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Background Avian haemosporidian parasites can cause severe disease in their hosts due to excessive exo-erythrocytic merogony and anaemia caused by blood stages. Notably, the development of megalomeronts by species of Haemoproteus and Leucocytozoon has been associated with mortalities in birds. Diagnosis of lethal infections is currently accomplished by the detection of parasites’ tissue stages in histological sections combined with PCR and sequencing. However, sequences frequently are not reliably obtained and the generic discrimination of exo-erythrocytic tissue stages based on morphological characters is challenging. Therefore, the present study aimed at developing specific molecular probes for the identification of Haemoproteus spp. and Leucocytozoon spp. in histological sections using chromogenic in situ hybridization. Methods Parasite subgenus-specific oligonucleotide probes were designed to target the 18S ribosomal RNA of Haemoproteus species (subgenus Parahaemoproteus ) and Leucocytozoon spp. (subgenus Leucocytozoon ) and were in situ hybridized to sections from formalin-fixed, paraffin-embedded tissue samples determined positive for these parasites by PCR and histopathology. To confirm the presence of parasites at sites of probe hybridization, consecutive sections were stained with haematoxylin–eosin and examined. Results Parahaemoproteus - and Leucocytozoon -specific probes labelled erythrocytic and exo-erythrocytic stages of Haemoproteus spp. and Leucocytozoon spp., respectively. Binding of probes to parasites was consistent with detection of the same exo-erythrocytic meronts in consecutive haematoxylin–eosin-stained sections. Cross-reactivity of the probes was ruled out by negative chromogenic in situ hybridization when applied to samples positive for a parasite of a genus different from the probes’ target. Conclusions Chromogenic in situ hybridization using 18S ribosomal RNA-specific oligonucleotide probes reliably identifies and discriminates Haemoproteus and Leucocytozoon parasites in tissue sections and enables unequivocal diagnosis of haemosporidioses.

Cryptosporidium is a major cause of diarrhoeal disease and mortality in young children in resource-poor countries, for which no vaccines or adequate therapeutic options are available. Infection in humans is primarily caused by two species: C. hominis and C. parvum . Despite C. hominis being the dominant species infecting humans in most countries, very little is known about its growth characteristics and life cycle in vitro, given that the majority of our knowledge of the in vitro development of Cryptosporidium has been based on C. parvum . In the present study, the growth and development of two C. parvum isolates (subtypes Iowa-IIaA17G2R1 and IIaA18G3R1) and one C. hominis isolate (subtype IdA15G1) in HCT-8 cells were examined and compared at 24 h and 48 h using morphological data acquired with scanning electron microscopy. Our data indicated no significant differences in the proportion of meronts or merozoites between species or subtypes at either time-point. Sexual development was observed at the 48-h time-point across both species through observations of both microgamonts and macrogamonts, with a higher frequency of macrogamont observations in C. hominis (IdA15G1) cultures at 48-h post-infection compared to both C. parvum subtypes. This corresponded to differences in the proportion of trophozoites observed at the same time point. No differences in proportion of microgamonts were observed between the three subtypes, which were rarely observed across all cultures. In summary, our data indicate that asexual development of C. hominis is similar to that of C. parvum, while sexual development is accelerated in C. hominis. This study provides new insights into differences in the in vitro growth characteristics of C. hominis when compared to C. parvum , which will facilitate our understanding of the sexual development of both species.

Eimeriosis, an infection with Eimeria spp. that affects poultry, causes huge economic losses. Silver nanoparticles (AgNPs) have antibacterial and antifungal properties, but their action against Eimeria infection has not yet been elucidated. This study demonstrates the action of AgNPs in the treatment of mice infected with Eimeria papillata . AgNPs were prepared from Zingiber officinale rhizomes. Phytochemical screening by gas chromatography–mass spectrometry analysis (GC–MS) was used to detect active compounds. Mice were divided into five groups: uninfected mice, uninfected mice that were administered AgNPs, untreated mice infected with 10 3 sporulated oocysts of E. papillata , infected mice treated with AgNPs, and infected mice treated with amprolium. Characterization of the samples showed the AgNPs to have nanoscale sizes and aspherical shape. Phytochemical screening by GC–MS demonstrated the presence of 38 phytochemical compounds in the extract of Z. officinale . Mice infected with E. papillata -sporulated oocysts were observed to have many histopathological damages in the jejuna, including a decrease in the goblet cell numbers affecting the jejunal mucosa. Additionally, an increased oocyst output was also observed. The treatment of infected mice with AgNPs resulted in the improvement of the jejunal mucosa, increase in the number of goblet cell, and decrease in the number of meronts, gamonts, and developing oocysts in the jejuna. Moreover, AgNPs also led to decreased oocyst shedding in feces. The results revealed AgNPs to have an anticoccidial effect in the jejunum of E. papillata -infected mice and, thus, could be a potential treatment for eimeriosis.

Based on morphological and genetic characteristics, we describe a new species of Hepatozoon in the European wild cat (Felis silvestris silvestris), herein named Hepatozoon silvestris sp. nov. The study also provides the first data on the occurrence of H. felis in this wild felid. Hepatozoon meronts were observed in multiple cross-sections of different organs of four (44%) cats. Additionally, extracellular forms, resembling mature gamonts of Hepatozoon, were found in the spleen and myocardium of two cats. Furthermore, tissues of six animals (67%) were positive by PCR. Hepatozoon felis was identified infecting one cat (11%), whereas the 18S rRNA sequences of the remaining five cats (56%) were identical, but distinct from the sequences of H. felis. Phylogenetic analyses revealed that those sequences form a highly supported clade distant from other Hepatozoon spp. Future studies should include domestic cats from the areas where the wild cats positive for H. silvestris sp. nov. were found, in order to investigate their potential role to serve as intermediate hosts of this newly described species. Identification of its definitive host(s) and experimental transmission studies are required for elucidating the full life cycle of this parasite and the possible alternative routes of its transmission.

A new microsporidian species, Globosporidium paramecii gen. nov., sp. nov., from Paramecium primaurelia is described on the basis of morphology, fine structure, and SSU rRNA gene sequence. This is the first case of microsporidiosis in Paramecium reported so far. All observed stages of the life cycle are monokaryotic. The parasites develop in the cytoplasm, at least some part of the population in endoplasmic reticulum and its derivates. Meronts divide by binary fission. Sporogonial plasmodium divides by rosette-like budding. Early sporoblasts demonstrate a well-developed exospore forming blister-like structures. Spores with distinctive spherical shape are dimorphic in size (3.7 ± 0.2 and 1.9 ± 0.2 μm). Both types of spores are characterized by a thin endospore, a short isofilar polar tube making one incomplete coil, a bipartite polaroplast, and a large posterior vacuole. Experimental infection was successful for 5 of 10 tested strains of the Paramecium aurelia species complex. All susceptible strains belong to closely related P. primaurelia and P. pentaurelia species. Phylogenetic analysis placed the new species in the Clade 4 of Microsporidia and revealed its close relationship to Euplotespora binucleata (a microsporidium from the ciliate Euplotes woodruffi), to Helmichia lacustris and Mrazekia macrocyclopis, microsporidia from aquatic invertebrates.

Habitat modification may facilitate the emergence of novel pathogens, and the expansion of agricultural frontiers make domestic animals important sources of pathogen spillover to wild animals. We demonstrate for the first time that Plasmodium juxtanucleare, a widespread parasite from domestic chickens, naturally infects free-living passerines. We sampled 68 wild birds within and at the border of conservation units in central Brazil composed by Cerrado, a highly threatened biome. Seven out of 10 passerines captured in the limits of a protected area with a small farm were infected by P. juxtanucleare as was confirmed by sequencing a fragment of the parasite's cytochrome b. Blood smears from these positive passerines presented trophozoites, meronts and gametocytes compatible with P. juxtanucleare, meaning these birds are competent hosts for this parasite. After these intriguing results, we sampled 30 backyard chickens managed at the area where P. juxtanucleare-infected passerines were captured, revealing one chicken infected by the same parasite lineage. We sequenced the almost complete mitochondrial genome from all positive passerines, revealing that Brazilian and Asian parasites are closely related. P. juxtanucleare can be lethal to non-domestic hosts under captive and rehabilitation conditions, suggesting that this novel spillover may pose a real threat to wild birds.

Avian haemosporidian parasites (Haemosporida, Apicomplexa) are globally distributed and infect birds of many orders. These pathogens have been much investigated in domestic and wild passeriform birds, in which they are relatively easy to access. In birds belonging to other orders, including owls (order Strigiformes), these parasites have been studied fragmentarily. Particularly little is known about the exo-erythrocytic development of avian haemosporidians. The goal of this study was to gain new knowledge about the parasites infecting owls in Europe and investigate their exo-erythrocytic stages. Tissue samples of 121 deceased owls were collected in Austria and Lithuania, and examined using polymerase chain reactions (PCR), histology, and chromogenic in situ hybridization (CISH). PCR-based diagnostics showed a total prevalence of 73.6%, revealing two previously unreported Haemoproteus and five novel Leucocytozoon lineages. By CISH and histology, meronts of several Leucocytozoon lineages (lASOT06, lSTAL5, lSTAL7) were discovered in the brains, heart muscles, and kidneys of infected birds. Further, megalomeronts of Haemoproteus syrnii (lineage hSTAL2) were discovered. This study contributes new knowledge to a better understanding of the biodiversity of avian haemosporidian parasites infecting owls in Europe, provides information on tissue stages of the parasites, and calls for further research of these under-investigated pathogens relevant to bird health.

Background Hepatozoon silvestris is an emerging apicomplexan parasite discovered in European wild cats from Bosnia and Herzegovina and blood samples of a domestic cat from Southern Italy in 2017. It has also been identified in Ixodes ricinus collected from a domestic cat in Wales, UK, in 2018. The clinical relevance, pathogenesis and epidemiology of this novel Hepatozoon species are not yet understood. Thus, the objective of this paper was to report and describe the first fatal case of an H. silvestris infection in a domestic cat. Results The cat, which originated from Switzerland, died shortly after presenting clinical signs of lethargy, weakness and anorexia. At necropsy, no specific lesions were observed. Histopathology of the heart revealed a severe lympho-plasmacytic and histiocytic myocarditis. Mature and developing protozoal meronts morphologically compatible with Hepatozoon species were observed associated with the myocardial inflammation. No other lesions were present in any other organ evaluated, and the cat tested negative for retroviral and other immunosuppressive infectious agents. Polymerase chain reaction from the myocardium resulted in a specific amplicon of the Hepatozoon 18S rRNA gene. Sequencing and BLAST analysis revealed 100% sequence identity with H. silvestris. Conclusions The severity of the infection with fatal outcome in an otherwise healthy animal suggests a high virulence of H. silvestris for domestic cats. The presence of this emerging parasite in a domestic cat in Switzerland with no travel history provides further evidence for a geographical distribution throughout Europe.

Background Cryptosporidium parvum is a zoonotic parasite and member of the phylum Apicomplexa with unique secretory organelles, including a rhoptry, micronemes and dense granules that discharge their contents during parasite invasion. The mucin-like glycoprotein GP900 with a single transmembrane domain is an immunodominant antigen and micronemal protein. It is relocated to the surface of excysted sporozoites and shed to form trails by sporozoites exhibiting gliding motility (gliding sporozoites). However, the biological process underlying its relocation and shedding remains unclear. The primary aim of this study was to determine whether GP900 is present as a transmembrane protein anchored to the plasma membrane on the surface of sporozoites and whether it is cleaved before being shed from the sporozoites. Methods Two anti-GP900 antibodies, a mouse monoclonal antibody (mAb) to the long N-terminal domain (GP900-N) and a rabbit polyclonal antibody (pAb) to the short C-terminal domain (GP900-C), were produced for the detection of intact and cleaved GP900 proteins in sporozoites and other parasite developmental stages by microscopic immunofluorescence assay and in discharged molecules by enzyme-linked immunosorbent assay. Results Both anti-GP900 antibodies recognized the apical region of unexcysted and excysted sporozoites. However, anti-GP900-N (but not anti-GP900-C) also stained both the pellicles/surface of excysted sporozoites and the trails of gliding sporozoites. Both antibodies stained the intracellular meronts, both developing and developed, but not the macro- and microgamonts. Additionally, the epitope was recognized by anti-GP900-N (but not anti-GP900-C) and detected in the secretions of excysted sporozoites and intracellular parasites. Conclusions GP900 is present in sporozoites and intracellular meronts, but absent in sexual stages. It is stored in the micronemes of sporozoites, but enters the secretory pathway during excystation and invasion. The short cytoplasmic domain of GP900 is cleaved in the secretory pathway before it reaches the extracellular space. The molecular features and behavior of GP900 imply that it plays mainly a lubrication role. Graphical Abstract

Species of Plasmodium (Plasmodiidae, Haemosporida) are widespread and cause malaria, which can be severe in avian hosts. Molecular markers are essential to detect and identify parasites, but still absent for many avian malaria and related haemosporidian species. Here, we provide first molecular characterization of Plasmodium matutinum, a common agent of avian malaria. This parasite was isolated from a naturally infected thrush nightingale Luscinia luscinia (Muscicapidae). Fragments of mitochondrial, apicoplast and nuclear genomes were obtained. Domestic canaries Serinus canaria were susceptible after inoculation of infected blood, and the long-lasting light parasitemia developed in two exposed birds. Clinical signs of illness were not reported. Illustrations of blood stages of P. matutinum (pLINN1) are given, and phylogenetic analysis identified the closely related avian Plasmodium species. The phylogeny based on partial cytochrome b (cyt b) sequences suggests that this parasite is most closely related to Plasmodium tejerai (cyt b lineage pSPMAG01), a common malaria parasite of American birds. Both these parasites belong to subgenus Haemamoeba, and their blood stages are similar morphologically, particularly due to marked vacuolization of the cytoplasm in growing erythrocytic meronts. Molecular data show that transmission of P. matutinum (pLINN1) occurs broadly in the Holarctic, and the parasite likely is of cosmopolitan distribution. Passeriform birds and Culex mosquitoes are common hosts. This study provides first molecular markers for detection of P. matutinum.