Pamela E. Ginn

Immunoconversion against Sarcocystis neurona in normal and dexamethasone-treated horses challenged with S. neurona sporocysts.

Equine protozoal myeloencephalitis is a common neurologic disease of horses in the Americas usually caused by Sarcocystis neurona. To date, the disease has not been induced in horses using characterized sporocysts from Didelphis virginiana, the definitive host. S. neurona sporocysts from 15 naturally infected opossums were fed to horses seronegative for antibodies against S. neurona. Eight horses were given 5x10(5) sporocysts daily for 7 days. Horses were examined for abnormal clinical signs, and blood and cerebrospinal fluid were harvested at intervals for 90 days after the first day of challenge and analyzed both qualitatively (western blot) and quantitatively (anti-17kDa) for anti-S. neurona IgG. Four of the challenged horses were given dexamethasone (0.1mg/kg orally once daily) for the duration of the experiment. All challenged horses immunoconverted against S. neurona in blood within 32 days of challenge and in CSF within 61 days. There was a trend (P = 0.057) for horses given dexamethasone to immunoconvert earlier than horses that were not immunosuppressed. Anti-17kDa was detected in the CSF of all challenged horses by day 61. This response was statistically greater at day 32 in horses given dexamethasone. Control horses remained seronegative throughout the period in which all challenged horses converted. One control horse immunoconverted in blood at day 75 and in CSF at day 89. Signs of neurologic disease were mild to equivocal in challenged horses. Horses given dexamethasone had more severe signs of limb weakness than did horses not given dexamethasone; however, we could not determine whether these signs were due to spinal cord disease or to effects of systemic illness. At necropsy, mild-moderate multifocal gliosis and neurophagia were found histologically in the spinal cords of 7/8 challenged horses. No organisms were seen either in routinely processed sections or by immunohistochemistry. Although neurologic disease comparable to naturally occurring equine protozoal myeloencephalitis (EPM) was not produced, we had clear evidence of an immune response to challenge both systemically and in the CNS. Broad immunosuppression with dexamethasone did not increase the severity of histologic changes in the CNS of challenged horses. Future work must focus on defining the factors that govern progression of inapparent S. neurona infection to EPM.

Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge.

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease of the horse. The causative agent. Sarcocystis neurona, has been suggested to be synonymous with Sarcocystis falcatula, implying a role for birds as intermediate hosts. To test this hypothesis, opossums (Didelphis virginiana) were fed muscles containing S. falcatula sarcocysts from naturally infected brown-headed cowbirds (Molothrus ater). Ten horses were tested extensively to ensure no previous exposure to S. neurona and were quarantined for 14 days, and then 5 of the horses were each administered 10(6) S. falcatula sporocysts collected from laboratory opossums. Over a 12-wk period, 4 challenged horses remained clinically normal and all tests for S. neurona antibody and DNA in serum and cerebrospinal fluid were negative. Rechallenge of the 4 seronegative horses had identical results. Although 1 horse developed EPM, presence of S. neurona antibody prior to challenge strongly indicated that infection occurred before sporocyst administration. Viability of sporocysts was confirmed by observing excystation in equine bile in vitro and by successful infection of naive brown-headed cowbirds. These data suggest that S. falcatula and S. neurona are not synonymous. One defining distinction is the apparent inability of S. falcatula to infect horses, in contrast to S. neurona, which was named when cultured from equine spinal cord.

INTESTINAL VOLVULUS AND STRICTURE ASSOCIATED WITH A LEIOMYOMA IN A GREEN TURTLE (CHELONIA MYDAS)

Abstract A previously stranded 30-kg female green turtle (Chelonia mydas) was referred to the Veterinary Medical Teaching Hospital at the University of Florida following a 2-mo history of anorexia, intermittent regurgitation, decreased fecal production, and positive buoyancy of the right side. Radiographs confirmed gaseous distension of bowel loops suggestive of intestinal obstruction. The coelom was surgically approached through a plastron osteotomy, and a 540° volvulus of the small intestine was identified and derotated. An intestinal stricture was present at the site of the volvulus, and segments of bowel orad to the stricture were greatly distended. Following resection of abnormal bowel, the osteotomy was stabilized using self-tapping screws and figure-eight wire, and the defect was sealed with fiberglass cloth and fast-drying epoxy resin. A leiomyoma associated with the focal stricture was identified by histology. Appetite and defecation returned to normal. Six months after surgery, the turtle had regained normal buoyancy and showed no further clinical signs of gastrointestinal obstruction.

Effect of a single dose of ponazuril on neural infection and clinical disease in Sarcocystis neurona-challenged interferon-gamma knockout mice

Interferon gamma-knockout mice were challenged with 5000 Sarcocystis neurona sporocysts acquired from a naturally infected opossum. Ponazuril was administered once, by gavage, at day 1, 3, 7, 10, or 14 post-infection (pi). Ponazuril was given at either 20 or 200mg/kg. Mice that survived to day 30 pi were euthanized. Severity of CNS infection was quantified as schizont density in the cerebellum. Unchallenged mice in treatment and non-treatment groups remained free of disease and gained weight throughout the experiment. All challenged mice, regardless of treatment, developed histologic evidence of CNS infection even though clinical signs were prevented in some groups. The greatest treatment benefits were seen in mice given 200mg/kg ponazuril between days 4 and 14 pi. Weight gain over the course of the experiment occurred only in mice that were given 200mg/kg ponazuril on day 7 or 10 pi. With the exception of groups given 200mg/kg ponazuril on day 7 or 14 pi, mice in groups that got sporocysts developed abnormal neurologic signs. No deaths before day 30 pi occurred in mice given ponazuril at 20mg/kg on day 7 pi or 200mg/kg on day 1, 7, 10, or 14 pi. This effect was not significant. Mice given 200mg/kg on day 7 pi had significantly fewer cerebellar schizonts than did those of the control group that was not given ponazuril. These results indicate that single-dose administration of ponazuril for prevention of CNS infection is partially protective when given between days 4 and 14 pi.

Primary Mesenchymal Chondrosarcoma in the Pericardium of a Dog

hemogram was normal except for a monocytosis (2,820/ ml), a mild lymphopenia (880/ml), and an increase in fibrinogen (500 mg/dl). The chemistry profile was normal except for an increase in alkaline phosphatase (851 IU/liter). Survey thoracic radiographs revealed a greatly enlarged, globoid cardiac silhouette with a small volume of pleural effusion. Moderate hepatomegaly with smooth margination was noted in the abdominal radiographs. Differential diagnoses at this time included benign pericardial effusion, atrial hemangiosarcoma with pericardial hemorrhage, dilated cardiomyopathy and a thoracic or cardiac tumor. An ultrasound-guided fine needle aspirate of the mass was obtained. Wright‐Giemsa-stained fine needle aspirates of the pericardial mass revealed moderate numbers of neoplastic round to spindle-shaped cells that were arranged individually and in small clumps within a lightly stippled, eosinophilic background. These cells were characterized by round to oval nuclei with a high nuclear to cytoplasm ratio, coarse nuclear chromatin pattern, and prominent often multiple nucleoli. Anisokaryosis and anisocytosis was moderate to marked. The cytoplasm was deeply basophilic and some of the cells contained variably sized purple granules. Multinucleated cells were also noted, containing from 2 to more than 100 nuclei (Fig. 1). Amorphous, pink, extracellular material was noted around a few of the cellular clumps. A cytologic interpretation of malignant mesenchymal neoplasia was made. The owners declined further evaluation and the dog was euthanatized. A complete necropsy was performed. Approximately two-thirds of the thoracic cavity was occupied by a large, firm intrapericardial mass that displaced the lungs dorsad and caudad. Incision of the pericardium revealed posterior displacement of the heart by a 14

Viability of Sarcocystis neurona sporocysts and dose titration in gamma-interferon knockout mice

Gamma-interferon knockout mice have become the model animal used for studies on Sarcocystis neurona. In order to determine the viability of S. neurona sporocysts and to evaluate the course of the disease in these mice, sporocysts were collected from opossums (Didelphis virginiana), processed, and stored for varying periods of time. Gamma-interferon knockout mice were then inoculated orally with different isolates at different doses. These animals were observed daily for clinical signs until they died or it appeared necessary to humanely euthanize them. 15 of 17 (88%) mice died or showed clinical signs consistent with neurologic disease. The clinical neurologic symptoms observed in these mice appeared to be similar to those observed in horses. 15 of 17 (88%) mice were euthanized or dead by day 35 and organisms were observed in the brains of 13 of 17 (77%) mice. Dose appeared not to effect clinical signs, but did effect the amount of time in which the course of disease was completed with some isolates. The minimum effective dose in this study was 500 orally inoculated sporocysts. Efforts to titrate to smaller doses were not attempted. Direct correlation can be made between molecularly characterized S. neurona sporocysts and their ability to cause neurologic disease in gamma-interferon knockout mice.

Evaluation of the shedding of Sarcocystis falcatula sporocysts in experimentally infected Virginia opossums (Didelphis virginiana)

Five Virginia opossums (Didelphis virginiana) were fed muscles of brown-headed cowbirds (Molothrus ater) containing sarcocysts of Sarcocystis falcatula. Shedding of sporocysts was confirmed in all five opossums by fecal flotation. Counts were conducted daily for 2 weeks and then biweekly until the animals were euthanized and necropsied. The average prepatent period was 9.8 (7-16) days. The number of sporocysts shed varied greatly between the opossums with maximum mean shedding occurring at 71.6 (26-112) days post-infection (DPI). Average sporocyst production was 1480 sporocysts/gram of feces (SPG). Maximum output was 37,000 SPG. Average fecal yield in captivity was 17.5g of feces/day. Opossums shed 25,900 sporocysts/day (average) and a maximum of 647,500 sporocysts/day. All opossums shed sporocysts until time of euthanasia (46-200 DPI). Histologically, numerous sporocysts were present in the lamina propria at necropsy, primarily in the proximal half of the small intestine. Sporocysts were generally in clusters within the lamina propria of the luminal two-thirds of the villi. Sporocysts were found less frequently in the epithelium. No evidence of ongoing gametogony or other development was evident.

A case of fatal anaphylaxis in a dog associated with a dexamethasone suppression test

Objective: To describe a case of fatal anaphylaxis in a dog associated with a ‘routine’ dexamethasone suppression test. Case summary: An 8-year-old, spayed female dog, was treated with parenteral dexamethasone for a diagnosis of immune-mediated thrombocytopenia. The dog had responded to treatment, but 9 months later was evaluated for endogenous hyperadrenocorticism, prior to surgery for a ruptured anterior cruciate ligament. A normal ACTH stimulation test was followed by a high-dose dexamethasone suppression test. Immediately following the intravenous injection of dexamethasone, the dog developed severe anaphylactic shock and died. The postmortem examination findings supported the diagnosis of anaphylaxis. New information provided: The anaphylaxis in this dog was fulminating and by-passed the usual early signs of drug hypersensitivity. This is the first case in the veterinary literature reporting on dexamethasone as the cause of this dogs catastrophic event.

T-cell-rich B-cell lymphoma in a ring-tailed lemur (Lemur catta).

Abstract A 13-yr-old ring-tailed lemur (Lemur catta) was evaluated for depression, anorexia, polyuria, and polydipsia. The lemur was in poor body condition and was anemic, hypoalbuminemic, and hyponatremic. Cytologic examination of aspirates of the spleen, liver, and bone marrow and histopathologic examination of liver and bone marrow biopsies revealed a disseminated round cell tumor. After euthanasia, necropsy revealed hepatomegaly, splenomegaly, and mesenteric lymphadenomegaly. Neoplastic cells were present within the spleen, liver, kidneys, multiple lymph nodes, bone marrow, lung, small intestine, pancreas, and testicle and were composed of large anaplastic round cells in a background of small well-differentiated lymphocytes. Immunohistochemical analysis revealed that the small well-differentiated lymphocytes labeled for the anti-human T-cell marker, CD3, and the large anaplastic round cells labeled with the anti-human B-cell marker, CD79a. On the basis of the immunohistochemical staining results and morphologic appearance, a diagnosis of a T-cell–rich B-cell lymphoma was made.

Neurologic disease in gamma-interferon gene knockout mice caused by Sarcocystis neurona sporocysts collected from opossums fed armadillo muscle.

Fifteen gamma-interferon gene knockout mice were each orally inoculated with 5 x 10(3) Sarcocystis sporocysts derived from Virginia opossums (Didelphis virginiana) fed nine-banded armadillo (Dasypus novemcinctus) muscle containing sarcocysts. Three mice were inoculated with similarly obtained homogenates, but in which no sporocysts were detected. Mouse M8 was pregnant when inoculated and gave birth during the trial. Fifteen of 15 (100%) mice inoculated with sporocysts developed neurologic signs and/or died by day 30 d.p.i. One of 3 (33.3%) mice inoculated with homogenates in which no sporocysts were detected developed clinical signs and died at 34 d.p.i. All young of mouse M8 had maternally acquired antibodies to Sarcocystis neurona, but none developed clinical neurologic signs or had protozoal parasites in their tissues. All brains from mice that developed clinical signs contained merozoites that reacted positively to S. neurona antibodies using immunohistochemical techniques. Evidence from this study further supports the nine-banded armadillo being an intermediate host of S. neurona.