363 J Vet Diagn Invest 14:363–370 (2002) Turlock-like bunyavirus associated with encephalomyelitis and myocarditis in an ostrich chick H. L. Shivaprasad, P. R. Woolcock, M. D. McFarland, M. Curtis, N. Karabatsos Abstract. In the fall of 1995, a 20-day-old female ostrich chick, 1 of a group of 20, was presented live with clinical signs of 2 days duration characterized by unsteady gait, circling to the left, and walking backward. Another bird with similar clinical signs had died and another had recovered. The bird was euthanized and examined at necropsy. Twenty-five milliliters of serous fluid was in the abdominal cavity and there was increased pericardial fluid. Histopathology of the brain revealed mild to moderate nonsuppurative encephalitis character- ized by mild multifocal malacia, perivascular cuffing by lymphocytes, and gliosis. The heart had multifocal infiltrations of lymphocytes mixed with macrophages and a few plasma cells throughout the myocardium. Cytopathic effects were observed in primary chicken embryo liver cells following inoculation with a tissue homogenate prepared from the brain of the affected ostrich. Virus particles the size and morphology of the family Bunyaviridae were observed in cell culture lysate by negative-stain electron microscopy. Viral charac- terization demonstrated that the virus isolate is a previously unknown serotypic variant (subtype) of Turlock virus. Twelve of 65 sera collected over a 3-year period from ostriches aged from 1 month to 4 years were positive for neutralizing antibody to both the Turlock prototype strain and the new subtype of Turlock virus described in this report. The Turlock virus group is one of several antigenic groups comprising the genus Bunyavirus. The proto- type Turlock virus was first isolated in 1954 from mos- quitos (Culex tarsalis) collected near Turlock, Califor- nia. 6–8 Experimental infection of chickens, bob white quail, chukar partridges, ring-necked pheasants, and Japanese quail with Turlock virus did not cause any clinical signs or morbidity. 16 Crimean-Congo hemor- rhagic fever virus, a nairovirus, is another member of family Bunyavirdae that has been associated with vi- remia but no clinical disease in ostriches. 18,21 Turlock virus has not previously been associated with clinical disease in a vertebrate host. In this article, we report the isolation of a Turlock-like virus from the brain of an ostrich chick with encephalomyelitis and myocar- ditis. Sera collected over a 3-year period from ostrich- es were screened for antibodies to the Turlock-like vi- rus and also to the prototype Turlock virus. Brief re- ports of this case have been made previously (Shiva- prasad HI, et al.: 1996, Proc AAVLD, Grand Rapids, Michigan, p. 20). 19 Materials and methods History and pathology In the fall of 1995, a live 20-day-old female ostrich chick, 1,760 g, was submitted with 2-day-duration neurological From the California Animal Health and Food Safety Laboratory System, Fresno Branch, University of California, Davis, CA 93725 (Shivaprasad, Woolcock, McFarland, Curtis) and the Division of Vector-Borne Infectious Diseases, CDC, Fort Collins, CO 80522 (Karabatsos). Received for publication September 13, 2001. signs that included circling to the left, unsteady gait, and walking backward. The bird was one of several in a group of 20 chicks with similar clinical signs, 1 of which had died and another that had recovered. All of the birds originated in the Central San Joaquin Valley of California and were kept in a large pen together with 1 breeding pair. The chicks were given ratite pellets containing 19% protein and alfalfa. The birds had not been vaccinated for any disease. The ostrich chick was euthanized with carbon dioxide and examined at necropsy. Blood for serologic tests was col- lected from the femoral vein. Tissue samples were collected for virus isolation, negative-stain electron microscopy, bac- teriology, and toxicology. Samples of trachea, lung, heart, liver, kidney, spleen, esophagus, proventriculus, gizzard, pancreas, intestine, thymus, thyroid, parathyroid, adrenal, skin, conjunctiva, eye, bursa of Fabricius, bone, spinal cord, peripheral nerves, and tympanic bulla were fixed in 10% neutral buffered formalin and routinely processed for histo- pathologic examination. Selected tissues were stained with periodic acid–Schiff (PAS), Von Kossa, and Gram stains us- ing standard methods. Virus isolation Virus isolation in egg embryos. Brain tissue was triturated with sterile silica using a mortar and pestle and then sus- pended in buffered saline (10% w:v) containing 100 g/ml Gentamicin and 2.5 g/ml Amphotericin B. The tissue sus- pension was clarified by centrifugation at 1,780 g for 10 min at 4 C and then passed through a 0.2-m membrane filter. The filtered brain suspension was inoculated into the chorioallantoic sac (CAS) of 10-day-old specific pathogen- free (SPF) embryonating chicken eggs (ECEs), the yolk sac (YS) of 7-day-old SPF ECEs, and the chorioallantoic mem- brane (CAM) of 10-day-old SPF ECEs. 17 The eggs were in-
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