A QPCR ASSAY AND TESTING GUIDELINES FOR THE MOLECULAR DIAGNOSIS OF SYSTEMIC ISOSPOROSIS (FORMERLY ATOXPLASMOSIS) IN PASSERINE BIRDS.

Systemic isosporosis (formerly atoxoplasmosis), is a protozoal infection that causes death in nestling and fledgling passerine birds impacting ex situ breeding and reintroduction programs. Because current antemortem diagnostic tests lack sensitivity, a qPCR was developed for detection of Isospora spp. using primers and a fluorescent-tagged MGB probe targeting the large subunit (28s) ribosomal RNA gene (assay efficiency = >100%; sensitivity = <1 dsDNA copy). The assay was used to screen postmortem frozen or formalin-fixed paraffin-embedded tissue samples from passerine birds (n = 24; 12 with confirmed systemic isosporosis), whole blood and feces (n = 38) from live passerines, and other tissues infected with phylogenetically similar protozoa. The qPCR identified Isospora sp. DNA in tissues from 21/24 birds including 12/12 birds with cytologically-histologically confirmed infection (100% sensitivity) and 9/12 birds lacking microscopic organisms. The assay also amplified Eimeria sp. DNA; however, sequence analysis ruled out infection in the passerine cases. Blood and/or feces were positive in 30/38 birds, and in only 7/38 birds, blood and feces both contained Isospora sp. DNA. Finally, the qPCR was utilized to screen 30 consecutive daily fecal samples from live passerines (n = 20) to determine optimal sampling protocols. One or more of the daily fecal samples were positive in all 20 birds. In individual birds, the interval between positive qPCR amplification results ranged from 0 to 23 days, with an average of 5.85 days. Simulated application of 13 potential sample collection schedules was used to identify the sensitivity of repeated testing for identification of infected birds. Increased sampling days resulted in higher sensitivity but increased both cost and animal handling requirements. Based on statistical analysis and clinical considerations, the testing recommendation for detection of fecal shedding was collection and assay of five consecutive daily fecal samples, which had an average diagnostic sensitivity of 0.86.

Investigation of systemic isosporosis outbreaks in an aviary of greenfinch (Carduelis chloris) and goldfinch (Carduelis carduelis) and a possible link with local wild sparrows (Passer domesticus).

CASE REPORT: An outbreak of systemic isosporosis caused mortalities in greenfinches (Carduelis chloris) and goldfinches (Carduelis carduelis) kept in an aviary in the western suburbs of Melbourne. The following year, a further outbreak in the same aviary occurred in a different flock of goldfinches. At the time of the second outbreak, dead and sick common sparrows (Passer domesticus) discovered near the aviary were also found to have systemic isosporosis. METHOD: The systemic isosporosis was investigated and described using histopathology, electron microscopy and sequence analysis of the 18s gene. RESULTS: Isospora spp. infecting the greenfinch and the goldfinch caused significant thickening of the duodenal lamina propria. Measurements in the goldfinches showed an inverse correlation coefficient between the thickening of the duodenum and the weightof the birds. Electron microscopy confirmed the presence of Isospora spp. within lymphocytes migrating into the lamina propria of the duodenum. Analysis of the 18s sequence discovered two different gene sequences across the three species of birds that didn't completely match any sequences previously deposited in GenBank. CONCLUSION: Although the sparrows were found to have died from causes other than systemic Isospora, molecular studies of samples from their liver revealed the presence of an Isospora with 18s gene sequence identical to that found in the captive greenfinches.

Pathological and molecular characterization of systemic isosporosis (atoxoplasmosis) in captive green-winged saltator (Saltator similis).

Systemic isosporosis, also called atoxoplasmosis or visceral coccidiosis, is a disease that affects birds in general. Pathogenesis of systemic isosporosis and its etiologic agent have not been well characterized, but taxonomically Atoxoplasma is currently considered a junior objective synonym of Isospora. The present report aimed to describe pathological and molecular findings of systemic isosporosis in captive green-winged saltators (Saltator similis) from the State of Espírito Santo, Brazil. In a commercial breeding facility eleven birds with two to nine months of age died from 2015 to 2016. These birds developed nonspecific clinical signs, including bristly feathers, hyporexia, loss of weight, and apathy. Two birds were necropsied, and grossly there were hepatomegaly, splenomegaly, necrosis of lymphoid follicles, hepatic necrosis, and severe enteritis. Merozoites were observed in the heart, small intestine, proventriculus, brain, liver, spleen, and kidneys. 23 S RNA PCR amplicons from DNA extracted from the liver and the intestinal contents had 99% identity with Atoxoplasma sp., whereas amplicons of mitochondrial cytochrome c oxidase subunit 1 ha d 97% identity with Isospora greineri. In conclusion, this report indicates that systemic isosporosis in green-winged saltator is a disease that affects the spleen, liver, and small intestine, with high mortality for young birds, resulting in significant loses to commercial breeding facilities.

Extraintestinal isosporoid coccidian causing atoxoplasmosis in captive green-winged saltators: clinical and hematological features / Coccidiose extraintestinal causando atoxoplasmose em trinca-ferros de cativeiro: aspectos clínicos e hematológicos

Populations of green-winged saltators, Saltator similis, are decreasing especially because of illegal trade and infectious diseases. We describe natural cases of an extraintestinal isosporoid coccidian in caged S. similis, and suggest the need of preventive measures in handling these birds. Nonspecific clinical signs were seen in all of them, however, intracytoplasmic Atoxoplasma sp. was found in peripheral blood, reinforcing the idea of systemic isosporosis. Leukocytosis with high number of heterophils and monocytes suggested that atoxoplasmosis in green-winged saltators can progress as an acute disease. The birds showed clinical improvement after treatment. Handling recommendations were proposed to upgrade hygienic conditions of the facilities. We concluded that nonspecific symptoms and an acute inflammatory process can be associated with atoxoplasmosis in young S. similis. We emphasize the importance of blood smear to detect merozoites.(AU)

As populações de trinca-ferro, Saltator similis, estão declinando especialmente em função do comércio illegal e das doenças infecciosas. Descrevem-se casos naturais de coccidiose extraintestinal em S. similis de cativeiro, sugerindo a necessidade de medidas preventivas no manejo desses animais. Sinais clínicos inespecíficos foram observados em todos os animais, contudo, formas intracitoplasmáticas de Atoxoplasma sp. foram vistas no sangue periférico, reforçando a ideia de isosporose sistêmica. Leucocitose com aumento de heterófilos e monócitos sugeriram que a atoxoplasmose em trinca-ferro pode progredir como uma doença aguda. As aves apresentaram melhora clínica após tratamento. Recomendações de manejo foram propostas a fim de melhorar as condições higiênicas das instalações das aves. Concluiu-se que sinais inespecíficos e processo inflamatório agudo podem ser associados com a atoxoplasmose em S. similis jovens. Enfatizamos a importância da avaliação do esfregaço sanguíneo para detector os merozoítos.(AU)

Infección por Isospora belli en pacientes con infección por VIH: presentación de dos casos y revisión de la literatura / Isospora belli infection in HIV positive patients: report of two cases and literature review

Isosporosis is an infection caused by parasitic protozoa of the genus Isospora, coccidia affecting various different vertebrate species, including humans. It is an uncommon infection in our country and it is not a zoonosis. We present two cases oí Isospora belli infection in HIV positive patients from the Valparaiso region. We discuss the clinical events caused by this agent, its epidemiology, cases published in the local and foreign literature, as well as its treatment and prevention measures.

La isosporosis es una infección parasitaria provocada por protozoos del género Isospora, coccidio que afecta con diversas especies a diferentes vertebrados, entre los que se encuentra el ser humano. Es una infección poco frecuente en nuestro país y no constituye una zoonosis. Se presentan dos casos con infección por Isospora belli en pacientes infectados con el virus de inmunodeficiencia humana procedentes de la Región de Valparaíso. Se analizan las manifestaciones producidas por este agente, la epidemiología de la infección, los casos publicados en la literatura nacional e internacional, así como su tratamiento y las medidas de prevención.