Molecular characterization of isosporoid coccidia (Isospora and Atoxoplasma spp.) in passerine birds

Prevalence and disease caused by isosporoid coccidia in passerine birds are well recognized, but confusion about the life cycles of the parasites has led to taxonomic inconsistencies. In this study, we characterized segments of the chromosomal small and large-subunit ribosomal RNA (rRNA) genes of coccidial parasites from 23 species of passerine birds, as well as heat shock protein 70, apicoplast rRNA, and chromosomal 5.8s rRNA genes from a subgroup of these animals, and we correlated genetic data with morphologic findings for different parasite developmental stages, host phylogeny, and overall taxonomic relations within the phylum Apicomplexa. Our findings indicate that isosporoid coccidia of passerine birds are monophyletic but exhibit substantial diversity, with most avian species having one or several unique parasite lineages that underwent synchronous speciation with their hosts, interrupted by sporadic episodes of lateral transmission across species and families. Molecular analyses support a homoxenous life cycle, with sexual forms occurring chiefly in the intestines and asexual merozoites present systemically. Rarely, extraintestinal sexual stages can occur. The passerine coccidia are genetically most closely related to species of Eimeria rather than Isospora. We suggest that these parasites, whether identified from blood merozoite stages or fecal oocysts, be provisionally grouped as a homogeneous clade of individual species in a single taxon and formally named when reliable criteria allowing reclassification of related genera in the suborder Eimeriina are clarified.     

Disseminated visceral coccidiosis in a wild white-naped crane (Grus vipio)

Disseminated visceral coccidiosis (DVC) was unexpectedly recognized in a wild white-naped crane (Grus vipio) killed by phosphamidon insecticide. On gross pathologic examination, widely disseminated white nodules were found on the serosa of the pro-ventriculus, gizzard, and intestine, as well as on the surface and in the parenchyma of liver, spleen, and cardiac muscle. Microscopically, asexual stages of a coccidia were observed in some nodules. However, the species of coccidia could not be determined because no oocysts were found on fecal examination. This is believed to be the first reported case of DVC in a wild white-naped crane infected with Eimeria spp.     

Intestinal Cryptosporidium sp. infection in the Egyptian tortoise, Testudo kleinmanni

An adult Egyptian tortoise (Testudo kleinmanni) presented with clinical signs of enteritis and died 5 weeks after initiation of antibiotic therapy. Histological examination of the small intestine revealed heavy infection with Cryptosporidium sp.; over 80% of epithelial cells harboured the pathogen. No Cryptosporidium developmental stages were present in the stomach or the lungs. The intestinal lamina propria and mucosa were infiltrated by heterophils, lymphocytes and macrophages. The present study constitutes the first report of Cryptosporidium sp. infection in T. kleinmanni, and the first histological documentation of intestinal cryptosporidiosis in Chelonia.     

Prevalence and pathological study on rabbit hepatic coccidiosis in Taiwan.

Five breeds of rabbits, which included the New Zealand, Californian, Spot, Rex and Angora rabbit, were found from a survey of 1,152 rabbits in Taiwan. The prevalence of coccidia in young rabbits (weaning-2 months old) was 95% to 100%. Adult female rabbits usually acted as carriers within the farm and transmitted the parasite to young rabbits, which caused severe infection with clinical signs and even death. Parasitism of hepatic coccidia (Eimeria stiedai) in the rabbit led to severe mortality. Numerous and scattered white nodules about 0.1 to 0.5 cm in diameter were seen on the liver surface and dark greenish mucoid exudate was found in intestinal lumen. Histopathologic lesions included hyperplasia of the bile duct epithelium with different developmental stages of coccidia within. Oocysts could be seen in the lumen, and granuloma tissues encircle the bile duct with infiltration of inflammatory cells. The other organs were not infected.     

Cytochemical study of various stages of life cycle of Toxoplasma gondii. 10. Phosphatases in the parasites during developmental stages in cat's intestines]

Several methods of acid and alkaline phosphatese and ATPase detection using both natural and artificial substrates were applied to the intestinal stages of Toxoplasma gondii with negative results to reveal enzymatic activity in all the stages except the microgametocyte. Possible explanation of this unexpected phenomenon is discussed taking into account host-parasite relationships with intestinal stages of Toxoplasma and with coccidia in general.     

The site of action of the anticoccidial salinomycin (Coxistac).

The anticoccidial salinomycin has a cidal effect against chicken coccidia. Restricted and unrestricted medication studies and histopathological examinations of chicks infected with Eimeria acervulina, E. maxima, or E. tenella showed that parasites were destroyed within host cells during asexual development. Most sporozoites failed to become trophozoites and were destroyed 30--72 hr after ingestion of oocysts. The drug also affected schizonts during initial nuclear replication by either destroying or significantly delaying their maturation. Parasites affected by the drug were distorted grossly. Drug action against gametogony was not observed histologically, but when medication was restricted to this period of the life cycle, subsequent oocyst shedding of all 3 species was reduced by 20--70% compared to unmedicated controls. When drug was provided during the entire parasite life cycle, activity against asexual stages was so complete that only a limited number of parasites survived to form gamonts, and oocyst shedding was reduced by 80--90% relative to controls. As with other ionophores, salinomycin had no effect upon rate of oocyst sporulation.     

Isospora as an Extraintestinal Parasite of Passerine Birds1

Toxoplasma-like avian parasites inhabiting mononuclear phagocytes have been called Haemogregarina, Toxoplasma. avian Toxoplasma, Atoxoplasma, and Lankesterella by various authors. My attempts to transmit the parasites by bloodsucking mites or by transfer of blood and tissues of infected sparrows and canaries were unsuccessful. However, it was noted that the infection was exacerbated under conditions that favored transmission of coccidiosis: crowding and lack of cleanliness. Oral inoculation of sporulated oocysts of Isospora resulted in death from overwhelming macrophage infection with Toxoplasma-like organisms. Experiments using sparrows and canaries showed that the Isospora species involved was not cross infectious. Further investigations using canaries demonstrated that after oral oocyst inoculation, infection of macrophages spread from the submucosa of the duodenum to the liver. spleen, and lungs. After several generations in the internal organs, asexual multiplication, occured in the intestinal epithelium of the small intestinc. Fecal oocysts first appeared at the end of 9–10 days. Oocysts continued to be passed in the feces for months after infection. This chronicity may be explained by the relatively long life of the macrophages that serve as host cells for the asexual stages as compared to the intestinal epithelium which is the cell type parasitized by conventional coccidia.     

The cathepsin-D expression in the cyto-invasive stages of coccidia in intestinal mucosa of chickens,and the relevance of this proteinase for intracellular penetration and spreading of coccidia in host tissues

The Cathepsin-D proteinase was demonstrated immunohistochemically in cases of intestinal coccidiosis in chickens; its expression concerned almost the cyto-invasive stages of reproductive cycle of several strains of coccidia (merozoites of the II-nd generation and also microgametes). The authors consider Cathepsin-D as a potentional "penetration/spreading factor", being significant for intracellular invasion and spreading of coccidia into host cells. In diagnostic practice, an immunohistochemical demonstration of Cathepsin-D is a relatively simple and sensitive method, which brings information on the extent of parasitic lesions, and on participation of particular evolutive forms of coccidial life cycle; it may also be useful in proving isolated coccidia in the intestinal epithelium, poorly recognized in routine HE preparation. Some Cathepsin-D protease reactivity in intestinal Cryptosporidiosis is also briefly mentioned. The examined cases of cerebral toxoplasmosis and primary acanthamoebic meningoencephalitis (PAME) showed in above mentioned methods negative results.     

斑马鱼食源性肠炎模型及其免疫细胞成像分析方法的建立

为了研究豆粕替代鱼粉对鱼类肠黏膜免疫的影响和建立缓解肠炎药物的筛选及功能性饲料添加剂的平台,研究采用固有免疫或适应性免疫细胞荧光标记的斑马鱼品系,通过50％的豆粕添加量替代鱼粉作为蛋白源,共设计了两组饲料,分别在幼鱼固有免疫或适应性免疫的发育阶段中饲喂荧光标记的斑马鱼,构建了斑马鱼豆粕诱导的肠炎模型,对构建的食源性肠炎...     

Discovery of three novel coccidian parasites infecting California sea lions (Zalophus californianus), with evidence of sexual replication and interspecies pathogenicity

Enteric protozoal infection was identified in 5 stranded California sea lions (Zalophus californianus). Microscopically, the apical cytoplasm of distal jejunal enterocytes contained multiple stages of coccidian parasites, including schizonts with merozoites and spherical gametocytes, which were morphologically similar to coccidians. By histopathology, organisms appeared to be confined to the intestine and accompanied by only mild enteritis. Using electron microscopy, both sexual (microgametocytes, macrogamonts) and asexual (schizonts, merozoites) coccidian stages were identified in enterocytes within parasitophorous vacuoles, consistent with apicomplexan development in a definitive host. Serology was negative for tissue cyst-forming coccidians, and immunohistochemistry for Toxoplasma gondii was inconclusive and negative for Neospora caninum and Sarcocystis neurona. Analysis of ITS-1 gene sequences amplified from frozen or formalin-fixed paraffin-embedded intestinal sections identified DNA sequences with closest homology to Neospora sp. (80%); these novel sequences were referred to as belonging to coccidian parasites "A," "B," and "C." Subsequent molecular analyses completed on a neonatal harbor seal (Phoca vitulina) with protozoal lymphadenitis, hepatitis, myocarditis, and encephalitis showed that it was infected with a coccidian parasite bearing the "C" sequence type. Our results indicate that sea lions likely serve as definitive hosts for 3 newly described coccidian parasites, at least 1 of which is pathogenic in a marine mammal intermediate host species.     

Pathogenesis of type II avian adenovirus infection in turkeys: in vivo immune cell tropism and tissue distribution of the virus.

Hemorrhagic enteritis virus (HEV), a type II avian adenovirus, causes intestinal hemorrhages and immunosuppression in turkeys. In this study, we exposed turkeys to virulent HEV and examined fractionated spleen cells for the presence of viral DNA by in situ hybridization and amplification of DNA extracted from virus-infected cells by PCR. HEV replication was detected only in the immunoglobulin M-bearing B lymphocytes and macrophage-like cells but not in the CD4+ or CD8+ T lymphocytes. The inability to infect T cells distinguishes type II avian adenoviruses from lymphotropic mammalian adenoviruses which infect and replicate in T cells. Furthermore, these data suggested that HEV-induced immunosuppression in turkeys may be due to the effect of the virus on B lymphocytes and macrophages. We also examined tissue tropism of HEV by in situ hybridization conducted on sections of lymphoid and nonlymphoid tissues. Large numbers of HEV-positive cells were detected in spleen and cecal tonsils. Diminutive viral activity was present in the intestines, the principal site of HEV-induced pathology. Thus, intestinal pathology was not associated with local cytopathic viral replication. This result and our previous observation that cyclosporin A abrogated intestinal hemorrhaging in HEV-infected turkeys strongly suggested that intestinal lesion induced by this virus may be immune system mediated.     

Gene expression profile in human late radiation enteritis obtained by high-density cDNA array hybridization

Late radiation enteritis is a sequela of radiation therapy to the abdomen. The pathogenic process is poorly understood at the molecular level. cDNA array analysis was used to provide new insights into the pathogenesis of this disorder. Gene profiles of six samples of fibrotic bowel tissue from patients with radiation enteritis and six healthy bowel tissue samples from patients without radiation enteritis were compared using membrane-based arrays containing 1314 cDNAs. Results were confirmed with real-time RT-PCR and Western blot analysis. Array analysis identified many differentially expressed genes involved in fibrosis, stress response, inflammation, cell adhesion, intracellular and nuclear signaling, and metabolic pathways. Increased expression of genes coding for proteins involved in the composition and remodeling of the extracellular matrix, along with altered expression of genes involved in cell- to-cell and cell-to-matrix interactions, were observed mainly in radiation enteritis samples. Stress, inflammatory responses, and antioxidant metabolism were altered in radiation enteritis as were genes coding for recruitment of lymphocytes and macrophages. The Rho/HSP27 (HSPB1)/zyxin pathway, involved in tissue contraction and myofibroblast transdifferentiation, was also altered in radiation enteritis, suggesting that this pathway could be related to the fibrogenic process. Our results provide a global and integrated view of the alteration of gene expression associated with radiation enteritis. They suggest that radiation enteritis is a dynamic process involving constant remodeling of each structural component of the intestinal tissue, i.e. the mucosa, the mesenchyme, and blood vessels. Functional studies will be necessary to validate the present results.     

Intralymphocytic schizonts associated with an initial infection of Saurocytozoon tupinambi in Tupinambis teguixin

An initial natural infection of Saurocytozoon tupinambi in a juvenile Tupinambis teguixin from Venezuela was studied for 131 days following capture of the host. Intralymphocytic parasites appeared in this sequence: small uninucleate and binucleate stages (days 1–31 and again on day 41); schizonts with 3–102 nuclei (days 8–14 and 29–35); immature gametocytes (days 29–35) and apparently mature gametocytes of Saurocytozoon tupinambi from day 41. Maximum parasitemia of trophozoites and binucleate schizonts occurred on day 4 when 11% of lymphocytes were infected. Maximum parasitemia by larger schizonts occurred on day 8 at 0.13% of lymphocytes, while maximum gametocytemia was found on day 49 with 16.4% of lymphocytes parasitized. Two types of schizonts were observed: intralymphocytic and the same type free of host cells, and fragments of varying size which may have been torn from capillary endothelium.Due to presence of concurrent infection by a small Plasmodium species, identity of intralymphocytic asexual stages with S. tupinambi cannot be established. Presence of asexual and sexual stages in the same type of host cells (lymphocytes and close derivatives), sequential appearance of trophozoites, schizonts and gametocytes over a period of 40 days, and correlated fluctuations in lymphocyte density suggest they are conspecific, and that Saurocytozoon, which has a plasmodiid type of sporogony may prove to further differ from leucocytozoids by presence of an asexual cycle in circulating blood cells.     

Early events in the life cycle of the mouse coccidium Eimeria falciformis (Eimer, 1870) Schneider, 1875 in naive and immune hosts

The initial phases of invasion of mammalian coccidia of the genus Eimeria into host tissue are still poorly known. This process, including the passage of oocysts through the intestinal lumen, excystation of sporozoites, their penetration into epithelial cells and migration to the target site was studied in both naive and immune mice infected with Eimeria falciformis. After oral infection, the intact oocysts were transported with enteral contents to the large intestine, where the excystation of sporozoites and their penetration into superficial epithelium took place. The sporozoites subsequently migrated into the epithelium of crypts, which is the specific site of asexual multiplication. The immune status of the hosts did not affect the passage of oocysts, excystation and penetration of sporozoites. However, the migration of sporozoites towards their target site (crypts) was impeded in immune mice and sporozoites tended to remain in superficial mucosa rather than migrate to the crypts.     

Cryptosporidium parvum life cycle in suckling mice: a Nomarski interference-contrast study of a human-derived strain.

Cryptosporidiosis has emerged as one of the life-threatening opportunistic enteric infections in HIV-infected persons. To date, Cryptosporidium parvum is known to infect man via person-to-person or zoonotic transmission. We studied the sequential stages of the life cycle of C. parvum by Normarski interference-contrast microscopy in fresh gut specimens of newborn mice, infected with a strain derived from an AIDS patient with cryptosporidial diarrheal enteritis. Many 4- to 5-day-old suckling BALB/C mice were orally inoculated with 1 x 10(6) oocysts, obtained by acid flocculation of the patient's stools. The animals were sacrificed from 4 to 96 h post-infection and the ileum was examined microscopically. All stages of the asexual life cycle of C. parvum, from excysted sporozoites in the intestinal lumen through the development of type II mature meronts, 12- to 72-h post-infection, were documented by extemporaneous microscopic evaluation of fresh gut samples. The sexual cycle, characterized by the appearance of micro- and macrogametocytes, followed by a zygote developing into a sporulated oocyst, was documented as early 48-h post-infection. Our Nomarski interference-contrast observations on the life cycle of C. parvum yielded data comparable with those originally published by Current and Reese, and confirm the results of previous electron microscopic studies performed by several other authors.     

Rotaviral enteritis in a raccoon

A hand-reared raccoon (Procyon lotor) kit had severe diarrhea and died within 24 hr. Gross and histopathologic findings were compatible with a diagnosis of viral enteritis. The immunoperoxidase test revealed rotavirus group A antigen in the intestinal mucosa. This is the first record of rotaviral enteritis in a raccoon.     

Hybridoma antibody characterization of stage-specific and stage-cross-reactive antigens of Eimeria tenella

Hybridoma antibodies (HAb) have been raised against the sporozoite stage of 3 species of avian coccidia. These HAb were utilized in Western blot analysis, resulting in the immunoenzymatic detection of sporozoite and merozoite antigens of 1 species, Eimeria tenella. The 5 HAb specific for the sporozoite stage showed either single bands at 22 and 28 kDa or a large diffuse band in the 7-10-kDa range. The 4 HAb that cross-reacted with both asexual stages recognized either a single sporozoite or merozoite antigen of 90 kDa, or multiple antigens (47-69 kDa) for both stages. The 9 HAb demonstrated 5 different immunofluorescent antibody (IFA) patterns, and the 4 cross-reactive HAb showed similar IFA patterns with both asexual stages of E. tenella. The sporozoite-specific HAb which identified the 22, 7-10, and 7-8 kDa antigens showed surface, surface-internal, or internal IFA patterns. The other sporozoite-specific HAb, which labeled the 28-kDa antigen, stained the refractile body. The IFA of the 4 stage-cross-reactive HAb, which recognized the 45-60-kDa and the 90- or 47-69-kDa antigens, localized these antigens to the surface and tip, respectively. Rabbit anti-sporozoite serum appeared to recognize all of the sporozoite and merozoite antigens identified by the HAb as well as a variety of additional stage-cross-reactive antigens.     

Cellular apoptosis]

The interrelations between lymphocytes and intestinal epithelium cells after total X-ray irradiation (15 Gy) were investigated autoradiographically in CBA mice. The labelled lymphocytes injected in the irradiated animals migrated in intestinal epithelium, the label being present within vacuoles of the crypt cells and later in their nuclei. The trophic function of lymphocytes and the nature of the so-called apoptosis are discussed.     

Determination of the activity of diclazuril against Sarcocystis neurona and Sarcocystis falcatula in cell cultures

Diclazuril is a benzeneacetonitril anticoccidial that has excellent activity against the extraintestinal stages of Toxoplasma gondii and Neospora caninum. It also is highly active against intestinal coccidia of poultry. The present study examined the efficacy of diclazuril in inhibiting merozoite production of Sarcocystis neurona and Sarcocystis falcatula in bovine turbinate cell cultures. Diclazuril inhibited merozoite production by more than 80% in cultures of S. neurona or S. falcatula treated with 0.1 ng/ml diclazuril and greater than 95% inhibition of merozoite production was observed when infected cultures were treated with 1.0 ng/ml diclazuril. Diclazuril may have promise as a therapeutic agent in the treatment of S. neurona-induced equine protozoal myeloencephalitis in horses and S. falcatula infections in birds.