SARCOCYSTOSIS IN A CAPTIVE FLOCK OF THICK-BILLED PARROTS (RHYNCHOPSITTA PACHYRHYNCHA) FROM 2005 TO 2016: MORBIDITY, MORTALITY, DIAGNOSTICS, AND MANAGEMENT STRATEGIES.

Sarcocystosis was diagnosed in a captive flock of thick-billed parrots (Rhynchopsitta pachyrhyncha) at the Wildlife Conservation Society's Queens Zoo. Since the index case in 2005, 45% of mortalities in birds over 30 days of age were due to sarcocystosis. Sarcocystis falcatula was repeatedly identified as the causative agent. The disease predominantly affected younger adult parrots. Administration of antiparasitic medications prior to development of respiratory signs prolonged life in infected birds, but disease was fatal until utilization of a three-drug combination (pyrimethamine, trimethoprim-sulfamethoxazole, and ponazuril). This protocol may require in excess of 6 mo of therapy to achieve clinical resolution of active disease. Plasma creatine kinase activity was found to be the most useful test in diagnosing infection and monitoring response to therapy. Polymerase chain reaction (PCR) for apicomplexan organisms on antemortem whole blood, blood smears, or dried blood spots helped confirm suspected cases, but due to the poor sensitivity was sometimes misleading when assessing response to therapy or resolution of clinical disease. Preventive measures, focusing on exclusion and removal of Virginia opossums (Didelphis virginiana) from zoo grounds failed to curtail the occurrence of sarcocystosis in the flock. Other preventative steps, such as modification of feeding stations to exclude potential arthropod paratenic hosts and prophylaxis trials with diclazuril, appeared to successfully mitigate new infections. Given the diagnostic and therapeutic challenges, prevention of exposure to S. falcatula is essential to ex-situ conservation efforts for thick-billed parrots.

Molecularly Confirmed Acute, Fatal Sarcocystis falcatula Infection in the Rainbow Lorikeets ( Trichoglossus moluccanus) at the Philadelphia Zoo.

The protozoan parasite Sarcocystis falcatula is an important cause of clinical disease in several avian intermediate hosts. The host range of S. falcatula is wide, and numerous outbreaks of acute sarcocystosis have been reported in passerine and psittacine birds in captivity in the Americas. Previous diagnosis was performed by serologic methods, light, and/or electron microscopic examinations with limited molecular confirmation. Here, we report histological and molecular diagnosis of acute, fatal S. falcatula infections in rainbow lorikeets ( Trichoglossus moluccanus) at the Philadelphia Zoo. Pulmonary sarcocystosis was suspected antemortem in 3 lorikeets (3-5 yr old); these birds died despite antiprotozoal therapy. The predominant lesion was pneumonia associated with S. falcatula-like schizonts in pulmonary vascular endothelium. The multilocus PCR-DNA sequencing ( 18S rDNA, 28S rDNA, ITS-1, and cox1) of frozen lung tissue confirmed S. falcatula infections in all 3 birds. Our results and previous studies suggest that acute pulmonary form of sarcocystosis is a major contributor of death to Old World psittacine birds.

Protozoal-related mortalities in endangered Hawaiian monk seals Neomonachus schauinslandi.

Protozoal infections have been widely documented in marine mammals and may cause morbidity and mortality at levels that result in population level effects. The presence and potential impact on the recovery of endangered Hawaiian monk seals Neomonachus schauinslandi by protozoal pathogens was first identified in the carcass of a stranded adult male with disseminated toxoplasmosis and a captive monk seal with hepatitis. We report 7 additional cases and 2 suspect cases of protozoal-related mortality in Hawaiian monk seals between 2001 and 2015, including the first record of vertical transmission in this species. This study establishes case definitions for classification of protozoal infections in Hawaiian monk seals. Histopathology and immunohistochemistry were the primary diagnostic modalities used to define cases, given that these analyses establish a direct link between disease and pathogen presence. Findings were supported by serology and molecular data when available. Toxoplasma gondii was the predominant apicomplexan parasite identified and was associated with 100% of mortalities (n = 8) and 50% of suspect cases (n = 2). Incidental identification of sarcocysts in the skeletal muscle without tissue inflammation occurred in 4 seals, including one co-infected with T. gondii. In 2015, 2 cases of toxoplasmosis were identified ante-mortem and shared similar clinical findings, including hematological abnormalities and histopathology. Protozoal-related mortalities, specifically due to toxoplasmosis, are emerging as a threat to the recovery of this endangered pinniped and other native Hawaiian taxa. By establishing case definitions, this study provides a foundation for measuring the impact of these diseases on Hawaiian monk seals.

Acute, fatal Sarcocystis calchasi-associated hepatitis in Roller pigeons (Columba livia f. dom.) at Philadelphia Zoo.

Four Roller pigeons (Columba livia f. dom.) at the Philadelphia Zoo died suddenly. Necropsy examination revealed macroscopic hepatitis. Microscopically, the predominant lesions were in liver, characterized with necrosis and mixed cell inflammatory response. Sarcocystis calchasi-like schizonts and free merozoites were identified in liver. Transmission electron microscopy confirmed that schizonts were in hepatocytes. A few schizonts were in spleen. PCR using S. calchasi-specific primers confirmed the diagnosis. Neither lesions nor protozoa were found in brain and muscles. This is the first report of acute visceral S. calchasi-associated sarcocystosis in naturally infected avian hosts.

Temporal association between land-based runoff events and California sea otter (Enhydra lutris nereis) protozoal mortalities.

Toxoplasma gondii and Sarcocystis neurona have caused significant morbidity and mortality in threatened Southern sea otters (Enhydra lutris nereis) along the central California coast. Because only terrestrial animals are known to serve as definitive hosts for T. gondii and S. neurona, infections in otters suggest a land to sea flow of these protozoan pathogens. To better characterize the role of overland runoff in delivery of terrestrially derived fecal pathogens to the near shore, we assessed the temporal association between indicators of runoff and the timing of sea otter deaths due to T. gondii and S. neurona. Sea otter stranding records 1998-2004, from Monterey and Estero bays were reviewed and cases identified for which T. gondii or S. neurona were determined to be a primary or contributing cause of death. Precipitation and stream flow data from both study sites were used as indicators of land-based runoff. Logistic regression was applied to determine if a temporal association could be detected between protozoal mortalities and runoff indicators that occur in the 2 mo preceding mortality events. A significant association was found between S. neurona otter deaths at Estero Bay and increased stream flow that occurred 30-60 days prior to mortality events. At this site, the cause of otter mortality following increased river flows was 12 times more likely to be S. neurona infection compared with nonprotozoal causes of death. There were no significant associations between the timing of T. gondii otter deaths and indicators of overland runoff. Our results indicate that the association between overland runoff and otter mortalities is affected by geography as well as parasite type, and highlight the complex mechanisms that influence transmission of terrestrially derived pathogens to marine wildlife. Policy and management practices that aim to mitigate discharges of contaminated overland runoff can aid conservation efforts by reducing pathogen pollution of coastal waters, which impacts the health of threatened marine wildlife and humans.

A protozoal-associated epizootic impacting marine wildlife: mass-mortality of southern sea otters (Enhydra lutris nereis) due to Sarcocystis neurona infection.

During April 2004, 40 sick and dead southern sea otters (Enhydra lutris nereis) were recovered over 18km of coastline near Morro Bay, California. This event represented the single largest monthly spike in mortality ever recorded during 30 years of southern sea otter stranding data collection. Because of the point-source nature of the event and clinical signs consistent with severe, acute neurological disease, exposure to a chemical or marine toxin was initially considered. However, detailed postmortem examinations revealed lesions consistent with an infectious etiology, and further investigation confirmed the protozoan parasite Sarcocystis neurona as the underlying cause. Tissues from 94% of examined otters were PCR-positive for S. neurona, based on DNA amplification and sequencing at the ITS-1 locus, and 100% of tested animals (n=14) had elevated IgM and IgG titers to S. neurona. Evidence to support the point-source character of this event include the striking spatial and temporal clustering of cases and detection of high concentrations of anti-S. neurona IgM in serum of stranded animals. Concurrent exposure to the marine biotoxin domoic acid may have enhanced susceptibility of affected otters to S. neurona and exacerbated the neurological signs exhibited by stranded animals. Other factors that may have contributed to the severity of this epizootic include a large rainstorm that preceded the event and an abundance of razor clams near local beaches, attracting numerous otters close to shore within the affected area. This is the first report of a localized epizootic in marine wildlife caused by apicomplexan protozoa.

Sarcocystis species lethal for domestic pigeons.

A large number of Sarcocystis spp. infect birds as intermediate hosts, but pigeons are rarely affected. We identified a novel Sarcocystis sp. that causes lethal neurologic disease in domestic pigeons in Germany. Experimental infections indicated transmission by northern goshawks, and sequence analyses indicated transnational distribution. Worldwide spread is possible.

Natural fatal Sarcocystis falcatula infections in free-ranging eagles in North America.

Three bald eagles (Haliaeetus leucocephalus) and 1 golden eagle (Aquila chrysaetos) were admitted to rehabilitation facilities with emaciation, lethargy, and an inability to fly. Intravascular schizonts and merozoites were present in 2 bald eagles, mainly in the lung tissue, whereas the third bald eagle and the golden eagle had lymphohistiocytic encephalitis with intralesional schizonts and merozoites. In all eagles, protozoal tissue cysts were present in skeletal musculature or heart. The protozoal organisms were morphologically compatible with a Sarcocystis sp. By immunohistochemistry, the protozoal merozoites were positive for Sarcocystis falcatula antigen in all cases when using polyclonal antisera. Furthermore, the protozoa were confirmed to be most similar to S. falcatula by polymerase chain reaction in 3 of the 4 cases. To the authors' knowledge, this report presents the first cases of natural infection in eagles with S. falcatula as a cause of mortality.

Sarcocystis neurona-associated meningoencephalitis and description of intramuscular sarcocysts in a fisher (Martes pennanti).

A free-ranging juvenile fisher (Martes pennanti) with ataxia, lethargy, stupor, and intermittent, whole-body tremors was examined postmortem. Microscopically, the fisher had protozoal meningoencephalitis caused by Sarcocystis neurona, which was confirmed by immunohistochemistry, polymerase chain reaction (PCR) and restriction fragment length polymorphism testing, and genetic sequencing. Sarcocysts found in the skeletal muscle of the fisher were negative for S. neurona by PCR, but were morphologically similar to previous light and electron microscopy descriptions of S. neurona. This is the first report of clinical neural S. neurona infection in a fisher.

Evaluation of risk factors associated with clinical improvement and survival of horses with equine protozoal myeloencephalitis.

OBJECTIVE: To investigate risk factors for use in predicting clinical improvement and survival of horses with equine protozoal myeloencephalitis (EPM). DESIGN: Longitudinal epidemiologic study. ANIMALS: 251 horses with EPM. PROCEDURE: Between 1992 and 1995, 251 horses with EPM were admitted to our facility. A diagnosis of EPM was made on the basis of neurologic abnormalities and detection of antibody to Sarcocystis neurona or S neurona DNA in CSF. Data were obtained from hospital records and through telephone follow-up interviews. Factors associated with clinical improvement and survival were analyzed, using multivariable logistic regression. RESULTS: The likelihood of clinical improvement after diagnosis of EPM was lower in horses used for breeding and pleasure activities. Treatment for EPM increased the probability that a horse would have clinical improvement. The likelihood of survival among horses with EPM was lower among horses with more severe clinical signs and higher among horses that improved after EPM was diagnosed. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of horses with EPM is indicated in most situations; however, severity of clinical signs should be taken into consideration when making treatment decisions. Response to treatment is an important indicator of survival.

In vitro cultivation and experimental inoculation of Sarcocystis falcatula and Sarcocystis neurona merozoites into budgerigars (Melopsittacus undulatus).

This is a report of the isolation and in vitro propagation of merozoites of Sarcocystis falcatula. Culture-derived S. falcatula merozoites remained infectious after several passages in tissue culture as determined by the susceptibility of experimentally inoculated Melopsittacus undulatus that died of pulmonary sarcocystosis after experimental inoculation. There was no obvious clinical disease or lesion when Sarcocystis neurona merozoites were used to inoculate M. undulatus. On the basis of the biological infectivity data, S. falcatula and S. neurona are not synonymous as suggested from limited molecular data.

Sarcocystis sp. in wading birds (Ciconiiformes) from Florida.

Sarcocysts were found in striated muscle of 21 adult wading birds among 145 examined grossly and 70 examined histologically (calculated prevalence = 24%), and in none of 332 immature wading birds examined from Florida (USA). Six of 12 species of ciconiforms were infected (Ardea herodias, Casmerodius albus, Egretta caerulea, Nyctanassa violacea, Butorides striatus, Eudocimus albus). Cysts were filamentous, usually extended the entire length of the muscle fiber, and were visible grossly in 33% of the positive cases. We concluded from ultrastructural examination of cysts that the same species of Sarcocystis may occur in all species of wading birds in Florida; however, two cyst diameters were noted that appeared to differ in their distribution by host species.

Sarcocystis transmitted by blood transfusion.

Merozoites were found in blood smears from calves, lambs, and pigs exhibiting signs of acute sarcocystosis after oral infection with sporocysts of Sarcocystis bovicanis, Sarcocystis ovicanis, and Sarcocystis suihominis. In each of 3 experiments, whole blood containing merozoites was transfused from an infected host to 2 uninfected recipients of the same species; 2 additional animals of the same species served as uninfected nontransfused controls. The bovine, ovine, and porcine donors all died of acute sarcocystosis. Clinical signs of sarcocystosis were not seen in any transfusion recipients or controls. All recipients and controls were killed and histologic specimens of esophagus, diaphragm, heart, skeletal muscle, and tongue were examined microscopically. Large numbers of intramuscular cysts were present in transfusion recipients, whereas few or no cysts were present in controls, indicating that S. bovicanis, S. ovicanis, and S. suihominis had been transmitted between intermediate hosts of the same species by blood transfusion.