High Abundance of Haemoproteus Parasites in Culicoides (Diptera, Ceratopogonidae), with a Confirmation of Culicoides reconditus as a New Vector of These Avian Blood Parasites.

Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors.

First experimental observation on biology of the avian malaria parasite Plasmodium (Novyella) homonucleophilum (lineage pSW2), with remarks on virulence and distribution.

Species of subgenus Novyella remain most fragmentarily studied amongst avian malaria agents. Transmission of the recently described Plasmodium (Novyella) homonucleophilum (lineage pSW2) occurs broadly in the Old World, including Europe, however biology of this pathogen remains insufficiently investigated. This study provided the first data on the development of P. homonucleophilum in the experimentally infected Eurasian siskins Spinus spinus exposed by inoculation of infected blood. The parasite strain was isolated from a naturally infected song thrush Turdus philomelos, multiplied in vivo, and inoculated to six Eurasian siskins. The same number of birds were used as negative controls. All exposed birds were susceptible, and the controls remained uninfected during the entire study (172 days). Prepatent period was 8-12 days post exposure (dpe). Maximum parasitaemia reached 50-90 % of infected erythrocytes between 20 and 44 dpe. Then, parasitaemia decreased but remained relatively high during the entire observation. Three of six exposed birds died, indicating high virulence of this infection. The parasitaemia increase coincided with a decline of haematocrit value, indicating anaemia. Polychromasia was evident in all infected birds but not in controls. Body mass of exposed birds increased, coinciding with increased food intake. The latter probably is an adaptation to compensate energy loss of hosts due to the long-lasting parasitism. Exo-erythrocytic stages were not found, suggesting that long-lasting parasitaemia was entirely due to erythrocytic merogony. The lineage pSW2 has been reported broadly in the Old World and is likely a generalist infection. Neglected avian Novyella malaria parasites are worth more attention of researchers due to their cosmopolitan distribution and high virulence.

Brain parasites and misorientation of migratory birds.

Haemoproteus blood parasites of birds are thought to be relatively benign. Recent findings show that infections may develop in the brain of birds, possibly distorting their orientation sense. Misdirected migration may lead migrants outside their range where they are recognized as vagrants and can transmit parasites to novel hosts.

Unexpected absence of exo-erythrocytic merogony during high gametocytaemia in two species of Haemoproteus (Haemosporida: Haemoproteidae), including description of Haemoproteus angustus n. sp. (lineage hCWT7) and a report of previously unknown residual bodies during in vitro gametogenesis.

Neglected avian blood parasites of the genus Haemoproteus (Haemoproteidae) have recently attracted attention due to the application of molecular diagnostic tools, which unravelled remarkable diversity of their exo-erythrocytic (or tissue) stages both regarding morphology and organ tropism levels. The development of haemoproteids might result in pathologies of internal organs, however the exo-erythrocytic development (EED) of most Haemoproteus species remains unknown. Seven individual birds - Curruca communis (1) and Phylloscopus trochilus (6) - with high gametocytaemia (between 1% and 24%) of Haemoproteus angustus n. sp. (hCWT7) and Haemoproteus palloris (lineage hWW1) were sampled in Lithuania, and their internal organs were examined extensively by parallel application of histology and chromogenic in situ hybridization methods. Tissue stages were apparently absent, suggesting that the parasitaemia was not accompanied by detectable tissue merogony. Haemoproteus angustus n. sp. was described and characterized morphologically and molecularly. Sexual process and ookinete development of the new species readily occurred in vitro, and a unique character for Haemoproteus parasites was discovered - the obligatory development of several tiny residual bodies, which were associated with intracellular transformation of both macrogametocytes and microgametocytes before their escape from the host cells and formation of gametes. A DNA haplotype network was constructed with lineages that cluster in one clade with the lineage hCWT7. This clade consists of lineages mostly found in Curruca birds, indicating specificity for birds of this genus. The lineage hCWT7 is mainly a parasite of C. communis. Most reports of this lineage came from Turkey, with only a few records in Europe, mostly in birds wintering in Africa where transmission probably occurs. This study highlights unexpected difficulties in the research of EED even when using sensitive molecular diagnostic tools and extends information about transformation in early stages of gametogenesis in haemosporidian parasites.

Exo-erythrocytic development of Leucocytozoon parasites (Haemosporida, Leucocytozoidae) in song thrushes Turdus philomelos.

Leucocytozoon parasites (Haemosporida, Leucocytozoidae) are haemosporidians whose diversity, exo-erythrocytic development and potential vectors are the least studied. The knowledge about their exo-erythrocytic development and pathogenicity is fragmentary, resulting in an incomplete comprehension of the impact of these parasites on avian hosts. For a long time, Leucocytozoon infections were considered benign to wild birds, even though they were virulent in poultry and responsible for some wild bird population declines. This study aimed to investigate the presence of Leucocytozoon species exo-erythrocytic stages in song thrushes Turdus philomelos using conventional histological techniques (sections stained by H&E) and chromogenic in situ hybridization (CISH). Tissues from ten birds (seven naturally infected and three opportunistic samplings) were examined using both methods. Parasite lineages were identified from blood samples using PCR-based techniques. Leucocytozoon species meronts were found in five individuals (in four birds using H&E staining protocol, and in three in CISH-treated histological sections). Meronts were found mainly in the kidneys, but some meronts were also present in the lungs. It was possible to observe different maturation stages of meronts in the same bird individual, indicating an asynchronous development. Cytomeres were readily visible in developing meronts. One megalomeront-like structure was present close to a blood vessel in the heart. It was covered with a prominent capsular-like wall. No inflammatory reaction or necrosis was seen in the tissues surrounding the meronts or the megalomeront-like structure. We could confirm the transmission of three Leucocytozoon lineages (lTUPHI14, lSTUR1 and lTUPHI13) in Europe, and add evidence of the transmission of two Plasmodium lineages, including Plasmodium circumflexum (pTURDUS1), and Haemoproteus asymmetricus (hTUPHI01). We call for further research to better understand Leucocytozoon parasite exo-erythrocytic development.

Comparative Analysis of the Exo-Erythrocytic Development of Five Lineages of Haemoproteus majoris, a Common Haemosporidian Parasite of European Passeriform Birds.

Haemoproteus parasites (Apicomplexa, Haemosporida) are widespread pathogens of birds, with a rich genetic (about 1900 lineages) and morphospecies (178 species) diversity. Nonetheless, their life cycles are poorly understood. The exo-erythrocytic stages of three Haemoproteus majoris (widespread generalist parasite) lineages have been previously reported, each in a different bird species. We aimed to further study and compare the development of five H. majoris lineages-hCCF5, hCWT4, hPARUS1, hPHSIB1, and hWW2-in a wider selection of natural avian hosts. A total of 42 individuals belonging to 14 bird species were sampled. Morphospecies and parasitemia were determined by microscopy of blood films, lineages by DNA-barcoding a 478 bp section of the cytochrome b gene, and exo-erythrocytic stages by histology and chromogenic in situ hybridization. The lineage hCWT4 was morphologically characterized as H. majoris for the first time. All lineage infections exclusively featured megalomeronts. The exo-erythrocytic stages found in all examined bird species were similar, particularly for the lineages hCCF5, hPARUS1, and hPHSIB1. Megalomeronts of the lineages hWW2 and hCWT4 were more similar to each other than to the former three lineages. The kidneys and gizzard were most often affected, followed by lungs and intestines; the site of development showed variation depending on the lineage.

Exo-erythrocytic development of two Haemoproteus species (Haemosporida, Haemoproteidae), with description of Haemoproteus dumbbellus, a new blood parasite of bunting birds (Emberizidae).

Avian haemosporidians are widespread parasites categorized into four families of the order Haemosporida (Apicomplexa). Species of the subgenus Parahaemoproteus (genus Haemoproteus) belong to the Haemoproteidae and are transmitted by Culicoides biting midges. Reports of death due to tissue damage during haemoproteosis in non-adapted birds have raised concerns about these pathogens, especially as their exo-erythrocytic development is known for only a few Haemoproteus spp. More research is needed to better understand the patterns of the parasites' development in tissues and their impact on avian hosts. Yellowhammers Emberiza citrinella (Emberizidae) and common house martins Delichon urbicum (Hirundinidae) were screened for Haemoproteus parasites by microscopic examination of blood films and PCR-based testing. Individuals with single infection were selected for histological investigations. H & E-stained sections were screened for detection and characterization of the exo-erythrocytic stages, while chromogenic in situ hybridization (CISH) and phylogenetic analysis were performed to confirm the Haemoproteus origin and their phylogenetic relationships. Haemoproteus dumbbellus n. sp. was discovered in Emberiza citrinella single-infected with the lineage hEMCIR01. Meronts of H. dumbbellus n. sp. developed in various organs of five of six tested individuals, a pattern which was reported in other Haemoproteus species clustering in the same clade, suggesting this could be a phylogenetic trait. By contrast, in Delichon urbicum infected with the Haemoproteus lineage hDELURB2, which was linked to the more distantly related parasite Haemoproteus hirundinis, only megalomeronts were found in the pectoral muscles of two of six infected individuals. All exo-erythrocytic stages were confirmed to be Haemoproteus parasites by CISH using a Haemoproteus genus-specific probe. While the development of meronts seems to be typical for species of the clade containing H. dumbbellus, further investigations and data from more species are needed to explore whether a phylogenetic pattern occurs in meront or megalomeront formation.

Host Cells of Leucocytozoon (Haemosporida, Leucocytozoidae) Gametocytes, with Remarks on the Phylogenetic Importance of This Character.

Leucocytozoon parasites remain poorly investigated in comparison to other haemosporidians. The host cell inhabited by their blood stages (gametocytes) remains insufficiently known. This study aimed to determine the blood cells inhabited by Leucocytozoon gametocytes in different species of Passeriformes and to test if this feature has a phylogenetic importance. We microscopically analyzed blood films stained with Giemsa from six different bird species and individuals and used PCR-based methods for parasite lineage identification. The DNA sequences obtained were applied for phylogenetic analysis. Leucocytozoon parasite from the song thrush Turdus philomelos (cytochrome b lineage STUR1), the blackbird Turdus merula (undetermined lineage), the garden warbler Sylvia borin (unknown lineage) inhabited erythrocytes, a parasite from the blue tit Cyanistes caeruleus (PARUS4) infects lymphocytes, while in the wood warbler Phylloscopus sibilatrix (WW6) and the common chiffchaff Phylloscopus collybita (AFR205) they were found inhabiting thrombocytes. Parasites infecting thrombocytes were closely related, while the parasites infecting erythrocytes were placed in three different clades, and the one found in lymphocytes was placed in a separate clade. This shows that the determination of host cells inhabited by Leucocytozoon parasites can be phylogenetically important and should be considered in future species descriptions. Noteworthy, phylogenetic analysis might be used for the prediction of which host cells parasite lineages might inhabit.

Genomic sequence capture of Plasmodium relictum in experimentally infected birds.

BACKGROUND: Sequencing parasite genomes in the presence of host DNA is challenging. Sequence capture can overcome this problem by using RNA probes that hybridize with the parasite DNA and then are removed from solution, thus isolating the parasite DNA for efficient sequencing. METHODS: Here we describe a set of sequence capture probes designed to target 1035 genes (c. 2.5 Mbp) of the globally distributed avian haemosporidian parasite, Plasmodium relictum. Previous sequence capture studies of avian haemosporidians from the genus Haemoproteus have shown that sequencing success depends on parasitemia, with low-intensity, chronic infections (typical of most infected birds in the wild) often being difficult to sequence. We evaluate the relationship between parasitemia and sequencing success using birds experimentally infected with P. relictum and kept under laboratory conditions. RESULTS: We confirm the dependence of sequencing success on parasitemia. Sequencing success was low for birds with low levels of parasitemia (< 1% infected red blood cells) and high for birds with higher levels of parasitemia. Plasmodium relictum is composed of multiple lineages defined by their mitochondrial DNA haplotype including three that are widespread (SGS1, GRW11, and GRW4); the probes successfully isolated DNA from all three. Furthermore, we used data from 25 genes to describe both among- and within-lineage genetic variation. For example, two samples of SGS1 isolated from different host species differed by 11 substitutions across those 25 genes. CONCLUSIONS: The sequence capture approach we describe will allow for the generation of genomic data that will contribute to our understanding of the population genetic structure and evolutionary history of P. relictum, an extreme host generalist and widespread parasite.

Increase of avian Plasmodium circumflexum prevalence, but not of other malaria parasites and related haemosporidians in northern Europe during the past 40 years.

BACKGROUND: Malaria is a health problem not only in human and veterinary medicine, but also in wildlife. Several theoretical studies have suggested that avian malaria transmission might be increasing in Europe. However, there are few direct empirical observations. Research on the distribution of avian haemosporidian parasites was initiated around the Curonian Lagoon, Europe in 1976 and continues since. This has provided an opportunity to compare the prevalence and diversity of avian malaria parasites (genus Plasmodium) and related haemosporidians (genera Haemoproteus and Leucocytozoon) in the same bird species using similar methodology but examined in two groups 40 years apart. This study aimed to describe and discuss the available data on this subject. METHODS: Prevalence and diversity of haemosporidians was compared in two passeriform bird groups, which consisted of the same species that were sampled on the coast of the Curonian Lagoon (Russia, Lithuania) during the same season (September) in 1978-1983 (bird Group 1) and 2020 (bird Group 2). Blood films of the European robin, Coal tit, Great tit, Eurasian wren, and Eurasian jay were screened by microscopic examination. Parasites were identified using morphological characters of blood stages. PCR-based methods were applied to determine genetic lineages of the parasites found in birds of Group 2. RESULTS: No difference was discernible in the prevalence or diversity of haemosporidian parasites belonging to Haemoproteus, Leucocytozoon, Plasmodium (Haemamoeba) and Plasmodium (Novyella) between birds of Groups 1 and 2. This indicates a similar rate of transmission and relatively stable epidemiological situation in regard of these infections during the past 40 years. The prevalence of only one malaria parasite species, Plasmodium (Giovannolaia) circumflexum, increased remarkably, but only in Coal tit, Great tit, and Eurasian wren, with no significant prevalence change in European robin and Eurasian jay. CONCLUSION: Plasmodium circumflexum is spreading and seems to be a new invasive avian malaria pathogen in countries with cold climates. The exceptionally high prevalence of P. circumflexum in birds breeding in relatively close-nests suggests an important role of the nesting biology related to bird-vector interaction in this pathogen transmission. The epidemiological situation seems to be relatively stable in regard of other studied avian hosts and haemosporidian parasites in northern Europe.

Massive Infection of Lungs with Exo-Erythrocytic Meronts in European Robin Erithacus rubecula during Natural Haemoproteus attenuatus Haemoproteosis.

Haemoproteus species are widespread avian blood parasites belonging to Haemoproteidae (Haemosporida). Blood stages of these pathogens have been relatively well-investigated, though exo-erythrocytic (tissue) stages remain unidentified for the majority of species. However, recent histopathological studies show that haemoproteins markedly affect bird organs during tissue merogony. This study investigated the exo-erythrocytic development of Haemoproteus (Parahaemoproteus) attenuatus (lineage hROBIN1), the common parasite of flycatchers (Muscicapidae). Naturally infected European robins Erithacus rubecula were examined. Parasite species and lineage were identified using microscopic examination of blood stages and DNA sequence analysis. Parasitaemia intensity varied between 0.8 and 26.5% in seven host individuals. Organs of infected birds were collected and processed for histological examination. Tissues stages (meronts) were seen in six birds and were present only in the lungs. The parasites were usually located in groups and were at different stages of maturation, indicating asynchronous exo-erythrocytic development. In most parasitized individuals, 100 meronts were observed in 1 cm2 section of lungs. The largest meronts reached 108 µm in length. Mature meronts contained numerous roundish merozoites of approximately 0.8 µm in diameter. Megalomeronts were not observed. Massive merogony and resulting damage of lungs is a characteristic feature during H. attenuatus infections and might occur in related parasite lineages, causing haemoproteosis.

First Report of Haemoproteus (Haemosporida, Haemoproteidae) Megalomeronts in the Brain of an Avian Host, with Description of Megalomerogony of Haemoproteus Pastoris, the Blood Parasite of the Common Starling.

Species of Haemoproteus (Haemoproteidae, Haemosporida) are common bird pathogens. Recent molecular studies combined with histopathology research have reported development of megalomeronts of these parasites in various organs, sometimes resulting in the death of the avian host. Five Common starlings (Sturnus vulgaris) were found naturally infected with Haemoproteus pastoris lineage hLAMPUR01. The parasite was identified using microscopic examination of blood films and DNA sequences. Infected bird organs were investigated histologically for (i) the presence of exo-erythrocytic stages and (ii) the patterns of development (morphology and localization) in different host individuals. For the first time, megalomeronts of Haemoproteus parasites were seen developing in the brain, while numerous others at different stages of maturation were found in the intestine, pancreas, kidneys, lungs, esophagus, spleen, gizzard, and trachea. Megalomeronts were predominantly roundish or oval, up to 800 µm, they were surrounded by a capsular-like wall and developed asynchronously in the same bird individual. After megalomeront maturation and rupture, a massive infiltration of blood cells occurred, indicating the hemorrhagic processes. Review of available data showed that different Haemoproteus species produce markedly different megalomeronts, morphology of which can probably be predicted using phylogenetic analysis based on partial sequences of cytochrome b gene.

Low MSP-1 haplotype diversity in the West Palearctic population of the avian malaria parasite Plasmodium relictum.

BACKGROUND: Although avian Plasmodium species are widespread and common across the globe, limited data exist on how genetically variable their populations are. Here, the hypothesis that the avian blood parasite Plasmodium relictum exhibits very low genetic diversity in its Western Palearctic transmission area (from Morocco to Sweden in the north and Transcaucasia in the east) was tested. METHODS: The genetic diversity of Plasmodium relictum was investigated by sequencing a portion (block 14) of the fast-evolving merozoite surface protein 1 (MSP1) gene in 75 different P. relictum infections from 36 host species. Furthermore, the full-length MSP1 sequences representing the common block 14 allele was sequenced in order to investigate if additional variation could be found outside block 14. RESULTS: The majority (72 of 75) of the sequenced infections shared the same MSP1 allele. This common allele has previously been found to be the dominant allele transmitted in Europe. CONCLUSION: The results corroborate earlier findings derived from a limited dataset that the globally transmitted malaria parasite P. relictum exhibits very low genetic diversity in its Western Palearctic transmission area. This is likely the result of a recent introduction event or a selective sweep.

Diagnosis and Management of Severe Asthma in Switzerland: Analysis of Survey Results Conducted with Specialists and General Practitioners.

BACKGROUND: Severe asthma commonly affects 5-10% of the asthmatic population and accounts for approximately 50% of the overall asthma costs. OBJECTIVE: This analysis investigated how severe asthma is diagnosed, treated, and managed by specialists and general practitioners (GPs) in Switzerland. METHODS: Two surveys, one each among specialists (N = 44) and GPs (N = 153), were conducted to understand their self-perception on diagnosis, treatment, and management of severe asthma. RESULTS: Fifty-five percent of the specialists felt very confident and 43% confident in recognizing the symptoms of severe asthma and diagnosing severe asthma. In contrast, 9% of the GPs were very confident and 59% confident in diagnosing severe asthma. More specific diagnostic tests for severe asthma, like total and specific immunoglobulin E levels and measurement of the fraction of exhaled nitric oxide, were run by specialists (χ2 = 171.4; df = 15, p < 0.001). GPs and specialists were using different measurements to assess severe asthma (χ2 = 385.2; df = 13, p < 0.001) and their prescribing patterns differed significantly (χ2 = 189.8; df = 10, p < 0.001). GPs referred patients with severe asthma if the diagnosis was unclear (24%), if treatment failure occurred (26%), and if the patients were at high risk (41%). CONCLUSIONS: Oral corticosteroids (OCSs) are considered as background therapy for severe asthma by GPs and specialists. In order to reduce the OCS burden, there is a need to improve the awareness for other add-on therapies. A joint collaboration between GPs and specialists is the key to leverage therapeutic strategies together.

Description of Haemoproteus asymmetricus n. sp. (Haemoproteidae), with remarks on predictability of the DNA haplotype networks in haemosporidian parasite taxonomy research.

Haemoproteus species (Haemosporida, Haemoproteidae) are cosmopolitan blood parasites, which have been neglected for over 100-years, but attracted attention recently due to reports of severe and even lethal haemoproteosis in birds and vectors. Approximately 150 species of avian Haemoproteus have been described and named, but molecular data suggest that hundreds of independently evolving molecular lineages might occur, indicating the existence of a remarkable undescribed species diversity. It is timely to develop a methodology, which allow the application of available genetic data in taxonomy of haemosporidians on species levels. This study aimed to test a hypothesis suggesting that DNA haplotype networks might aid in targeting genetically distinct, but still undescribed parasites, and might be used to direct taxonomic studies on haemosporidian species levels. Mainly, we tested a prediction that the lineage hTUPHI01, a common Haemoproteus parasite of Turdus philomelos, might be a new species, which is morphologically similar and genetically closely related to the parasites of Haemoproteus minutus group. Blood samples of T. philomelos naturally infected with this parasite lineage were collected and studied using microscopic examination of blood films and PCR-based methods. Haemoproteus asymmetricus n. sp. was found in this bird, described and characterised molecularly using partial cytochrome b (cytb) sequences. The new species shared some features with parasites of the H. minutus group, as was predicted by the DNA haplotype network. Due to the visualisation of closely related lineages as well as the evaluation of their host and geographic distributions, DNA haplotype networks can be recommended as the helpful methodology, able to direct and speed practical work on parasite species taxonomy and pathogen biodiversity. The combined molecular phylogenetic and morphological approaches showed that the well-supported clades in Bayesian phylogenetic trees based on the partial cytb gene sequences contain morphologically remarkably different Haemoproteus parasite species, which however, share some basic biological features. Phylogenetic analysis can be used for prediction of these basic features in still undescribed parasites. This study calls for further fusion of advanced molecular and microscopy approaches for better understanding haemosporidian parasite biology.

Evolution of vector transmitted parasites by host switching revealed through sequencing of Haemoproteus parasite mitochondrial genomes.

Parasite species evolve by switching to new hosts, cospeciating with their current hosts, or speciating on their current hosts. Vector transmitted parasites are expected to speciate by host switching, but confirming this hypothesis has proved challenging. Parasite DNA can be difficult to sequence, thus well resolved parasite phylogenies that are needed to distinguish modes of parasite speciation are often lacking. Here, we studied speciation in vector transmitted avian haemosporidian parasites in the genus Haemoproteus and their warbler hosts (family Acrocephalidae). We overcome the difficulty of generating parasite genetic data by combining nested long-range PCR with next generation sequencing to sequence whole mitochondrial genomes from 19 parasite haplotypes confined to Acrocephalidae warblers, resulting in a well-supported parasite phylogeny. We also generated a well-supported host phylogeny using five genes from published sources. Our phylogenetic analyses confirm that these parasites have speciated by host switching. We also found that closely related host species shared parasites which themselves were not closely related. Sharing of parasites by closely related host species is not due to host geographic range overlap, but may be the result of phylogenetically conserved host immune systems.

Patterns of Haemoproteus majoris (Haemosporida, Haemoproteidae) megalomeront development.

Blood parasites of the genus Haemoproteus (Haemosporida, Haemoproteidae) are cosmopolitan and prevalent in birds. Numerous species and lineages of these pathogens have been identified. Some of the infections are lethal in avian hosts mainly due to damage of organs by tissue stages, which remain insufficiently investigated. Several closely related lineages of Haemoproteus majoris, a common parasite of passeriform birds, have been identified. One recent study described megalomeronts of unique morphology in the lineages hPHYBOR04 and hPARUS1 of H. majoris and suggested that the similar tissues stages might also be features in other phylogenetically closely related lineages of the same parasite species. This study aimed to test if (i) megalomeronts are present during the development of the lineage hPHSIB1 of H. majoris and if (ii) they are similar to the other investigated lineages of this species in regard of their morphology and location in organs. One adult wood warbler Phylloscopus sibilatrix, an Afrotropical migrant, naturally infected with H. majoris lineage hPHSIB1 was wild-caught after seasonal spring migration and screened using microscopic examination of blood films and histological sections of organs as well as using PCR-based testing. Bayesian phylogenetic analysis placed the lineages hPHSIB1, hPHYBOR04 and hPARUS1 in one, well-supported clade. Parasitaemia was high (6.5%) in the examined wood warbler, numerous megalomeronts were found in kidneys, and a few in the intestine. Megalomeronts of the lineage hPHSIB1 were morphologically hardly distinguishable from those of lineages hPHYBOR04 and hPARUS1; only negligible differences in the maturation stage of the cytomeres were seen. The kidneys were the main location site of the megalomeronts in all three lineages of this parasite species. This study shows that closely related lineages of H. majoris produce megalomeronts of similar morphology and predominant location in kidneys, while the normal function of this organ may be affected by the presence of numerous large megalomeronts. Megalomeronts of different avian Haemoproteus species are markedly variable in morphology and location, but phylogenetically closely related lineages possess cryptic megalomeronts. This finding suggests that phylogenies based on partial cytb gene could provide information for prediction of patterns of exo-erythrocytic development of closely related Haemoproteus parasites and are worthy of attention in planning haemosporidian parasite tissue stage research.

Parenteral is more efficient than mucosal immunization to induce regression of human papillomavirus-associated genital tumors.

Cervical cancer is a public health concern as it represents the second cause of cancer death in women worldwide. High-risk human papillomaviruses (HPV) are the etiologic agents, and HPV E6 and/or E7 oncogene-specific therapeutic vaccines are under development to treat HPV-related lesions in women. Whether the use of mucosal routes of immunization may be preferable for inducing cell-mediated immune responses able to eradicate genital tumors is still debated because of the uniqueness of the female genital mucosa (GM) and the limited experimentation. Here, we compared the protective activity resulting from immunization of mice via intranasal (i.n.), intravaginal (IVAG) or subcutaneous (s.c.) routes with an adjuvanted HPV type 16 E7 polypeptide vaccine. Our data show that s.c. and i.n. immunizations elicited similar frequencies and avidity of TetE71CD81 and E7-specific Interferon-gamma-secreting cells in the GM, whereas slightly lower immune responses were induced by IVAG immunization. In a novel orthotopic murine model, both s.c. and i.n. immunizations allowed for complete long-term protection against genital E7-expressing tumor challenge. However, only s.c. immunization induced complete regression of already established genital tumors. This suggests that the higher E7-specific systemic response observed after s.c. immunization may contribute to the regression of growing genital tumors, whereas local immune responses may be sufficient to impede genital challenges. Thus, our data show that for an efficiently adjuvanted protein-based vaccine, parenteral vaccination route is superior to mucosal vaccination route for inducing regression of established genital tumors in a murine model of HPV-associated genital cancer.

Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Antigen binding to secretory immunoglobulin A results in decreased sensitivity to intestinal proteases and increased binding to cellular Fc receptors.

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA x antigen immune complexes are selectively transported across Peyer's patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (Fc alphaRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.