THE PHARMACOKINETICS AND PHARMACODYNAMICS OF ORAL PONAZURIL IN THE TREATMENT OF SYSTEMIC ISOSPOROSIS IN PASSERINE BIRDS.

Systemic isosporosis, previously atoxoplasmosis, is a significant cause of mortality in juvenile passerine birds. Recommended treatment regimens are empiric and vary in efficacy. The goal of this study was to determine the pharmacokinetics and pharmacodynamics of ponazuril for treatment of systemic isosporosis. Ponazuril, diluted with water to create an oral suspension (50 mg/ml), was administered (100 mg/kg) to 72 European starlings (Sturnus vulgaris) by a single dose via direct oral gavage (n = 24), a single dose injected into superworm larvae (Zophobas morio; n = 24), or a daily dose mixed with commercial dog food to top-dress feed for 5 d (n = 24). Peak plasma concentrations were 5.84, 2.46, and 9.13 µg/ml for the direct gavage, injected larvae, and top-dressed feed groups, respectively. With repeated dosing, mean plasma concentrations from the top-dressed feed group were maintained between 8.12 to 13.11 µg/ml. Results suggested ponazuril at a dosage of 100 mg/kg administered via direct gavage or top-dressed feed, but not via injected larvae, would exceed the concentrations needed to inhibit merogony of other apicomplexan parasites in cell culture (5 µg/ml). To assess the pharmacodynamics of this dose, seven passerine birds, red-vented bulbuls (Pycnonotus cafer; n = 2), blue-grey tanager (Thraupis episcopus; n = 1), and red-capped cardinals (Paroaria gularis; n = 4), were identified as shedders of systemic Isospora spp. via fecal qPCR. Birds were then treated with ponazuril (100 mg/kg) daily on top-dressed feed for 14 d. Fecal shedding was assessed via qPCR for 6 wk from the initiation of treatment. Treatment was associated with reduction in proportions of fecal shedding during the treatment period and the week following treatment, but shedding resumed in all birds by the end of sampling. Results support that treatment of breeding birds with 100 mg/kg ponazuril could reduce the shedding of active oocysts and decrease risk of clinical infection in susceptible juveniles.

Unique Isospora-associated histologic lesions in white-rumped shama (Copsychus malabaricus).

Twenty-one white-rumped shamas (19 necropsied, 2 biopsied) (Copsychus malabaricus) housed at the San Diego Zoo between 1992 and 2020 were diagnosed with Isospora infection based on evaluation of histologic sections. Review of these cases revealed a consistent histologic lesion characterized by nodular aggregates of atypical epithelioid macrophages containing few intracytoplasmic protozoa, with or without lymphocytic infiltrates. Of the 19 necropsied cases, 16 (84%) had systemic lesions variably affecting the liver, spleen, gastrointestinal tract, lung, pancreas, connective tissues, or bone marrow, while all 21 diagnosed cases had skin involvement. The findings suggest that white-rumped shamas have a unique inflammatory response to isosporosis with a predilection for the skin. Skin may be a diagnostically sensitive sampling site for histologic diagnosis of Isospora in this species.

Cystoisospora belli, liver disease and hypothesis on the life cycle.

Cystoisospora belli causes chronic diarrhoea, acalculous cholecystitis, cholangiopathy and disseminated cystoisosporosis in patients with AIDS. Clinical manifestations and histological stages during C. belli infection in a patient with AIDS and liver disease were described. It was possible to identify sporozoite-like structures in the villus epithelium of the duodenum, close to the vascularization that underlies the basal membrane and unizoite tissue cysts near to the vascularization in the lamina propria. Unizoite tissue cysts were found inside of sinusoids in the liver communicating with the central vein and with a bile canaliculus and portal spaces. Based on these findings a hypothesis on C. belli life cycle could consider that the route of migration of unizoite tissue cysts up the liver is via the portal blood. The unizoite tissue cysts located in hepatic portal vein could migrated via sinusoid to central vein and general circulation through the venous system. The unizoite tissue cysts could also return via bile canaliculus to bile duct to portal triad. This hypothesis allows to understand the presence of unizoite stages in blood, the pathway by which the bile ducts become infected and unizoites in the liver being able to behave like hypnozoites that favour relapses and treatment failures.

A RETROSPECTIVE ANALYSIS OF MORBIDITY AND MORTALITY IN CAPTIVE BLUE-CROWNED LAUGHING THRUSHES (PTERORHINUS COURTOISI): 1998-2018.

The blue-crowned laughingthrush (BCLT) (Pterorhinus courtoisi) is a species of critically endangered passerine that is part of a Global Species Management Plan and experiences high mortality rates in some collections. Complete necropsy and medical records from 15 European institutions that housed BCLT between 1998 and 2018 were assessed to evaluate morbidity and mortality in the captive population. Medical records for 407 BCLT were received; a total of 191 morbidity events were recorded from 111 individual BCLT. The most common cause of morbidity was coccidial disease, including systemic isosporosis (30.4%, n = 58). Age was a significant factor contributing to mortality, with 53% of deaths occurring between 0 and 14 d old and 78% by 1 yr of age. The most common cause of death was trauma (22.8%, n = 31), followed by systemic isosporosis (22%, n = 30). Of the 343 deceased birds for which records were provided, 50.4% had a necropsy performed and 40.5% had histopathology carried out postmortem. Based on these findings, BCLT should be managed carefully to mitigate risk of trauma, and screening for isosporosis should be performed. Institutions should carry out standardized necropsies and histopathology on every BCLT that dies in their collection to elucidate the etiology of disease in this critically endangered species.

SYSTEMIC ISOSPORIASIS (ATOXOPLASMOSIS) IN PASSERINE BIRDS AT THE ZOOLOGICAL SOCIETY OF LONDON, LONDON ZOO.

Infection with systemic Isospora species (systemic isosporiasis [SI]) is common in passerine birds and may cause substantial mortality in zoological collections. Ten years of postmortem records of 26 species of captive, nonnative passerine birds maintained at the Zoological Society of London, London Zoo, plus seven free-ranging species found dead within the zoo, were reviewed to assess cause of death and occurrence of SI (presence of merozoites in tissue impression smears and/or polymerase chain reaction [PCR] testing for Isospora DNA). The records of 287 juveniles and adults were reviewed, of which 161 had SI test results. The most common cause of death was physical (trauma, predation, drowning, and hypothermia), diagnosed in 39.0% of cases. Virulent SI was considered the cause of death in only nine individuals from five species (3.1% of all cases, 5.6% of tested birds). However, merozoites were recorded in 36.0% of the 150 individuals examined cytologically (representing 18 of the 33 species), while 45.3% of 53 spleen samples (14 species) were positive for Isospora DNA. Test agreement for the 42 birds tested by both methods was 69.0%. Assuming that the PCR result was correct in these, 37.9% of the 161 birds (21 species) were positive for SI at the time of death. These figures might underestimate prevalence because of poor DNA preservation and low numbers of individuals of some species tested. Eight new 28S rDNA sequences and 12 new internal transcriber spacer 1/2 sequences were amplified. Sequences from individuals of the same host species clustered together, suggesting a single Isospora species, and there was no evidence of overlap among hosts. These results confirm that systemic infection with Isospora species in zoo passerines is generally of low pathogenicity and most likely coevolved with their hosts. Severe disease may occur, however, with overwhelming exposure, secondary to immunosuppression, or following coinfection with another pathogen.

Cystoisospora belli associated persistent diarrhea in an AIDS patient.

Cystoisospora belli infection is regarded as an indicator disease of AIDS in Japan; however, only a few case reports showing this association are present. Our case study involved a 49-year-old Thai woman living in Japan since her marriage to a Japanese man. She was repeatedly hospitalized owing to persistent diarrhea. Considering her native country, she was suggested of having AIDS. Serological examination for HIV-1 tested positive, and C. belli infection was diagnosed on detection of oocysts in her stool samples. She was treated successfully for the parasitic infection with oral trimethoprim-sulphamethoxazole therapy for 10 days. No AIDS-associated opportunistic infections other than cystoisosporiasis were detected. Thus, this study suggests that an immunocompromised individual with persistent and recurrent diarrhea should be examined to confirm for C. belli infection. Moreover, it is possible that a person in a high-latitude region will develop a parasitic infection common in tropical areas because of globalization.

Distribution, redescription, and molecular identification of Isospora striata McQuistion et al. 1997 (Eimeriidae), from woodcreepers (Dendrocolaptidae) in South America.

Woodcreepers are passerines of the family Dendrocolaptidae, which have a high forest dependency. The current work aimed to redescribe Isospora striata McQuistion et al. 1997, from two new hosts in protected areas in Brazil, revealing new localities of parasitism, in addition to providing preliminary genotypic identifications via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene from both host species. Isospora striata has oocysts that are subspheroidal to ovoidal, 19.4 × 16.8 µm with smooth wall. Oocyst residuum is absent, but micropyle and polar granules are present. Sporocysts are ovoidal, 13.6 × 8.3 µm, with both Stieda and sub-Stieda bodies. Sporocyst residuum is present and sporozoites with refractile body, nucleus, and striations. The morphological study and the 100% similarity in sequencing of the COI gene between samples of different dendrocolaptid species confirmed the identification of a single species, supporting the identification of I. striata in the Brazilian Atlantic forest and consequently the wide distribution of this coccidian species in the Neotropical Region.

Detection and characterization of the Isospora lunaris infection from different finch hosts in southern Iran.

This study was conducted to investigate the Isosporoid protozoan infections in finch types. Fecal samples were collected from marketed domestic Java sparrows (Lonchura oryzivora), colored and white Zebra finch (Taeniopygia guttata), and European goldfinch (Carduelis carduelis) in southern Iran. The coccidial oocysts were recovered and investigated according to the morphological features and the ribosomal gene markers. Additionally, a challenge infection was conducted with 5 × 104 and 5 × 103 sporulated oocysts in four java sparrows to estimate the clinical manifestations. Based on the morphology, the oocysts of Isospora lunaris were identified in all sampled bird types; however, the molecular method revealed the isolates had considerable similarities with some of Isospora and systemic Isospora-like organisms named as Atoxoplasma. Phylogenetic data also constructed an Atoxoplasma/Isospora clade with high sequence identities. High dose of the challenge with the parasite led to severe depression and sudden death, but it did not coincide with remarkable lesions and parasitic invasion in visceral organs. Contrary to molecular results, this feature is consistent with the common Isospora infections in passerines and differs from those described for Atoxoplasma species. Because of the prevalence, possibility of transmission, and clinical consequences, preventive measures are necessary to avoid outbreaks of isosporoid infections among finch type birds.

Morphological and genetic characterization of the first Isospora species (I. lugensae n. sp.) from a Kerguelen petrel (Lugensa brevirostris).

A new coccidian species, Isospora lugensae n. sp., was described from a single Kerguelen petrel (Lugensa brevirostris). Sporulated oocysts (n = 25) were characterized as subspheroidal to ellipsoidal measuring 24-25 µm × 21-23 µm (24.8 × 22.2 µm) in length/width (L/W), respectively, with a ratio of 1.07-1.14 µm (1.12). They contained a bi-layered wall with a thickness of 0.8-1.2 µm (1.0) and the outer layer smooth, with c.2/3 of total thickness. The oocyst contained two polar granules with both micropyle and oocyst residuum absent. Ovoidal sporocysts (n = 25) measured 15-16 µm × 10-11 µm (15.7 × 10.8 µm) in L/W, with a ratio of 1.41-1.49 µm (1.46). A flattened to knob-like Stieda body was present (c.0.5 µm deep × 2.5 µm wide) as well as a rounded to trapezoidal sub-Stieda (c.1.5 µm deep × 3.0 µm wide); however, no para-Stieda body was detected. The sporocyst residuum was composed of scattered spherules of different sizes, while vermiform sporozoites contained a refractile body, nucleus and visible striations. Analysis of the full-length mitochrondrial (mtDNA) genome revealed 3 protein-coding genes, (CytB, COI and COIII), 18 LSU and 14 small subunit (SSU) rDNA fragments, without transfer RNA genes with a total length of 6257 bp. Phylogenetic analysis of genomic SSU ribosomal sequences indicated that Isospora lugensae n. sp. is genetically similar to Eimeria reichenowi, isolated from a red-crowned crane (Grus japonensis) from Japan, with a 96.6% homology. The mtDNA sequence is most similar to Isospora serinuse with a 95.8% genetic similarity. Based on morphological and molecular data, this isolate is a new species of coccidian parasite that to date has only been found in a Kerguelen petrel.

A rare diarrheic parasite in a kidney transplant patient: Cystoisospora belli.

Cystoisospora belli colitis is a rare complication of immunosuppression in solid organ transplant recipients. We describe a case of Cystoisospora belli infection with colitis following renal transplantation.

Morphological and molecular characterization of Cystoisospora laidlawi oocysts (Apicomplexa: Eimeriidae) in farmed American mink (Neovison vison) in Denmark.

From a longitudinal survey conducted on 30 Danish mink farms in 2016, 11.0% of faecal samples (456/4140) were positive for Cystoisospora laidlawi oocysts by microscopy, with 60% (189/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts identified Cystoisospora oocysts measuring 34.3 × 29.5 µm with an oocyst length/width (L/W) ratio of 1.2. The morphological features of the oocysts were identical to Isospora laidlawi previously morphological identified in farmed mink from Denmark and elsewhere. Phylogenetic analysis of 18S rDNA sequences (1221 bp) from three positive mink indicated that Cystoisospora from mink shared the highest genetic similarity to C. canis from a Canadian dog (99.6%). The phylogenetic analysis placed Cystoisospora from mink in a clade with other Cystoisospora isolates.

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.

Isospora coronoideae n. sp. (Apicomplexa: Eimeriidae) from the Australian raven (Corvus coronoides) (Passeriformes: Corvidae) (Linnaeus, 1758) in Western Australia.

A new Isospora (Apicomplexa: Eimeriidae) species is described from an Australian raven (Corvus coronoides) in Western Australia. Sporulated oocysts (n = 21) are ovoid, 21.2 (18.4-23.9) µm in length and 18.8 (16.9-20.6) µm in width, with a shape index of 1.13. The bi-layered oocyst wall is smooth and colourless, 1.2 µm thick. A polar granule and oocyst residuum is present, but the micropyle is absent. The sporocysts are ovoid-shaped, 16.3 (13.7-18.9) × 10.7 (8.4-12.9) µm, with a shape index (length/width) of 1.52. Stieda and substieda bodies are present, the Stieda body being small and hemidome-shaped and the substieda being indistinct. Each sporocyst with four vermiform sporozoites arranged head to tail. The sporozoites are crescent-shaped, 9.0 (8.9-9.2) × 2.7 (2.3-3.0) µm, with a shape index (length/width) of 3.33. The sporocyst residuum is present. The isolated oocysts had different morphological characteristics when compared with all known Isospora spp. The coccidian parasite was analysed at the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) loci. At the 18S locus, I. coronoideae n. sp. exhibited 98.9% similarity to I. neochmiae from a captive-bred red-browed finch (KT224380) and Isospora sp. from domestic pigeons (Columba livia domestica) (AB757860), 98.5% similarity to I. gryphoni (AF080613) from an American goldfinch and 98.3% similarity to I. manorinae (KT224379) from a yellow-throated miner. At the 28S locus, it exhibited 95.4% and 94.8% similarity to I. manorinae (KT224381) and I. anthochaerae (KF766053), respectively. At the COI locus, it exhibited 99.8% and 99.7% similarity to I. butcherae (KY801687) and I. neochmiae (KT224378), respectively. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora coronoideae n. sp. after its host, the Australian raven (Corvus coronoides) (Passeriformes: Corvidae) (Linnaeus, 1758).

Isospora svecica sp. n. (Apicomplexa: Eimeriidae), a new species of coccidium from the white-spotted bluethroat Luscinia svecica cyanecula (Aves: Passeriformes: Muscicapidae).

Using a combination of morphological and molecular data, we describe a new apicomplexan parasite, Isospora svecica sp. n., from the white-spotted bluethroat, Luscinia svecica cyanecula, from the Czech Republic. Oocysts were found in its intestinal tract. Sporulation was exogenous and took 1-3 days. The oocysts were slightly ellipsoidal, of average size 26.17 × 20.33 µm, with a smooth bilayered wall. Micropyle, oocyst residuum, and polar granules were absent. Sporocysts were bottle-shaped, of an average size of 18.82 × 8.82 µm, with a thin, colourless wall. A conspicuous knob-like Stieda body was present. Substieda body was barely visible. Sporocyst residuum was present in the form of granules of various sizes. Sporozoites were banana-shaped and contained large anterior and small posterior refractile bodies. Partial DNA sequences of three genes were obtained from oocysts of Isospora svecica sp. n., being most closely related to other isosporans described from passerines. Little is known about the parasites of the avian family Muscicapidae, including coccidia, a highly prevalent parasitic protist group in all vertebrate classes. Only six species of the genus Isospora have so far been described in Muscicapidae, together with several "Isospora sp." that in fact most likely represent Isospora lacazei. The newly described Isospora svecica sp. n. differs morphologically from other coccidia reported from muscicapid birds, and represents the first coccidian species described from Luscinia svecica.

Clinical and Pathological Aspects of Systemic Isospora Infection in Blue-crowned Laughing Thrushes (Garrulax courtoisi) at Jersey Zoo.

Based on a review of species mortalities, systemic Isospora species was identified as the primary cause of death in 22% (19 of 87) of blue-crowned laughing thrushes (BCLTs; Garrulax courtoisi) at the Jersey Zoo between 1997 and 2016. Fifty-eight percent of the affected birds were between 1 and 2 years old, and in 89% of cases, death occurred between August and December. Abnormal clinical findings in BCLTs with Isospora species infections included hepatomegaly and pectoral muscle myositis in 79% of the cases. The results of diagnostic blood testing in 90% of infected BCLTs 30 days before death were consistent with a severe leukocytosis with greater than 20% of mononuclear cells infected by merozoites. The most common lesions identified during gross necropsy examination were splenomegaly (100%), hepatomegaly (95%), and multifocal, raised, white foci in pectoral (84%) and heart (79%) muscle. Lymphohistiocytic inflammation was identified in the liver, heart, spleen, lung, striated muscle, and kidney tissue of birds with positive results for Isospora species. Merozoites were often observed in spleen, liver, pectoral muscle, and hearts of infected BCLTs. Polymerase chain reaction diagnostic testing that targeted the cytochrome c oxidase subunit, followed by Sanger sequencing, was used to confirm Isospora species in all 14 birds tested. Of samples tested, the highest genetic correlation was with GenBank accession number KT203397 (Isospora species JRB-2016 mitochondrion).

Molecular and morphological description of Isospora sp. from the common mynah (Acridotheres tristis) and a preliminary survey of two anticoccidial drugs in natural infection.

Coccidian parasites, especially Isospora, are prevalent parasites in Passeriformes. Isosporan oocysts from common mynahs (Acridotheres tristis) are incompletely described. Detailed knowledge on biology, prevalence, pathogenesis and treatment of avian isosporiasis is scant. In this study, isosporan oocysts isolated from common mynahs were morphologically and molecularly characterized. The medication efficiencies of diclazuril and sulfadiazine-trimethoprim in isosporiasis in naturally infected mynahs were evaluated. Isosporan oocysts from common mynahs were described morphologically by microscopic imaging. The 18S rRNA and COI genes were amplified using PCR and the resultant products were sequenced and analysed phylogenetically. To evaluate the efficiencies of diclazuril and sulfadiazine-trimethoprim, two experimental treatment groups and a null control were assigned. General health status, weight and oocysts per gram of faeces were evaluated. Oocysts from all birds contained isosporan oocysts that were morphologically and dimensionally similar (P < 0.05). The oocysts were spherical; with no oocyst residuum, micropyle or polar granules. At both loci, phylogenetic analyses placed the Isospora isolate in the same clade with Isospora spp. from other Passeriformes. Both of the anticoccidials were well tolerated by the birds, a rapid reduction in oocyst excretion was noted at the commencement of treatment and 72 h after drug administration, oocyst excretion zeroed in all treated birds. Based on morphological and molecular data, this isolate does not resemble any previously described isosporas, hence Isospora tristum n. sp. is proposed for the current species. Both evaluated anticoccidials seemed to be efficient in reduction of oocyst production and can be recommended for the treatment of mynah isosporiasis.

Isospora sagittulae McQuistion & Capparella, 1992 (Apicomplexa: Eimeriidae) from antbirds (Passeriformes: Thamnophilidae) in the Amazon and Atlantic Forest of Brazil: with notes on its distribution and dispersion in the Neotropical region.

In the current study, Isospora sagittulae McQuistion and Capparella, 1992 (Protozoa: Apicomplexa: Eimeriidae) is reported from white-shouldered fire-eyes Pyriglena leucoptera (Vieillot, 1818) in the Atlantic Forest in southeastern Brazil. To date, this coccidian species was described from antbirds in Ecuador and Brazilian Amazon. In this sense, oocysts and measurements of the description of I. sagittulae from Amazonian antbirds were required from the deposit for comparison between samples from the Amazon and Atlantic Forest. The morphology was similar in all aspects, despite the polymorphism associated with the oocyst shape. DNA sequences for the mitochondrial cytochrome c oxidase subunit 1 (cox1) locus of the oocysts had similarity of 100%. Therefore, these strong morphological, molecular, and ecological equivalences ensure the unique identification of I. sagittulae. Finally, this finding reveals the wide distribution of I. sagittulae in the Neotropical region and indicates that other antbirds in the Brazilian Cerrado should disperse I. sagittulae to the Amazon and Atlantic Forest.

Intestinal parasitic infections in relation to CD4(+) T cell counts and diarrhea in HIV/AIDS patients with or without antiretroviral therapy in Cameroon.

BACKGROUND: Intestinal parasitic infections (IPI) are a major public health concern in HIV/AIDS patients particularly in resource-limited settings of Sub-Saharan Africa. Studies investigating the relationship between intestinal parasitic infections and CD4(+) T cell counts and diarrhea in HIV/AIDS patients with or without antiretroviral therapy in the region are not readily available hence the need to perform this study. METHODS: In a comparative cross-sectional study involving 52 pre-ART and 248 on-ART HIV patients. Stool samples were collected and analysed for intestinal parasites by wet and iodine mounts, Kato-Katz, formol ether, modified field staining, and modified Ziehl-Neelsen staining techniques. Blood samples were collected and analysed for CD4(+) T cell counts by flow cytometry. A pre-tested semi-structured questionnaire was used to collect data on socio-demographic and clinical presentation. Data were analysed using STATA version 12.1. Statistical tests performed included the Pearson Chi-square, logistic regression and student's t-test. P < 0.05 was considered to be statistically significant. RESULTS: The prevalence of intestinal parasitic infections in pre-ART and on-ART was 84.6% and 82.3% respectively with no significant difference observed with respect to age (p = 0.06), and gender (p = 0.736). All the opportunistic parasites including Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli and Microsporidium spp. were isolated from both groups, with only Microsporidium spp. significantly associated with CD4(+) T cell counts below 200 cells/µl in pre-ART (p = 0.006) while Cryptosporidium parvum, Microsporidium spp. and Isospora belli were associated with counts below 200 cells/µl in on-ART. Cryptosporidium parvum was significantly associated with diarrhea in pre-ART (p = 0.025) meanwhile it was significantly associated with diarrhea in on-ART (p = 0.057). The risk of diarrhea was highest in patients with CD4(+) T cell counts below 200 cells/µl (COR = 10.21, p = 0.000) for both pre- and on-ART treatment. CONCLUSION: A very high prevalence of intestinal parasitic infections was observed, which did not differ with respect to ART status. All known opportunistic parasites were isolated in both pre-ART and on-ART patients. Low CD4(+) T cell count may appear to be a factor for intestinal parasitic infections and development of diarrhea. Regular screening and treatment of intestinal parasitic infections is very vital in improving the overall quality of care of HIV/AIDS patients.

Morphological and molecular characterization of Isospora manorinae n. sp. in a yellow-throated miner (Manorina flavigula wayensis) (Gould, 1840).

A new Isospora (Apicomplexa:Eimeriidae) species is described from a single yellow-throated miner bird (Manorina flavigula) (subspecies M. f. wayensis) in Western Australia. Sporulated oocysts (n = 32) of this isolate are spherical to subspherical, 22.8 (20.3-23.8) × 18.3 (17.7-18.7) µm, with a shape index (length/width) of 1.25 (1.2-1.3); and a smooth and bilayered oocyst wall, 1.3 µm thick (outer layer 0.9 µm, inner 0.4 µm). A polar granule is present, but the micropyle and oocyst residuum are absent. The sporocysts are lemon-shaped, 15.5 (14.6-15.8) × 9.5 (9.5-10.2) µm, with a shape index of 1.6. Stieda and substieda bodies are present, the Stieda body being knob-like and the substieda body being subspherical-shaped. A sporocyst residuum is present and composed of numerous granules of different size scattered among the sporozoites, a spheroid or subspheroid refractile body is present in the sporozoite. Morphologically, the oocysts from this isolate are different from those of all known valid Isospora spp. Molecular analysis was conducted at 3 loci; the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) gene. At the 18S locus, this new isolate exhibited 99.2% similarity to Isospora gryphoni and three other Isospora spp. Further analysis of a subgroup of 300 bp long 18S sequences (8), including Isospora anthochaerae was conducted. This new isolate grouped in a clade with I. anthochaerae and exhibited 99.3% similarity. At the 28S locus, this new isolate grouped with I. anthochaerae with which it shared 99.1% similarity. At the COI locus, this new isolate exhibited 96.8% similarity to Isospora sp. JCI-2015 from a spectacled warbler (Sylvia conspicillata) in Spain. Further analysis from a subgroup of shorter COI sequences (n = 13) was performed and this new isolate exhibited 99.1% similarity to I. anthochaerae. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora manorinae n. sp. after its host, the yellow-throated miner (Manorina flavigula wayensis).

Exploration of fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for detection of Isospora suis oocysts.

Isospora suis is an intestinal protozoan parasite in pigs. The 2-3 weeks old piglets are most often infected by I. suis because their immune system is not fully developed. The infection exhibits clinical features such as diarrhea and dehydration and seriously affects the economic interests of farmers. The traditional method of identifying I. suis relies on the detection of fecal oocysts, which depends heavily on the accumulation of experience. Thus, missed detection, and false alarms often occur during detection. With the development of molecular-based detection methods, development of a simple, convenient and more sensitive method for the detection of I. suis is an urgent need. In this study, based on the 18S rRNA gene sequence, a fluorescence -based real-time loop-mediated isothermal amplification (LAMP) assay was established for the detection of I. suis. The results showed that the assay is highly specific and sensitive, with a detection limit of 2.74 × 10(2) copies/µL recombinant plasmid of I. suis, corresponding to 1 fg/µL plasmid when converted to DNA concentration. The sensitivity is about 100 times higher than conventional PCR. Additionally, DNA extracted from a certain number of oocysts was used for detection, and it showed that the LAMP assay had a detection limit of 5 oocysts, lower than that of 13 oocysts of conventional PCR. The established LAMP assay overcomes the shortage of the traditional microscopy-based method, and provides a valuable way for molecular detection of I. suis.