Cerebrospinal nematodiasis outbreak in an urban outdoor aviary of cockatiels (Nymphicus hollandicus) in southern California

455797JVDXXX10.1177/1040638712455797D iab et al.cerebrospinal nematodiasis outbreak Cerebrospinal nematodiasis outbreak in an urban outdoor aviary of cockatiels (Nymphicus hollandicus) in southern California Journal of Veterinary Diagnostic Investigation 24(5) 994 999 2012 The Author(s) Reprints and permission: sagepub.com/journalspermissions.nav DOI: 10.1177/1040638712455797 http://jvdi.sagepub.com Santiago S. Diab, 1 Francisco A. Uzal, Federico Giannitti, H.L. Shivaprasad Abstract. An outbreak of cerebrospinal nematodiasis due to Baylisascaris sp. was documented in an urban outdoor aviary in southern California. Thirty-four out of 35 cockatiels (Nymphicus hollandicus, 97%) showed a variety of neurological signs including ataxia, lateral recumbency, opisthotonus, and torticollis. Thirteen carcasses were submitted for necropsy; the histological lesions were restricted to the central nervous system (brain and spinal cord) and were predominantly degenerative, characterized by multifocal white matter vacuolation, gliosis, axonal swelling, gitter cell infiltration, and mild hemorrhage, rarely accompanied by mild granulomatous inflammation and mild lymphocytic perivascular cuffs. Nematode larvae morphologically compatible with Baylisascaris sp. were observed in the brain of 5 birds, away from the lesion site. Key words: Baylisascaris sp.; cerebrospinal nematodiasis; cockatiels; neural larva migrans; psittacines. Cerebrospinal nematodiasis (CSN) is produced by migration of nematode larvae through the brain and spinal cord causing severe neurological signs and lesions that often result in death. 7,8,11 Over a dozen human cases of CSN, 3 of which occurred in California, have been confirmed in the United States. 5,8,21 Cerebrospinal nematodiasis has also been reported in several mammalian and in more than 90 avian species including chickens, turkeys, ostriches, emus, pigeons, doves, quails, pheasants, partridges, blue jays, robins, and psittacines. 1,3,6,9,16,19,20,23-25 However, reports of CSN in psittacines are rare, limited to a single cockatiel housed in a cage in which raccoons (Procyon lotor) had been housed previously, and a group of macaws that had access to raccoon feces. 1,19 Larval stages of Baylisascaris procyonis, a primary intestinal ascarid of raccoons, are responsible for the majority of cases of CSN in mammals and birds. Baylisascaris columnaris of skunks (Mephitis mephitis) and Baylisascaris melis of badgers (Taxidea taxus) are incriminated much less frequently, although they may also cause CSN in animals. 13,14 The risk of animal, including human, exposure to B. procyonis is proportional to the number of raccoons, the prevalence of infected raccoons, and their latrine (site of raccoon defecation) density in a given area. 8 In North America, raccoons are extremely common in rural, suburban and urban settings, where they have become well adapted to living alongside people. Because B. procyonis eggs may remain infective for years, long after surrounding raccoon feces have degraded, contaminated areas can serve as long-term sources of infection for susceptible animals. 8 Cerebrospinal nematodiasis has been previously described, albeit rarely, in psittacines. The outbreak in cockatiels (Nymphicus hollandicus) described herein had previously undescribed epidemiological features, such as the urban setting, large number of birds involved, high disease prevalence and mortality, and high zoonotic potential. The aviary housed 35 cockatiels (25 adults and 10 juveniles), was 4.82 m long, 1.5 m wide, and 1.90 m tall, had a double-door entry way, dirt floor, and was totally enclosed with a solid galvanized iron sheet wall on one side and chicken wire reinforced with plastic siding elsewhere, including the roof. Two small cages for housing chicks were also within the aviary. The birds were fed with open-top wooden feeders hanging from the side wire walls approximately 1 m off the ground. Although insects, mice, and small rats could enter the aviary through small cracks, larger animals did not have access to it. Rats, mice, squirrels, cats, opossums, beetles, cockroaches, and flies were observed in the area surrounding the aviary. Raccoons were present in the neighborhood but had not been seen near the aviary around the time of the outbreak. Neurological clinical signs were noticed initially in a few adult and young cockatiels, and the disease progressed to From the California Animal Health and Food Safety Laboratory, University of California Davis, San Bernardino branch (Diab, Uzal), Davis branch (Giannitti) and Tulare branch (Shivaprasad), CA. 1 Corresponding Author: Santiago S. Diab, California Animal Health and Food Safety Laboratory, University of California Davis, 105 W Central Avenue, San Bernardino, CA 92408. ssdiab@cahfs.ucdavis.edu

Cerebrospinal nematodiasis outbreak 995 Figure 1. Clinical signs in a female cockatiel (Nymphicus hollandicus, case no. 3) with ataxia, torticollis, and inability to fly. involve 34 of the 35 (97%) birds over a period of 5 months. Except for 1, all affected birds died after varying times, usually several days or weeks after the onset of clinical disease. The most common clinical signs included ataxia, lateral recumbency, opisthotonus, and torticollis (Fig. 1). A few birds were lethargic, obtunded, lost the ability to fly, and repeatedly toppled over. Thirteen cockatiels of different ages and sex were submitted for necropsy and diagnostic workup. Details about clinical history, location of the histological lesions, presence or absence of nematode larvae in brain sections, and the results of laboratory ancillary tests are shown in Table 1. No gross lesions were observed in any of the birds. Representative samples from various visceral organs (lungs, heart, liver, spleen, kidneys, and gastrointestinal tract), brain from all the birds, and spinal cord, ears, and eyes from 11 of the 13 birds were fixed in 10% buffered formalin and processed routinely for histopathology to obtain 4-µm thick sections that were stained with hematoxylin and eosin. Significant histological lesions were limited to the brain and spinal cord. All 13 birds had moderate to severe encephalomalacia occasionally accompanied by mild encephalitis. Four of the 11 birds in which the spinal cord was examined had myelomalacia in addition to encephalomalacia (birds 3, 7, 12, 13). In 5 out of the 13 birds (birds 1, 3, 7, 9, 10), cross or tangential sections of nematode larvae were identified after examining up to 30 serial coronal or parasagittal sections of the brain. The lesions in the brain were multifocal and were found most commonly within the white matter of the medulla oblongata, cerebellar peduncles, and cerebellum. Moderate to severe encephalomalacia with Wallerian degeneration characterized by multifocal white matter vacuolation, myelin sheath dilation, axonal swelling (spheroids), occasional gemistocytic astrocytes, infiltration of a few gitter cells, rare multinucleate giant cells, and mild hemorrhage was observed. Accompanying some of these foci of malacia in the brain was a mild inflammatory response characterized by perivascular lymphocytic cuffs and mild gliosis. The lesions in the white matter of the spinal cord were characterized by mild spongiosis, with myelin sheath dilation, axonal swelling with occasional gitter cells, and occasional mild, focal to multifocal gliosis (Figs. 2 5). Larvae were observed in 5 birds that also had lesions in the brain or spinal cord; however, the larvae were not associated with histologic lesions. These larvae, morphologically compatible with Baylisascaris sp., were 50 57 µm in diameter, had an approximately 5-µm thick cuticle with prominent lateral chords and lateral alae, coelomyarian polymyarian muscles, a pseudocoelom, esophagus, and an intestine lined by several uninucleate columnar cells with a brush border (Fig. 6). Several ancillary laboratory tests were performed in select birds, and results were either negative or within normal reference ranges. Briefly, the tests included virus isolation from pooled tissues (liver, lung, kidney, spleen, and brain) in 4 birds (birds 1 4), hepatic levels of heavy metals (lead, manganese, iron, mercury, arsenic, molybdenum, zinc, copper, and cadmium) and selenium in 4 birds (birds 1 4), routine aerobic bacterial cultures from liver and small intestine in 4 birds (birds 1 4), fecal float in 4 birds (birds 1 4), avian Influenza A virus (AIV) and Newcastle disease virus quantitative reverse transcription polymerase chain reaction (qrt- PCR) from oropharyngeal and cloacal swabs in 5 birds (birds 1 3, 7, and 8), brain salt screen by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and brain acetyl cholinesterase activity by the modified Ellman method in 2 birds (birds 3 and 4), and Newcastle disease virus serology by hemagglutination inhibition (HI) assay in 2 birds (birds 1 and 3). All laboratory testing was performed at the California Animal Health and Food Safety Laboratory System, University of California, Davis, following standard operating procedures. Based on the morphological characteristics of the parasite larvae on histological sections and the epidemiological characteristics of the outbreak, the species involved was considered most likely to be B. procyonis. 2 After the initial diagnosis of CSN was made, the owner of the aviary noted groups of dry and fresh feces ( latrines ) on the top of the aviary s roof that were suspected, but not confirmed, to be from raccoons. The feces were dark brown, tubular, approximately 5 15 cm long by 2 cm in diameter, and contained a variety of seeds and unrecognizable foodstuffs. Raccoons were frequently observed in the neighborhood individually or in small groups, while other mid-sized mammals such as skunks and badgers were not seen. The eggs of Baylisascaris sp. are extremely resistant to environmental degradation and, given adequate moisture levels, can remain viable and potentially infective in soil for many years, long after raccoon latrines have ceased to be active and feces have degraded. In addition, B. procyonis eggs are sticky and tend to adhere to animal fur and a variety of objects, including children s toys and presumably human fingers. Specific risk factors in people include exposure to raccoons and raccoon feces, indoor

996 Diab et al. Table 1. Summary of history, central nervous system (CNS) lesion location, presence of larvae in brain histological sections, and laboratory test results in 13 cockatiels with neurologic clinical signs due to Baylisascaris sp. cerebrospinal nematodiasis.* Case Sex Age CNS lesion location Larvae in CNS Ancillary laboratory tests 1 M 9 years Cerebral white matter Yes Negative virus isolation Negative AIV and NDV qrt-pcr Negative NDV serology (HI) at 1:4 2 M 9 years Cerebral white matter No Negative virus isolation Negative AIV and NDV qrt-pcr 3 F Adult Cerebrospinal white matter Yes Negative virus isolation Negative AIV qrt-pcr Brain sodium within normal range No evidence of organophosphate or carbamate intoxication Negative NDV serology (HI) at 1:4 4 M Adult Cerebral white matter No Negative virus isolation Brain sodium within normal range No evidence of organophosphate or carbamate intoxication 5 U Adult Cerebral white matter No Not performed 6 U Adult Cerebral white matter No Not performed 7 F 7 years Cerebrospinal white matter Yes Negative AIV and NDV qrt-pcr 8 F 4 years Cerebral white matter No Negative AIV and NDV qrt-pcr 9 M 7 years Cerebral white matter Yes Not performed 10 F 2 years Cerebral white matter Yes Not performed 11 U Adult Cerebral No Not performed 12 U Chick Cerebrospinal white matter No Not performed 13 M 7 years Cerebrospinal white matter No Not performed * F = female; M = male; U = unknown; SI = small intestine; AIV = avian Influenza A virus; NDV = Newcastle disease virus; qrt-pcr = quantitative reverse transcription polymerase chain reaction; HI = hemagglutination inhibition assay. Pooled tissues (liver, lung, kidneys, spleen, and brain). Cloacal and pharyngeal swabs. Brain acetyl cholinesterase activity determination. storage of contaminated downed timber, wood chips or bark for firewood, indoor contamination by raccoon dens in chimneys and fireplaces, and contamination of outdoor children s playgrounds by raccoon feces. Human baylisascariasis has at least 3 clinical presentations: visceral larva migrans, neural larva migrans, and ocular larva migrans. Most human cases of neural larva migrans have occurred in toddlers or young children, which is probably related to a combination of 2 important risk factors: contaminated soil from play areas and children that ingest or place potentially infectious materials, such as dirt or wood chips, in their mouth. 8 There is abundant scientific evidence of a high prevalence of B. procyonis eggs within raccoon feces in urban, suburban, and rural areas of southern California. Further, at least 3

Cerebrospinal nematodiasis outbreak 997 Figure 2. Case no. 3; cockatiel (Nymphicus hollandicus); anterior cerebellar peduncle, subgross view. Vacuolation (spongiosis) and hypercellularity of the white matter; fourth ventricle (*). Hematoxylin and eosin. Bar = 200 µm. Figure 5. Case no. 3; cockatiel (Nymphicus hollandicus); brainstem (pons). A finely fibrillar pale eosinophilic matrix branching between dilated myelin sheaths and astrocytes (glial scarring), a gemistocytic astrocyte with abundant cytoplasm (arrow) and cross sections of swollen hypereosinophilic axons (spheroids). Hematoxylin and eosin. Bar = 20 µm. Figure 3. Case no. 3; cockatiel (Nymphicus hollandicus); cervical spinal cord. Degenerative myelopathy with bilateral symmetrical white matter vacuolation (spongiosis) and myelin sheath dilation in the ventral funiculi. Bar = 100 µm. Inset: close up view of a ventral funiculus showing vacuolation of the white matter and a gitter cell within a vacuole (digestion chamber). Hematoxylin and eosin. Bar = 20 µm. Figure 6. Case no. 10; cockatiel (Nymphicus hollandicus); brainstem (pons). Longitudinal/tangential section of a 50 60 µm wide nematode larva consistent with Baylisascaris sp., characterized by a 2 4 µm -thick cuticle with lateral alae (la), a pseudocoelom, lateral cords (lc), coelomyarian polymyarian musculature, and intestines lined by uninucleate columnar cells (i). Note the lack of inflammatory reaction around the larva. Hematoxylin and eosin. Bar = 20 µm. Figure 4. Case no. 3; cockatiel (Nymphicus hollandicus); brainstem. Marked white matter vacuolation and hypercellularity (gliosis). Hematoxylin and eosin. Bar = 50 µm. human cases of neural larva migrans caused by B. procyonis have been reported in California, 5,6,8,18,21 where outdoor aviaries housing cockatiels and other psittacines are common. For the clinician and pathologist, the diagnosis of CSN in cockatiels can be challenging. Neurologic signs due to Baylisascaris sp. infection in birds are nonspecific, and differential diagnoses include zinc and lead toxicoses, insecticide toxicosis, hepatic encephalopathy, otitis media and interna, trauma, and viral, bacterial, fungal, protozoal, and other verminous encephalitides. 9 A definitive etiologic diagnosis is based on the central nervous system lesions (encephalomalacia with/without encephalomyelitis) accompanied

998 Diab et al. by the finding of Baylisascaris sp. larvae in brain or spinal cord sections. In the current outbreak, the disease was initially suspected based on clinical signs and histologic lesions and confirmed with the finding of Baylisascaris sp. larvae in brain sections. However, up to 30 brain sections were necessary to visualize larvae in 5 of the 13 necropsied birds. Unfortunately, making many serial tissue sections is often not possible in a diagnostic laboratory setting because it is costly and time consuming. However, even in the absence of nematode larvae in histological sections of the brain, the presence of malacia and Wallerian degeneration in the white matter of the brainstem and cerebellum of birds with a history of neurologic signs and the presence of raccoons in the area are highly suggestive of CSN and the disease should be considered a top differential diagnosis. In these instances, a recently described PCR assay for the detection of B. procyonis eggs and larvae from fecal, environmental, and tissue samples would greatly aid in the confirmation of baylisascariasis in a laboratory setting. 4 The finding of Baylisascaris sp. larvae in nervous tissue without inflammation or degenerative changes may suggest that the larvae continue to migrate through the brain after the host is dead. The absence of larvae and/or histologic lesions in extraneural tissues of the cockatiels of the current outbreak is consistent with previous reports of this infection in avian species and is different from the situation in mammals in which larvae are commonly found in inflammatory lesions within skeletal muscles, eyes, and visceral organs. 1,7,8,14-17,22 However, in contrast to what has been previously described in CSN in many other avian species in which there were marked inflammatory lesions, such as perivascular cuffing, eosinophilic infiltration, and eosinophilic granulomas, 1,7,12,13 lesions in the central nervous system of cockatiels in the present study were predominantly degenerative and/or necrotizing, and only mild inflammation was observed. Naturally occurring case reports of CSN in avian species have been described in over 90 bird species, usually involving a limited number of birds mostly in the wild or in a zoological setting 1,3,7,10,17,19,22,23,25 and occasionally as large outbreaks in commercial operations. 16,20 In previous studies, raccoon or skunk feces contaminated with Baylisascaris sp. eggs have been implicated as the source of the infection directly by ground-feeding birds feeding on non-digested seeds in dried feces or indirectly through contaminated bedding, soil, feed, or feeding bowls. 1,3,7,16,17,20,23,24 In the current outbreak, the aviary was totally enclosed with no possible access for midsized animals. Therefore, it is speculated that the soil, feed, or open-top feed bowls may have been indirectly contaminated with Baylisascaris sp. eggs carried into the aviary by small rodents or insects or that the soil had been contaminated with Baylisascaris sp. eggs before the aviary was built. In summary, the current study describes the epidemiology, clinical signs, and pathology of a large outbreak of CSN due to Baylisascaris sp. (most likely B. procyonis) in cockatiels. The findings highlight the diagnostic challenge that Baylisascaris sp. infection in cockatiels and other birds poses to clinicians and pathologists, and alerts diagnosticians to a possible increase in the prevalence of this potentially devastating zoonotic disease in southern California. Acknowledgements The authors thank Ms. Bonita Pereyra for providing valuable and detailed history on the outbreak; Liz Thornburgh, Wendy Byrd, Tawnya Rapier, Jillian Van De Merghel, and Meridith Rhea for their technical assistance; and Drs. Robert Poppenga and Birgit Puschner for the toxicology testing. Declaration of conflicting interests The author(s) declare that they do not have any conflict of interest with respect to the research, authorship, and/or publication of this article. 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