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.

Pitfalls in Morphologic Diagnosis of Pathogens: Lessons Learned From the Pseudo-Cystoisospora Epidemic.

Multiple groups have recently reported involvement of the gallbladder mucosa of immunocompetent patients by cystoisospora organisms. However, this has recently been disproved with the support of molecular and ultrastructural studies. Here we present a summary of these events, recounting how this pseudo-Cystoisospora epidemic began and ended. This review also highlights the important role played by ancillary techniques in supplementing the morphologic diagnosis of pathogens.

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.

Ultrastructural Characteristics of Gallbladder Epithelial Inclusions Mimicking Cystoisospora.

OBJECTIVES: There is recently reported increased prevalence of Isospora organisms in cholecystectomy specimens from immunocompetent patients, especially in acalculous cholecystectomies. We performed an ultrastructural and molecular evaluation of these specimens. METHODS: From 28 gallbladders with intraepithelial inclusions, two specimens with diffuse involvement of the gallbladder epithelium were analyzed by electron microscopy. Polymerase chain reaction was performed on five samples for the ITS2 region of C belli and eukaryotic 18S region. The 18S products were sequenced by next-generation sequencing. RESULTS: Electron microscopic analysis showed cytoplasmic condensations leading to vacuole formation. In contrast with true C belli, there were no identifiable organelles or organization. None of these cases showed amplified products other than human on molecular analysis. CONCLUSIONS: Electron microscopic analysis demonstrates that the inclusions are condensed cytoplasmic material and not true organisms.

Epithelial Inclusions in Gallbladder Specimens Mimic Parasite Infection: Histologic and Molecular Examination of Reported Cystoisospora belli Infection in Gallbladders of Immunocompetent Patients.

Recent publications have described epithelial cytoplasmic vacuoles and inclusions incidentally noted within gallbladder epithelium and concluded that they represent coccidian parasite infection, in particular, Cystoisospora belli. We identified 8 gallbladder specimens from our institution in the past 3 years in which this diagnosis was suggested or in which similar epithelial alterations were prominent. Molecular analysis was performed on the 8 gallbladder specimens and on 3 positive control specimens: small bowel biopsies from acquired immunodeficiency syndrome patients with diarrhea. Polymerase chain reaction using primers designed to amplify an internal transcribed spacer (ITS2) in the C. belli ribosomal gene cluster was performed on the DNA samples. All 8 gallbladder specimens were negative for amplification, while a product consistent with C. belli was amplified from all 3 positive controls. Histologically, the gallbladder cytoplasmic inclusions stained diffusely positive for Grocott-Gomori's methenamine silver and Periodic acid-Schiff with diastase. In contrast, sections from a positive control small bowel biopsy demonstrated organisms that were negative for Grocott-Gomori's methenamine silver and showed a distinct capsular and punctate internal staining on Periodic acid-Schiff with diastase in various parasite forms. Together, the lack of molecular evidence of C. belli and the distinct morphologic and special staining patterns in these gallbladders compared with positive control small bowel suggest that these epithelial changes do not represent true C. belli infection. Our results suggest that gallbladders of immunocompetent patients may occasionally show epithelial changes that can morphologically mimic C. belli infection. Pathologists should be aware of this histologic variant to minimize unnecessary treatment, testing, and patient anxiety.

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.

Clinical cystoisosporosis associated to porcine cytomegalovirus (PCMV, Suid herpesvirus 2) infection in fattening pigs.

Cystoisospora (syn. Isospora) suis is the causative agent of neonatal porcine coccidiosis and one of the main causes of diarrhoea in suckling piglets worldwide. Infection with porcine cytomegalovirus (PCMV, Suid herpesvirus 2) causes inclusion body rhinitis in pigs. In a Swiss pig herd (n=2 boars, 7 sows, 2 gilts, 18 finishing pigs, 30 fattening pigs, 54 suckling piglets), an outbreak of PCMV infection with high morbidity in all age categories, characterized by fever, anorexia, reduced general condition, respiratory signs and increased piglet mortality, was diagnosed by histopathology and molecular methods. Five fattening pigs (age~17weeks) additionally showed diarrhoea, not typical for PCMV infections, and one fattener had to be euthanized due to poor condition. Histopathologically, severe fibrinopurulent jejunoileitis with extensive atrophy and fusion of intestinal villi, loss of goblet cells and crypt abscesses associated to C. suis infection were present. In the liver, herpesvirus intranuclear inclusion bodies were observed and PCMV was confirmed by PCR/sequencing. No further infectious causes of diarrhoea (i.e. Rotavirus A; TGEV; PEDV; PCV-2; enterotoxigenic Escherichia coli or Lawsonia intracellularis) were detected in the euthanized fattener. Coproscopically, C. suis oocysts were identified in the faeces from further fatteners with diarrhoea. While C. suis usually produces disease only in suckling piglets, its association with severe intestinal lesions and diarrhoea in ~17-week-old fatteners was surprising. It is supposed that the underlying PCMV infection might have contributed to the presentation of clinical cystoisosporosis in fattening pigs. The interaction mechanisms between these two pathogens are unknown.

Cystoisospora belli Infection of the Gallbladder in Immunocompetent Patients: A Clinicopathologic Review of 18 Cases.

Cystoisospora belli, previously known as Isospora belli, is an obligate intracellular coccidian parasite that is most often associated with gastrointestinal disease in immunocompromised patients. In this study, we detail the clinicopathologic features of 18 cases of Cystoisospora infection affecting the gallbladder in immunocompetent individuals and compare them with a control group. Each case was reviewed for cholecystitis (none, acute, chronic), epithelial disarray, presence of intraepithelial lymphocytes (none, rare [≤5 per 20 epithelial cells], present [>5 per 20 epithelial cells]), architectural distortion, intramucosal eosinophilia, and mural thickening/serositis. The mean age of patients with Cystoisospora infection was 33 years and the male to female ratio 1:4.3. Cholecystectomy was performed for biliary dyskinesia (n=7), abdominal pain (n=7), suspected cholelithiasis (n=5), and cholecystitis (n=3). In 2 cases, Cystoisospora was found in donor gallbladders resected at the time of liver transplantation. Each case was characterized by eosinophilic, oval or banana-shaped intraepithelial parasites within perinuclear parasitophorous vacuoles. Most cases showed epithelial disarray and minimal intraepithelial lymphocytosis. Of the 11 cases with an average follow-up of 15 months, none had evidence of disease related to Cystoisospora infection within the biliary tract or elsewhere in the gastrointestinal tract. We present the largest series of gallbladder cystoisosporiasis in immunocompetent patients to date. Cystoisospora infection is underrecognized in the gallbladders of immunocompetent patients, in part due to the subtle findings in routine cholecystectomy specimens. On the basis of the clinical follow-up, gallbladder cystoisosporiasis in immunocompetent individuals appears to be a self-limited infection.

Isospora belli Infection with Chronic Diarrhea in an Alcoholic Patient.

Chronic diarrhea with a 35 kg weight loss (75 kg to 40 kg) occurred during 2 years in an alcoholic patient was diagnosed with Isospora belli infection in the Republic of Korea. The patient, a 70-year old Korean male, had been a heavy drinker for more than 30 years. He was admitted to the Seoul National University Hospital because of long-standing diarrhea and severe weight loss. He had an increased white blood cell (WBC) count with high peripheral blood eosinophilia (36.8-39.9%) and lowered protein and albumin levels but without any evidence of immunosuppression. A parasitic infection was suspected and fecal examination was repeated 3 times with negative results. Peroral endoscopy with mural biopsy was performed in the upper jejunum. The biopsy specimens revealed villous atrophy with loss of villi together with various life cycle stages of I. belli, including trophozoites, schizonts, merozoites, macrogamonts, and microgamonts. The patient was treated successfully with oral doses of trimethoprim 160-320 mg and sulfamethoxazole 800-1,600 mg daily for 4 weeks. A follow-up evaluation at 2.5 years later revealed marked improvement of body weight (68 kg), increased protein and albumin levels, and normal WBC count with low eosinophils (3.1%). This is the first clinical case of isoporiasis with demonstration of various parasitic stages in the Republic of Korea.

Molecular diagnosis of cystoisosporiasis using extended-range PCR screening.

The differential diagnosis of diarrhea in immunocompromised patients encompasses many intestinal parasites including the coccidian Cystoisospora belli. Gastrointestinal infection with C. belli leads to cystoisosporiasis with diarrhea and, depending on host immune status, can cause extraintestinal disease. C. belli is usually diagnosed by examination of stool or intestinal biopsy specimens; however, the organism may be undetected using these test methods. Thus, more sensitive molecular tools for detection of pathogenic parasites are desirable. Herein is described a patient with AIDS who had persistent diarrhea of unknown cause. Microscopic examinations of stool and ileal biopsy specimens were initially unremarkable for any specific pathogen. Screening of DNA extracted from biopsy material using extended-range PCR primers recognizing conserved DNA sequences found in many fungi and parasites revealed infection with C. belli, which was confirmed at repeat histologic analysis. Extended-range PCR screening was used because the differential diagnosis was broad and other tools were not applied, yet this molecular approach led to the appropriate diagnosis and treatment of the condition. Thus, this approach offers a promising test for diagnosis of parasitic diseases that elude diagnosis using conventional methods.

Characterization of the host response in systemic isosporosis (atoxoplasmosis) in a colony of captive American goldfinches (Spinus tristis) and house sparrows (Passer domesticus).

Systemic isosporosis, also known as atoxoplasmosis, is a common parasitic disease of passerines. Infection is thought to be endemic in wild birds with fulminant, fatal disease occurring under the influence of stress, concurrent infections, or immunosuppression. Here, we describe the histologic and immunohistochemical characteristics of the cellular infiltrate occurring in captive colonies of American goldfinches and house sparrows. Necropsies were performed on 9 birds, and histologic examination was performed on the intestines of 7 additional birds. Lesions were most severe in the proximal small intestines. Histologically, the changes ranged from variably intense infiltrates of lymphocytes that filled the lamina propria to sheets of large, atypical cells that expanded and obliterated normal mucosal epithelium and invaded through the wall of the intestine and into the ceolomic cavity. Both the smaller lymphocytes and large atypical cells were immunoreactive for CD3. Intracellular parasites consistent with Isospora were detected in the large atypical cells, but they were more easily detectable in the more differentiated lymphocytes. Polymerase chain reaction and virus isolation performed on tissues from 7 birds were negative for retroviruses and herpesvirus. The immunohistochemical results of this study and the destructive nature of the cellular infiltrate suggest that the lesion represents T-cell lymphoma. In birds, lymphomas are most often associated with herpes and retroviruses; the absence of these viruses suggests that the parasite initiated neoplastic transformation. Though much work needs to be done to prove the transformative nature of the lesions, these preliminary results suggest that passerine birds may be susceptible to parasite-associated lymphomas.

Age, not infection dose, determines the outcome of Isospora suis infections in suckling piglets.

Data from 13 trials involving 124 suckling piglets experimentally infected with Isospora suis were evaluated for the effects of infection dose and age on the clinical and parasitological outcome of infection in four different models, infections with 1,000 oocysts on the 1(st) day of life (d.o.l.) (model 1; 9 piglets/3 litters), 1,000 oocysts on the 4(th) d.o.l. (model 2; 25 piglets/11 litters), 1,500 oocysts on the 4(th) d.o.l. (model 3; 40 piglets/20 litters) and 10,000 oocysts on the 4(th) d.o.l. (model 4; 50 animals/10 litters). Weights were determined on the day of birth and in weekly intervals. Faecal consistency and quantitative oocysts excretion were evaluated for 2 weeks starting 4 days after infection (d.p.i.). The weight gain depression was most noticeable in model 2 (infection on the 1(st) d.o.l.), where animals only gained 2.08 x their birth weight until the 22(nd) d.o.l., compared to 2.31-2.52 x in the other groups. This correlated with the occurrence of watery diarrhoea which was found in 37 % of the samples in the acute phase (4-11 d.p.i.) in model 2 but only in 12-20 % of the samples in the other models. Median oocyst excretion peaked earlier in the models with higher infection doses but reached the highest values in model 2 (early infection). As in previous studies, this cross-sectional analysis of a larger number of animals confirms the influence of age on the outcome of isosporosis in suckling piglets, stressing the need to control the infection at an early life phase.

Efficacy of various anticoccidials against experimental porcine neonatal isosporosis.

The efficacies of 20 mg/kg body weight (BW) of toltrazuril (Toltra) 2 days post-infection (dpi), 2 mg/kg BW of diclazuril 2 and 3 dpi and 200 mg/kg BW of sulphadimidine 2, 3 and 4 dpi were compared in a model for piglet isosporosis. Weight gain (first 4 weeks of life) and diarrhoea and oocyst excretion from 4 to 11 dpi were evaluated (10-12 piglets/group). Additionally, animals were killed and examined for pathohistological changes of the small intestines 5, 7, 11 and 14 dpi (n = 3 per group and time point) and lengths of the intestinal villi. Diarrhoea (semi-liquid or liquid faeces) was seen from 5 dpi in all groups except Toltra, and the differences in prevalence and intensity of diarrhoea were statistically significant (p < 0.05) between Toltra and the other groups, which were similar (trial 1). Oocyst excretion was greatly reduced in the Toltra group, which was also statistically significant for the mean and median excretion rates and the percentage of excreting piglets between Toltra and the other groups (p < 0.05). Weight gain was highest in Toltra (p < 0.05). Histopathology revealed mostly villous necrosis and atrophy in the small intestines except the duodenum, which peaked at 7 dpi, in all groups except Toltra. Between 5 and 11 dpi, the Toltra group had significantly longer villi than the other groups. Reduced weight gain and diarrhoea caused by Isospora suis was controlled by a single application of Toltra in the pre-patent period, while neither diclazuril nor sulphadimidine improved the clinical picture of isosporosis.

Isosporiasis in Venezuelan adults infected with human immunodeficiency virus: clinical characterization.

A cross-sectional study was undertaken to determine the prevalence of isosporiasis and its clinical and laboratory pattern in Venezuelan patients infected with human immunodeficiency virus (HIV) (n = 397). At enrollment, they underwent a thorough clinical history and physical examination, and provided stool specimens for the identification of Isospora belli and other parasites. Isospora belli was identified in 56 subjects (14%) and diarrhea, either acute or chronic, was present in 98% of these cases (P < 0.001). Eosinophilia was strongly associated with isosporiasis (P = 0.01). It was also found that the presence of eosinophilia was more common in I. belli-infected patients without weight loss (P < 0.001). Twenty-six (81.25%) subjects with I. belli infection had CD4+ cell counts < 200 cells/mm3 (P = 0.03). In addition, the data and its description shows the association to be < 100 cells/mm3. This infection seems to be seasonal because the recovery of oocysts occurred mainly in months with significant rainfall. In fact, isosporiasis should be suspected in HIV-infected patients from tropical countries with diarrhea, weight loss, eosinophilia, and low CD4+ cell counts.

Parasitological and morphological findings in porcine isosporosis after treatment with symmetrical triazintriones.

Neonatal porcine isosporosis is known to cause serious economic losses in piglet farms by causing severe enteritis with dehydration, weight loss and reduced development in the affected animals, predominantly during the first weeks of life. In the present study, piglets experimentally infected with Isospora suis were treated with Bay Vi 9143, a symmetrical triazintrione, at different days post-infection. As shown by clinical and pathological examinations, Bay Vi 9143 is effective against the asexual and sexual stages of I. suis at all selected treatment times. However, the therapeutic use at an early stage of asexual multiplication is most effective before the onset of clinical symptoms.

Lectin histochemistry for glycoconjugates in the small intestines of piglets naturally infected with Isospora suis.

Composition of glycoconjugates was examined in small intestines naturally infected with Isospora suis in preweaned pigs by use of 21 biotinylated-labeled lectins with avidin-biotin-peroxidase complex method. As compared with control pig, staining of 18 lectins altered in jejunal villus brush border and goblet cells of pigs naturally infected with I. suis. These results indicate that I. suis infection alters carbohydrate residues on the jejunal intestines.

Isospora belli infection: observation of unicellular cysts in mesenteric lymphoid tissues of a Brazilian patient with AIDS and animal inoculation.

We describe the finding of unizoic cysts of Isospora belli in lymphoid tissues of a Brazilian patient with AIDS, and discuss the possibilities of their drug resistance, they being the cause of relapses, and of being an indication for the existence of intermediary or paratenic animal hosts.

An outbreak of adenoviral infection in inland bearded dragons (Pogona vitticeps) coinfected with dependovirus and coccidial protozoa (Isospora sp.).

Thirty of 200 (15%) hatchling inland bearded dragons were found dead after a short period (48 hours) of weakness and lethargy. The most common clinical signs were head tilt and circling. Six bearded dragons with neurological signs were euthanized, and postmortem examination revealed no gross abnormalities. Microscopically, severe, randomly distributed hepatocellular necrosis with large basophilic intranuclear inclusion bodies in numerous hepatocytes was noted. Small-intestinal enterocytes contained intracytoplasmic coccidial protozoa (Isospora sp.) and occasional enterocytes had basophilic intranuclear inclusion bodies. Transmission electron microscopy revealed both 80- and 20-nm-diameter viral particles, which were consistent with adenoviruses and dependoviruses, respectively. Adenoviral outbreaks in groups of animals are uncommon. An adverse synergistic effect of the coccidiosis with the adenoviral infection may have played a critical role in the high morbidity and mortality in this case.