chickens were euthanized 59 dpi. All experiments were performed under governmental approval (No. Reg 0111/08).
Gross and Histopathologic Examination
A complete necropsy was performed on all pigeons and chickens. Tissue samples from the skin, skeletal muscle (pectoral, gastrocnemius and neck muscles), bone marrow, oral cavity, oesophagus, crop, lung, heart, liver, pancreas, spleen, kidneys, proventriculus, ventriculus, intestine and brain were fixed in phosphate-buffered formalin and processed as described above.
Fecal Examination and Microbiology
After infection, fecal material of pigeons and chickens was tested twice daily between the 1st and 7th dpi for sporocysts using flotation with saturated sodium chloride solution (Rommel et al., 1995). For detection of Salmonella and Paramyxovirus, liver, gut, lung, brain, kidney and spleen samples from all pigeons were analysed as described (Olias et al., 2009a).
Identification of Parasites
For molecular detection of the D2 region of the 28S rRNA gene and characterization of potentially developed muscle cysts, 25 mg of pectoral muscle and liver of each pigeon was minced into small pieces and further processed for PCR as described above.
Pigeons of groups 1 to 4 ( 3 Ã- 106 to 8 Ã- 104 sporocysts) died within 12 dpi with severe apathy and polyuria (table 1; figure 1, 3A). The sentinel pigeon kept in group 1 developed mild apathy and polyuria 13 dpi but clinical signs of this animal resolved after three days. Pigeons of groups 5 and 6 (104 to 103 sporocysts) developed a biphasic disease with mild to moderate apathy and polyuria about 10 dpi, with signs resolving 12-18 dpi as a first phase. Eight weeks after infection, pigeons of group 5 developed severe and pigeons of group 6 moderate neurological signs including torticollis, opisthotonus, trembling and paralysis as the second phase of the disease (figure 2). Pigeons of group 7 infected with 102 sporocysts had mild to moderate neurological signs 57 to 64 dpi without earlier clinical signs. Control pigeons of group 8 remained healthy throughout the entire trial. None of the chickens developed any clinical signs.
Fecal Examination and Microbiology
Pigeons did not shed parasites in their feces at any time. No Salmonella spp. were cultured and no hemagglutinating agent was detected in all samples examined.
Pathological and Histopathological Lesions after Infection
At necropsy, pigeons of group 1 to 4 that died 7 to 12 dpi (first phase) had enlarged, discoloured livers with multifocal to coalescing necroses (figures 3). Two pigeons additionally showed a severe diffuse, fibrinous perihepatitis. Microscopically, livers and spleens had multifocal necroses and moderate to severe infiltration with lymphocytes, plasma cells and macrophages (figures 4B,C; table 1). Only a few pigeons had necrosis and inflammation in the lungs, kidneys, bone marrows and connective tissues of several organs adjacent to blood vessels. Parasitic stages were predominantly observed in the liver, spleen and in or next to endothelial cells of several other organs (figures 4C,D; table 1). Pigeons of group 5 to 7 and the sentinel pigeon kept in group 1 that were euthanized 51 to 65 dpi (second phase) had marked encephalitis, myositis and Sarcocystis cysts in all skeletal muscles examined (pectoral, gastrocnemius and neck muscles). Both control pigeons of group 8 had no gross or microscopical lesions. None of the chickens had any gross or histological lesions.
Comparison of the D2 region of the 28S rRNA gene revealed identical gene sequences of cysts in the skeletal muscle of the experimentally infected pigeons compared to the sporocysts from the Northern goshawks and the cysts of the naturally infected pigeons (Olias et al., 2009a).
The lifecycle of a new Sarcocystis species named S. calchasi has previously been described to include the Northern goshawk (Accipiter gentilis) as definitive (final) host and the domestic pigeon (Columba livia f. domestica) as intermediate host (Olias et al., 2009b,c). Oral administration with different doses of sporocysts of the parasite confirmed that domestic pigeons are highly susceptible as intermediate hosts. The character and severity of clinical signs varied, depending on the infectious dose and suggest a biphasic course of the disease.
Clinically, affected pigeons developed apathy and polyuria within 6 to 12 dpi of which pigeons of groups 1 to 4 infected with high doses died. Only pigeons infected with 102 sporocysts (group 7) developed no clinical signs in this first phase of the disease (figure 1). Histopathology and electron microscopy of deceased pigeons revealed many schizonts in all developmental stages to be mainly located in the liver and in or next to endothelial cells in several other organs associated with a moderate to severe inflammatory response. All surviving pigeons (groups 5 to 7) suddenly developed moderate to severe central nervous signs 51 to 65 dpi as a second phase. Histopathology of the brain and the muscle tissue revealed identical lesions when compared to previously described naturally infected pigeons (Olias et al., 2009a).
Of note, the sentinel animal developed identical lesions when compared to those of low dose infected pigeons, suggesting a mechanical transmission of sporocysts from freshly infected to the uninfected pigeon. Gastrointestinal passage or shedding of sporocyst was not detected in all pigeons inoculated. Transmission via contaminated drinking water similar to transmission of Trichomonas gallinae in pigeon flocks (Kocan, 1969) therefore seems to be likely. However, gastrointestinal passage....(Schafe...
Unlike pigeons, chickens were unsusceptible to oral infection with sporocysts. However, it cannot be excluded that the parasite is infectious for avian species other than chicken. This assumption is supported by a close sequence homology with a Sarcocystis species of the White-fronted goose (Anser albifrons) in Lithuania as described previously (Butkauskas et al., 2007; Olias et al., 2009b). In general, bird-infecting Sarcocystis species are suggested to have a low host-specifity and therefore might exhibit a high potential to cause disease in incidental and aberrant host species (Box & Smith, 1982; Mansfield et al., 2008; Elsheikha, 2009). In particular, S. falcatula is known to cause an acute fatal pulmonary disease in non-American psittacines (Clubb & Frenkel, 1992). Although domestic pigeons were susceptible to experimental infection with S. falcatula, chickens failed to exhibit any clinical disease or histopathological lesions (Box & Smith, 1982). It is therefore tempting to speculate that a similar host range might also exist for the S. calchasi. Previously, several other cases of acute deaths in birds have been reported due to schizogony in the lungs of as yet unidentified Sarcocystis species (Suedmeyer et al., 2001; Villar et al., 2008; Ecco et al., 2008). Furthermore, several cases of fatal central nervous sarcocystosis have been reported in birds, however, genetic characterizations of the causative Sarcocystis species were lacking (Gustafsson et al., 1997; Dubey et al., 2001; Siegal-Willott et al., 2005). The range of Northern goshawks extends over most of the northern hemisphere and several other birds of prey have recently been reported to harbour a great variety of genetically and morphologically different Sarcocystis species (Ferguson-Lees, 2001; Yabsley et al., 2009). Thus, despite S. calchasi, it seems reasonable to assume that several other as yet unidentified species might demonstrate an unrecognised threat for several avian species.
In summary, we have shown that S. calchasi which cycles between Northern goshawks and domestic pigeons is highly pathogenic for domestic pigeons. Remarkabely, a biphasic disease developed in domestic pigeons after experimental infection with polyuria and apathy as a first phase and central nervous signs as a second phase of the clinical course.
We are grateful for the technical assistance provided by G. Hahn, A. Harder, P. Nehrig and C. Sabl.