LETTERS feline strains from Italy and Australia 2. De Grazia S, Giammanco GM, Potgieter Avian grouped together, as did the P[9] AC, Matthijnssens J. Bányai KN, Platia MA, et al. Unusual assortment of seg- Deaths in Parrots, strains from the United States. ments in 2 rare human rotavirus genomes. Thus, both the VP7 and VP4 Emerg Infect Dis. 2010;16:859–62. Europe genes of G3P[9] strain identifi ed in 3. Griffi n DD, Nakagomi T, Hoshino Y, this study were most closely related Nakagomi O, Kirkwood CD, Parashar To the Editor: Avian malaria is UD, et al. Characterization of nontype- to a racoon dog rotavirus strain (RAC- an insect-borne disease induced by a able rotavirus strains from the United so far unknown number of protozoan DG5), suggesting an animal origin of States: identifi cation of a new rotavirus this rotavirus strain. These strains are reassortant (P2A[6],G12) and rare P3[9] blood parasites of the genera likely an example of an animal strain strains related to bovine rotaviruses. Vi- and rology. 2002;294:256–69. doi:10.1006/ causing limited disease in humans, (hematozoa) (1,2). The unintentional viro.2001.1333 introduction of P. relictum to the rather than existence of a true strain, 4. Inoue Y, Kitahori Y. Rare group a rota- which has entered and adapted to the virus P[9]G3 isolated in Nara Prefecture, Hawaiian Islands, USA, has had human environment. Recent whole- Japan. Jpn J Infect Dis. 2006;59:139–40. fatal effects for the native bird 5. Khamrin P, Maneekarn N, Peerakome S, genome sequencing of 2 G3P[9] (3). In Europe, asymptomatic blood Tonusin S, Phan TG, Okitsu S, et al. Mo- by hematozoa have been strains isolated from children in Italy lecular characterization of rare G3P[9] showed they were composed of genes rotavirus strains isolated from children regularly observed, with an especially of human, bovine, and feline origin hospitalized with acute gastroenteritis. J high in songbirds (4). Med Virol. 2007;79:843–51. doi:10.1002/ (2); whether the G3P[9] strains from However, numerous outbreaks of jmv.20840 fatal protozoan infections have been Malaysia identifi ed in this study are 6. Wang Y-H, Kobayashi N, Zhou X, Na- also human/animal reassortant strains gashima S, Zhu Z-R, Peng J-S, et al. Phy- reported over the past 40 years, requires further study. logenetic analysis of rotaviruses with pre- mainly among psittacines of Australia dominant G3 and emerging G9 genotypes Identifi cation of G3P[9] strains that have been kept in aviaries (5,6). from adults and children in Wuhan, China. Diagnosis in all these cases was in Malaysia continues to highlight the J Med Virol. 2009;81:382–9. doi:10.1002/ presence of these rare strains in Asian jmv.21387 based on histopathologic detection communities. The close similarity of 7. Rasool NB, Larralde G, Gorziglia MI. of protozoan cyst-like structures Determination of human rotavirus VP4 the strains to a G3P[9] strain from a of unexplained origin in the heart using serotype-specifi c cDNA probes. and skeletal muscles and, to a lesser raccoon dog further highlights the Arch Virol. 1993;133:275–82. Medline transmission of rotavirus strains doi:10.1007/BF01313768 extent, in other organs. In most cases, between animal and human sources. 8. Iturriza-Gómara MI, Cubitt D, Dessel- the protozoans were identifi ed as berger U, Gray J. Amino acid substitution Whether this strain can establish members of the genus within the VP7 protein of G2 rotavirus because of their morphologic features. itself in humans and cause disease strains associated with failure to serotype. is unknown, but the identifi cation of J Clin Microbiol. 2001;39:3796–8.doi: Recent studies suggest that these cases rare strains illustrates that movement 10.1128/JCM.39.10.3796-3798.2001 may, in fact, have been infections of 9. Simmonds MK, Armah G, Asmah R, of rotaviruses between various hosts Besnoitia spp. () or other Banerjee I, Damanka S, Esona M, et al. unknown hematozoa (5); however, does occur from time to time. New nucleotide primers for p-typing of rotavirus strains: strategies for typ- genetic evidence is lacking. Ling-Sing Ch’ng, Way S. Lee, ing previously untypeable strains. J Clin In August 2010, sudden deaths and Carl D. Kirkwood Virol. 2008;42:368–73. doi:10.1016/j. of parrots were noticed in 2 separate Author affi liations: Murdoch Childrens jcv.2008.02.011 aviaries in northern Germany and Research Institute, Parkville, Victoria, 10. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genet- Switzerland (online Technical Australia (L.-S. Ch’ng, C.D. Kirkwood); ics Analysis (MEGA) software version Appendix Table, www.cdc.gov/EID/ University of Melbourne, Melbourne, 4.0. Mol Biol Evol. 2007;24:1596–9. content/17/5/950-Techapp.pdf). Victoria, Australia (L.-S. Ch’ng, C.D. doi:10.1093/molbev/msm092 Nine yellow-crowned parakeets Kirkwood); and University of Malaya, Kuala (Cyanoramphus auriceps), 3 barred Address for correspondence: Carl D. Kirkwood, Lumpur, Malaysia (W.S. Lee) parakeets (Bolborhynchus lineola), Enteric Virus Group, Murdoch Children’s and 2 budgerigars (Melopsittacus DOI: 10.3201/eid1705.101652 Research Institute, Royal Children’s Hospital, undulatus) died within 2–5 days Flemington Rd, Parkville, Victoria, 3052, References after a history of reduced general Australia; email: [email protected] 1. Parashar UD, Burton A, Lanata C, Boschi- conditions and reduced activity and Pinto C, Shibuya K, Steele D, et al. Global food intake before death. In addition, mortality associated with rotavirus dis- 2 budgerigars and 1 barred parakeet ease among children in 2004. J Infect Dis. in the aviary in Germany showed 2009;200:S9–15. doi:10.1086/605025

950 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 5, May 2011 LETTERS lethargy and reduced food intake for spp. Because all affected parrots Haemoproteus spp. of birds 2 weeks but fully recovered. About had been bred in Europe and had no in Europe (2). half of the birds were juvenile. No contact to imported birds, these results In conclusion, we identifi ed the new birds had been introduced into suggest that was the result cause of a previously unexplained lethal the aviaries during the previous 24 of previously unknown cross- disease of captive parrots in Europe, months. transmission of Haemoproteus spp. induced by numerous large cyst-like Necropsy and histologic examin- between birds of only distantly related megalomeronts in several organs, ation of 7 animals with fatal disease orders (8,9). including the heart. Morphologically, showed numerous large cyst-like Blood samples from surviving, the parasitic structures were strikingly protozoan structures (size up to 800 asymptomatic animals from the German similar to yet undetermined parasites μm in diameter; online Technical outbreak were tested cytologically of numerous previous outbreaks Appendix Figure) in myocardial and and by nested PCR for the presence (5,6). Genetically, the parasites had skeletal muscles and, to a lesser extent, of Haemoproteus spp. PCR identifi ed 99%–100% homology to known in the lung and the smooth muscles of Haemoproteus sequences in the blood Haemoproteus spp. from wild the intestinal tract without obvious of 3 of 26 psittacines, although parasitic European songbirds. The avian malaria signs of infl ammation. The cyst-like structures were not identifi able in blood parasites identifi ed are highly prevalent structures had a thick eosinophilic smears. Retrieved sequences were in the native songbird population but outer wall, were partly compartmented identical with that of MEUND1, except generally do not cause overt disease or by internal septae, and were fi lled for a single-nucleotide polymorphism death in their natural hosts. In contrast, with many merozoites. Surrounding in 1 sequence (MEUND3; Figure). the cases reported here suggest that these muscle fi bers were degenerated or A latent infection of these animals parasites that have adapted to European necrotic and, in some cases, associated therefore seems possible and may songbirds may cause fatal outbreaks in with hemorrhage. Blood smears of constitute a potential risk for further native psittacines of Australia, New clinically affected animals screened horizontal transmission in aviaries by Zealand, and South America that are for ≈5 × 105 cells each did not show blood-sucking insects such as biting raised in captivity. These fi ndings also parasites. midges (Culicoides), the vectors for show that preexisting pathogens may To further characterize the parasites, we carried out a nested PCR and subsequent DNA sequencing as described (7). Notably, phylogenetic comparison of 479 bp of the mitochondrial cytochrome b gene derived from protozoan cyst-like structures with known sequences of avian hematozoa found 99%–100% homology of parasites from both outbreaks with the avian malaria parasites (Haemoproteus spp.) of European songbirds (Figure). Identical cytochrome b sequences were detected in a yellow-crowned parakeet from Switzerland (CYAUR1), a budgerigar Figure. Phylogenetic relationships based on alignment of 479 bp of the cytochrome b gene from Germany (MEUND1), and of Haemoproteus spp. isolated from megalomeronts (m) of infected muscles and blood (b) a Haemoproteus sp. (TUPHI1) of parrots with related hematozoan parasites in GenBank and the database MalAvi (http:// previously found in the blood of a mbio-serv4.mbioekol.lu.se/avianmalaria; 10). Nucleotide distance values of the maximum song thrush (Turdus philomelos) in likelihood were calculated under the HKY substitution model. New sequences of Haemoproteus spp. from parrots of this study are shown in boldface. Two Bulgaria. The sequence derived from distinct species of the genus Leucocytozoon served as outgroup of the phylogenetic tree. the barred parakeet (BOLIN1) of the The branch lengths are proportional to the degree of inferred evolutionary change as shown German outbreak was identical with by the scale bar, and the numbers indicate bootstrap values (1,000 replicates). While the H. minutus of the common blackbird cytochrome b sequences CYAUR1, MEUND1, and BOLIN1, respectively, found matching (T. merula). In fact, different psittacine sequences, MEUND3 showed closest sequence similarities with Haemoproteus spp. of the lineage COLL2, which depict a wider host breadth among songbirds (http://mbio-serv4. species of the German outbreak were mbioekol.lu.se/avianmalaria). The isolates of Haemoproteus spp. from psittacine birds infected with different Haemoproteus were deposited into GenBank under accession nos. HQ398207–HQ398212.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 5, May 2011 951 LETTERS be a potential hazard for invading 7. Hellgren O, Waldenström J, Bensch S. A found antibodies to western (WEEV) species. Avian malaria should therefore new PCR assay for simultaneous stud- and eastern equine encephalitis ies of Leucocytozoon, Plasmodium, and be considered a threat for exotic parrots Haemoproteus from avian blood. J Para- viruses by using complement fi xation in Europe until results of further sitol. 2004;90:797–802. doi:10.1645/GE- and hemagglutination inhibition tests epidemiologic and experimental 184R1 in serum specimens from children (3). studies are available. Because many 8. Križanauskienė A, Hellgren O, Kosarev In 1972–1973, epizootics in horses V, Sokolov L, Bensch S, Valkiūnas G. European bird species have been Variation in host specifi city between spe- caused by WEEV were reported in introduced to the native range of the cies of avian hemosporidian parasites: Argentina and Uruguay, and WEEV psittacines studied here, a concern has evidence from parasite morphology and was isolated from a sick horse (4). been expressed that these parasites cytochrome b gene sequences. J Parasi- We report a fatal case of tol. 2006;92:1319–24. doi:10.1645/GE- already have become established in 873R.1 viral encephalitis in April 2009 in these areas and are affecting the natural 9. Ricklefs RE, Fallon SM. Diversifi cation Montevideo, Uruguay, in a previously populations. and host switching in avian malaria para- healthy 14-year-old boy. Four days sites. Proc Biol Sci. 2002;269:885–92. before he sought treatment, he had Philipp Olias, Maria Wegelin, doi:10.1098/rspb.2001.1940 10. Bensch S, Hellgren O, Pérez-Tris J. MalA- fever, asthenia, and headaches. At Wolfgang Zenker, vi: a public database of malaria parasites hospital admission (April 12, 2009), Sabrina Freter, and related haemosporidians in avian hosts he was febrile and without neurologic Achim D. Gruber, based on mitochondrial cytochrome b lin- signs; amoxicillin treatment was and Robert Klopfl eisch eages. Mol Ecol Resources. 2009;9:1353– 8. doi:10.1111/j.1755-0998.2009.02692.x initiated. Results of a computed Author affi liations: Freie Universität, Berlin, tomography scan of the brain were Germany (P. Olias, S. Freter, A.D. Gruber, Address for correspondence: Philipp Olias, normal. R. Klopfl eisch); IDEXX Diavet Labor, Bäch, Department of Veterinary Pathology, Freie On day 1, headache, vomiting, Switzerland (M. Wegelin); and Animal Clinic Universität Berlin, Robert-von-Ostertag-Str. 15, neck stiffness, and partial left Neuwiesen, Uster, Switzerland (W. Zenker) 14163 Berlin, Germany; email: olias.philipp@ seizures on the left side developed. DOI: 10.3201/eid1705.101618 vetmed.fu-berlin.de Also exhibited were consciousness depression (Glasgow Coma Scale 12 References points), hyperrefl exia, and bilateral Babinski sign. A cerebrospinal fl uid 1. Atkinson CT, van Riper C III. Pathoge- (CSF) sample was negative for bacteria nicity and epizootiology of avian haema- in cultures. An electroencephalogram tozoa: Plasmodium, Leucocytozoon, and Haemoproteus. In Loye JE, Zuk M, edi- showed diffuse brain suffering. The tors. Bird–parasite interactions. Ecology, patient was brought to the intensive evolution, and behavior. New York: Ox- Fatal Human Case care unit with a clinical diagnosis of ford University Press; 1991. p. 19–48. viral encephalitis. Over the next 24– 2. Martinsen ES, Perkins SL, Schall JJ. of Western Equine 36 hours, intracranial hypertension A three-genome phylogeny of malaria Encephalitis, parasites (Plasmodium and closely re- developed, and medical treatment lated genera): evolution of life-history Uruguay was given (sedation, hyperventilation, traits and host switches. Mol Phylogenet mannitol, and barbiturates). Con- Evol. 2008;47:261–73. doi:10.1016/j. To the Editor: The genus science depression progressed to ympev.2007.11.012 Alphavirus (family Togaviridae) a deeper level, and a computed 3. van Riper C III, van Riper SG, Goff ML, comprises 29 viral species (1), grouped Laird M. The epizootiology and ecologi- tomography scan of the brain showed in at least 7 antigenic complexes cal signifi cance of malaria in Hawaiian dilatation of the temporal ventricles land birds. Ecol Monogr. 1986;56:327–44. by their serologic cross-reactivity and compression of the peritroncal doi:10.2307/1942550 (2). They are maintained in nature and sylvian cisterns. During the next 4. Scheuerlein A, Ricklefs RE. Prevalence of through enzootic cycles involving blood parasites in European passeriform 48 hours, the coma level went deeper, arthropods as vectors with subsequent birds. Proc Biol Sci. 2004;271:1363–70. reaching 6 on the Glasgow Scale. doi:10.1098/rspb.2004.2726 amplifi cation in small mammals or Another CSF specimen was taken, 5. Bennett GF, Peirce MA, Ashford RW. birds, and epizootic cycles between and PCR results were negative for Avian haematozoa: mortality and patho- mosquitoes and large mammals such genicity. J Nat Hist. 1993;27:993–1001. herpesvirus and enterovirus. Glasgow as horses or humans. doi:10.1080/00222939300770621 Coma Scale level was 3 on April 15, 6. Stidworthy MF, Greenwood AG. Deaths Few reports have been made of the and a decompressive craniectomy in aviary birds associated with protozoal circulation of alphaviruses in Uruguay. was done. Seventy-two hours after megaloschizonts. Vet Rec. 2006;159:606. A serologic study conducted in 1970 doi:10.1136/vr.159.18.606 admission, the patient died.

952 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 5, May 2011 Article DOI: 10.3201/eid1705.101618 Avian Malaria Deaths in Parrots, Europe

Technical Appendix

Technical Appendix Table. Complete list of animals in the affected aviaries, northern Germany and Switzerland, August 2010 No. Histologic detection of Haemoproteus spp. PCR detection of animals Gametocytes in Megalomeronts in heart Haemoproteus Site location (species) died/total erythrocytes and skeletal muscle† spp. in blood Aviary 1 (Germany), 52°16′N, 13°39′E Budgeridgar (Melopsittacus undulatus) 2/20 0/18 1/1 3/18 Cockatiel (Nymphicus hollandicus) 0/6 0/2 ND 0/2 Barred parakeet (Bolborhynchus lineola) 3/6 0/5 1/1 1/5 Eastern rosella (Platycercus eximius) 0/2 0/2 ND 0/2 Chicken (Gallus gallus domesticus) 0/4 0/4 ND 0/4 Aviary 2 (Switzerland) 47°25′N, 8°50′E Yellow-crowned parakeet (Cyanoramphus auriceps) 9/18 ND 5/5 ND Cockatiel (Nymphicus hollandicus) 0/6 ND ND ND Domestic canary (Serinus canaria) 0/15 ND ND ND *ND, not done. †Confirmed by PCR.

Technical Appendix Figure. Myocardium of yellow-crowned parakeet that is severely infected with numerous large megalomeronts of Haemoproteus spp. Note the thick eosinophilic hyaline wall of megalomeront with internal septation and myriads of spherical basophilic merozoites. Surrounding muscle fibers are degenerated, necrotic, and hemorrhagic (hematoxylin and eosin stain). Scale bar = 150 µm.

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