Isospora and Lankesterella Parasites (Eimeriidae, Apicomplexa) of Passeriform Birds in Europe: Infection Rates, Phylogeny, and Pathogenicity.

Wild birds are common hosts to numerous intracellular parasites such as single-celled eukaryotes of the family Eimeriidae (order Eucoccidiorida, phylum Apicomplexa). We investigated the infection rates, phylogeny, and pathogenicity of Isospora and Lankesterella parasites in wild and captive passerine birds. Blood and tissue samples of 815 wild and 15 deceased captive birds from Europe were tested using polymerase chain reaction and partial sequencing of the mitochondrial cytochrome b and cytochrome c oxidase I and the nuclear 18S rRNA gene. The infection rate for Lankesterella in wild birds was 10.7% compared to 5.8% for Isospora. Chromogenic in situ hybridization with probes targeting the parasites' 18S rRNA was employed to identify the parasites' presence in multiple organs, and hematoxylin-eosin staining was performed to visualize the parasite stages and assess associated lesions. Isospora parasites were mainly identified in the intestine, spleen, and liver. Extraintestinal tissue stages of Isospora were accompanied by predominantly lymphohistiocytic inflammation of varying severity. Lankesterella was most frequently detected in the spleen, lung, and brain; however, infected birds presented only a low parasite burden without associated pathological changes. These findings contribute to our understanding of Isospora and Lankesterella parasites in wild birds.

RNAscope in situ hybridization reveals microvascular sequestration of Plasmodium relictum pSGS1 blood stages but absence of exo-erythrocytic dormant stages during latent infection of Serinus canaria.

BACKGROUND: Birds chronically infected with avian malaria parasites often show relapses of parasitaemia after latent stages marked by absence of parasites in the peripheral circulation. These relapses are assumed to result from the activation of dormant exo-erythrocytic stages produced during secondary (post-erythrocytic) merogony of avian Plasmodium spp. Yet, there is no morphological proof of persistent or dormant tissue stages in the avian host during latent infections. This study investigated persistence of Plasmodium relictum pSGS1 in birds with latent infections during winter, with the goal to detect presumed persisting tissue stages using a highly sensitive RNAscope® in situ hybridization technology. METHODS: Fourteen domestic canaries were infected with P. relictum pSGS1 by blood-inoculation in spring, and blood films examined during the first 4 months post infection, and during winter and spring of the following year. After parasitaemia was no longer detectable, half of the birds were dissected, and tissue samples investigated for persisting tissue stages using RNAscope ISH and histology. The remaining birds were blood-checked and dissected after re-appearance of parasitaemia, and their tissues equally examined. RESULTS: Systematic examination of tissues showed no exo-erythrocytic stages in birds exhibiting latent infections by blood-film microscopy, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, RNAscope ISH revealed rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. Birds examined after re-appearance of parasitemia showed higher numbers of P. relictum blood stages in both capillaries and larger blood vessels, indicating replication during early spring and re-appearance in the peripheral circulation. CONCLUSIONS: The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. Re-appearance of parasitaemia in spring seems to result from increased erythrocytic merogony, therefore representing recrudescence and not relapse in blood-inoculated canaries. Further, the study highlights strengths and limitations of the RNAscope ISH technology for the detection of rare parasite stages in tissues, providing directions for future research on persistence and tissue sequestration of avian malaria and related haemosporidian parasites.

Co-infecting Haemoproteus species (Haemosporida, Apicomplexa) show different host tissue tropism during exo-erythrocytic development in Fringilla coelebs (Fringillidae).

Avian haemosporidians of the genera Plasmodium, Haemoproteus, and Leucocytozoon are common blood parasites in wild birds all over the world. Despite their importance as pathogens potentially compromising host fitness and health, little is known about the exo-erythrocytic development of these parasites, particularly during co-infections which predominate in wildlife. This study aimed to address this issue using Haemoproteus parasites of Fringilla coelebs, a common bird species of the Western Palearctic and host to a variety of haemosporidian parasite lineages. Blood and tissue samples of 20 F. coelebs, positive for haemosporidians by blood film microscopy, were analysed by PCR and sequencing to determine cytochrome b lineages of the parasites. Tissue sections were examined for exo-erythrocytic stages by histology and in situ hybridization applying genus-, species-, and lineage-specific probes which target the 18S rRNA of the parasites. In addition, laser microdissection of tissue stages was performed to identify parasite lineages. Combined molecular results of PCR, laser microdissection, and in situ hybridization showed a high rate of co-infections, with Haemoproteus lineages dominating. Exo-erythrocytic meronts of five Haemoproteus spp. were described for the first known time, including Haemoproteus magnus hCCF6, Haemoproteus fringillae hCCF3, Haemoproteus majoris hCCF5, Haemoproteus sp. hROFI1, and Haemoproteus sp. hCCF2. Merogonic stages were observed in the vascular system, presenting a formerly unknown mode of exo-erythrocytic development in Haemoproteus parasites. Meronts and megalomeronts of these species were distinct regarding their morphology and organ distribution, indicating species-specific patterns of merogony and different host tissue tropism. New pathological aspects of haemoproteosis were reported. Furthermore, phylogenetic analysis of Haemoproteus spp. with regard to their exo-erythrocytic stages points towards separation of non-megalomeront-forming species from megalomeront-forming species, calling for further studies on exo-erythrocytic development of haemosporidian parasites to explore the phylogenetic character of this trait.

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.

Detection of Pneumocystis and Morphological Description of Fungal Distribution and Severity of Infection in Thirty-Six Mammal Species.

Pneumocystis spp. are thought to adapt to the lungs of potentially all mammals. However, the full host range, fungal burden and severity of infection are unknown for many species. In this study, lung tissue samples originating from 845 animals of 31 different families of eight mammal orders were screened by in situ hybridization (ISH) using a universal 18S rRNA probe for Pneumocystis, followed by hematoxylin and eosin (H&E) staining for determining histopathological lesions. A total of 216 (26%) samples were positive for Pneumocystis spp., encompassing 36 of 98 investigated mammal species, with 17 of them being described for the first time for the presence of Pneumocystis spp. The prevalence of Pneumocystis spp. as assessed by ISH varied greatly among different mammal species while the organism load was overall low, suggesting a status of colonization or subclinical infection. Severe Pneumocystis pneumonia seemed to be very rare. For most of the Pneumocystis-positive samples, comparative microscopic examination of H&E- and ISH-stained serial sections revealed an association of the fungus with minor lesions, consistent with an interstitial pneumonia. Colonization or subclinical infection of Pneumocystis in the lung might be important in many mammal species because the animals may serve as a reservoir.

Trichurosis on a Conventional Swine Fattening Farm with Extensive Husbandry-A Case Report.

Helminth infections of swine regain clinical and economic importance due to the increasing demand for pork from extensive husbandry. Infections with Trichuris suis in pigs can lead to wasting and diarrhoea. This was demonstrated by a case of clinical trichurosis on a conventional fattening farm, where pigs were kept on pasture. While all pre-fattening pigs, which had not been on the pasture yet, had a good body condition and firm faeces, diarrhoea and poor body condition were observed in approximately half of the fattening pigs kept on pasture. Rectally collected faecal samples from all animals were investigated using faecal flotation. High numbers of T. suis eggs were detected in 17 out of 32 faecal samples, while all samples from pre-fattening pigs were negative. The highest number of eggs per gram of faeces was 778,000. Two out of three environmental samples were also positive for T. suis in faecal flotation. This case demonstrates that T. suis must be considered as an enteropathogen in pigs kept on pasture, as favourable environmental conditions, and the lack of removal of faeces from a pasture can lead to the accumulation of large numbers of infective eggs in the pigs' surroundings.

Trichomonosis in Austrian Songbirds-Geographic Distribution, Pathological Lesions and Genetic Characterization over Nine Years.

In the early summer of 2012, sudden mass mortality among songbirds, particularly in greenfinches (Chloris chloris, syn: Carduelis chloris) was observed in Austria, which was caused by the protozoan parasite Trichomonas gallinae. This pathogen induced fibrinonecrotic ingluvitis and/or esophagitis, leading to impairment of food intake and ultimately death due to starvation. The pathogen was successfully detected within the lesions by polymerase chain reaction (PCR) and chromogenic in situ hybridization. The epizootic resulted in a significant decline in the Austrian greenfinch population. Continuing passive surveillance in the subsequent years (2013-2020) revealed that the condition occurred each year and was present in the entire country. Genetic characterization of the pathogen showed the presence of an identical strain irrespective of geographical location, bird species, and year.

Avian haemosporidian parasites of accipitriform raptors.

BACKGROUND: The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. METHODS: Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites' 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. RESULTS: Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon, 25 for Plasmodium, and 21 for Haemoproteus. CONCLUSION: The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.

Pneumocystis spp. in Pigs: A Longitudinal Quantitative Study and Co-Infection Assessment in Austrian Farms.

While Pneumocystis has been recognized as both a ubiquitous commensal fungus in immunocompetent mammalian hosts and a major opportunistic pathogen in humans responsible for severe pneumonias in immunocompromised patients, in pigs its epidemiology and association with pulmonary diseases have been rarely reported. Nevertheless, the fungus can be quite abundant in porcine populations with up to 51% of prevalence reported so far. The current study was undertaken to longitudinally quantify Pneumocystis carinii f. sp. suis and other pulmonary pathogens in a cohort of 50 pigs from five Austrian farms (i.e., 10 pigs per farm) with a history of respiratory disease at five time points between the first week and the fourth month of life. The fungus was present as early as the suckling period (16% and 26% of the animals in the first and the third week, respectively), yet not in a high amount. Over time, both the organism load (highest 4.4 × 105 copies/mL) and prevalence (up to 88% of positive animals in the third month) increased in each farm. The relative prevalence of various coinfection patterns was significantly different over time. The current study unravelled a complex co-infection history involving Pneumocystis and other pulmonary pathogens in pigs, suggesting a relevant role of the fungus in the respiratory disease scenario of this host.

A retrospective study on the presence of selected infectious agents in lung samples of cats with pneumonia.

The causative role of some infectious agents found in cases of feline pneumonia is under debate, because they are also part of the physiological microbiota of the respiratory tract of healthy animals. In this retrospective study, archived formalin-fixed and paraffin-wax-embedded lung samples of 69 severe and lethal cases of pneumonia in cats were examined by immunohistochemistry (IHC) for the detection of nine selected infectious agents: Pasteurella multocida, Bordetella bronchiseptica, Mycoplasma felis, M. gateae, Chlamydia felis, feline herpesvirus type 1, feline coronavirus, canine distemper virus, and Toxoplasma gondii. The intention was to elucidate their immediate involvement in pneumonia formation. Due to the cross-reactivity of the applied antibodies, a species-specific polymerase chain reaction (PCR) for both targeted Mycoplasma species was applied additionally. In the 42 cases (60.9%) positive for at least one pathogen, several agents were present in a high proportion of the samples (P. multocida - 34.8%, B. bronchiseptica - 29.0%), while others were present in a moderate (feline herpesvirus type 1 - 18.8%, M. gateae - 13.0%, M. felis - 10.1%) or low percentage (T. gondii - 1.4%). All samples were negative for C. felis, feline coronavirus and canine distemper virus. Mixed infections of up to four pathogens were more frequent than single infections. Mycoplasma preferably colonised lung tissue damaged by other pathogens because they never occurred as single infections. Pasteurella multocida, B. bronchiseptica, M. felis, feline herpesvirus type 1 and T. gondii showed abundant replication within lung lesions, thus suggesting a prominent role in pneumonia formation.

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden.

BACKGROUND: Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects. METHODS: For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin-eosin-stained sections. RESULTS: By PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses. CONCLUSION: This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

Hippocampal necrosis and sclerosis in cats: A retrospective study of 35 cases.

Hippocampal necrosis and hippocampal sclerosis in cats is a neuropathological entity which is a major concern in feline epilepsy. The aim of our study was to identify associated pathologic brain lesions possibly serving as aetiological triggers in this condition. Therefore, the formalin-fixed and paraffin waxembedded brain tissue of 35 cats diagnosed with hippocampal necrosis or sclerosis was examined retrospectively. In 26 cats inflammatory infiltrates could be found in the hippocampus or adjacent brain regions. Fifteen out of these animals demonstrated mild to moderate infiltrations by lymphocytes and complement deposition in the hippocampus similar to human limbic encephalitis, seven showed unspecific, predominantly non-suppurative inflammation, and two demonstrated suppurative inflammation of the hippocampus or adjacent brain regions. Additionally, one cat was diagnosed with central nervous manifestation of feline infectious peritonitis virus and another one with cerebral Toxoplasma gondii infection. Intracranial neoplasia was present in five cases altogether. Three of them comprised meningioma which was present additionally to lesions resembling limbic encephalitis in two cases, and a dentate gyrus alteration in one case. The other two tumour-associated cases comprised oligodendroglioma. Structural alterations of the dentate gyrus together with hippocampal sclerosis were encountered in three cases in total. Besides the case associated with a meningioma, one case demonstrated lesions resembling limbic encephalitis. A vascular infarct in the temporal lobe was encountered in one cat. In four cases no lesions other than hippocampal necrosis or sclerosis were found. The involvement of feline immunodeficiency virus infections, which may be able to produce hippocampal lesions, was not encountered in the cats examined.

Comparison of Pneumocystis nucleic acid and antibody profiles and their associations with other respiratory pathogens in two Austrian pig herds.

Pneumocystis carinii f. sp. suis (PCS) nucleic acid and antibody profiles on two Austrian-farrow-to-finish farms were investigated. Furthermore, associations with other respiratory pathogens were evaluated. Respiratory specimen and sera from pigs of five age classes between the 1st week and the 3rd month of life as well as samples from sows were analyzed. On Farm A, PCS infection occurred early in life. The suckling piglets were already infected in the 1st week of life and the pigs remained positive until the 3rd month of life. On Farm B, pigs were infected later, between 3 and 4 months of age. The maximum PCS nucleic acid load on Farm A was 8.3 log10 genome copies/mL BALF, whereas on Farm B the PCS burden was significantly lower, with 4.0 log10 genome copies/mL BALF. Anti-PCS antibodies were detected in sows, as maternal antibodies in suckling piglets and as an immunological reaction to infection. On both farms, PCS infection was accompanied by several co-infections. On Farm A, there were concurrent infections with PRRSV, a virulent strain of Haemophilus parasuis, and Mycoplasma hyopneumoniae. On Farm B, PCS was accompanied by infections with swine influenza virus, Mycoplasma hyopneumoniae, and a non-virulent strain of Haemophilus parasuis. The results clearly show that the PCS profiles can vary between farms. Younger pigs may be more susceptible as they had higher PCS burdens. It is possible that PCS may contribute to a respiratory disease in pigs and further investigation of its potential role is warranted.

Characterization of the T-cell receptor gamma chain gene rearrangements as an adjunct tool in the diagnosis of T-cell lymphomas in the gastrointestinal tract of cats.

Feline alimentary lymphoma is the most common hematopoietic neoplasia in cats. It affects mainly the small intestines and is most frequently of T-cell origin. Evaluation of a fine needle aspirate is often the first step in the diagnostic work-up. Differentiation between a resident mature lymphocyte population as encountered in inflammatory bowel disease and small cell lymphoma cannot be achieved by cytology alone. Even full thickness biopsies evaluated by histopathology can be inconclusive. These cases warrant the application of complementary tools like PCR-based T-cell receptor (TCR) clonality testing for confirmation. The aim of this study was to optimize the DNA extraction protocol for formalin fixed and paraffin embedded tissues (FFPE) and to establish a heteroduplex analysis to enhance resolution of the PCR fragments of the T-cell receptor gamma (TCRG) V-J gene. The new protocols resulted in improved quantity and quality of the extracted DNA. Heteroduplex analysis of the samples improved the resolution of the electrophoresis results so that rules for interpretation of the different patterns could be established. Application of this improved setup detected clonal rearrangements in at least one TCRG primer reaction in 31 of 36 of our feline intestinal lymphoma samples after DNA quality testing.

Retrospective Analysis of Bacterial and Viral Co-Infections in Pneumocystis spp. Positive Lung Samples of Austrian Pigs with Pneumonia.

Aim of this study was the retrospective investigation of viral (porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), torque teno sus virus type 1 and 2 (TTSuV1, TTSuV2)) and bacterial (Bordetella bronchiseptica (B. b.), Mycoplasma hyopneumoniae (M. h.), and Pasteurella multocida (P. m.)) co-infections in 110 Pneumocystis spp. positive lung samples of Austrian pigs with pneumonia. Fifty-one % were positive for PCV2, 7% for PRRSV, 22% for TTSuV1, 48% for TTSuV2, 6% for B. b., 29% for M. h., and 21% for P. m. In 38.2% only viral, in 3.6% only bacterial and in 40.0% both, viral and bacterial pathogens were detected. In 29.1% of the cases a co-infection with 1 pathogen, in 28.2% with 2, in 17.3% with 3, and in 7.3% with 4 different infectious agents were observed. The exposure to Pneumocystis significantly decreased the risk of a co-infection with PRRSV in weaning piglets; all other odds ratios were not significant. Four categories of results were compared: I = P. spp. + only viral co-infectants, II = P. spp. + both viral and bacterial co-infectants, III = P. spp. + only bacterial co-infectants, and IV = P. spp. single infection. The evaluation of all samples and the age class of the weaning piglets resulted in a predomination of the categories I and II. In contrast, the suckling piglets showed more samples of category I and IV. In the group of fattening pigs, category II predominated. Suckling piglets can be infected with P. spp. early in life. With increasing age this single infections can be complicated by co-infections with other respiratory diseases.

Mortality and pathology in birds due to Plasmodium (Giovannolaia) homocircumflexum infection, with emphasis on the exoerythrocytic development of avian malaria parasites.

BACKGROUND: Species of avian malaria parasites (Plasmodium) are widespread, but their virulence has been insufficiently investigated, particularly in wild birds. During avian malaria, several cycles of tissue merogony occur, and many Plasmodium spp. produce secondary exoerythrocytic meronts (phanerozoites), which are induced by merozoites developing in erythrocytic meronts. Phanerozoites markedly damage organs, but remain insufficiently investigated in the majority of described Plasmodium spp. Avian malaria parasite Plasmodium (Giovannolaia) homocircumflexum (lineage pCOLL4) is virulent and produces phanerozoites in domestic canaries Serinus canaria, but its pathogenicity in wild birds remains unknown. The aim of this study was to investigate the pathology caused by this infection in species of common European birds. METHODS: One individual of Eurasian siskin Carduelis spinus, common crossbill Loxia curvirostra and common starling Sturnus vulgaris were exposed to P. homocircumflexum infection by intramuscular sub-inoculation of infected blood. The birds were maintained in captivity and parasitaemia was monitored until their death due to malaria. Brain, heart, lungs, liver, spleen, kidney, and a piece of breast muscle were examined using histology and chromogenic in situ hybridization (ISH) methods. RESULTS: All exposed birds developed malaria infection, survived the peak of parasitaemia, but suddenly died between 30 and 38 days post exposure when parasitaemia markedly decreased. Numerous phanerozoites were visible in histological sections of all organs and were particularly easily visualized after ISH processing. Blockage of brain capillaries with phanerozoites may have led to cerebral ischaemia, causing cerebral paralysis and is most likely the main reason of sudden death of all infected individuals. Inflammatory response was not visible around the brain, heart and muscle phanerozoites, and it was mild in parenchymal organs. The endothelial damage likely causes dysfunction and failure of parenchymal organs. CONCLUSION: Plasmodium homocircumflexum caused death of experimental passerine birds due to marked damage of organs by phanerozoites. Patterns of phanerozoites development and pathology were similar in all exposed birds. Mortality was reported when parasitaemia decreased or even turned into chronic stage, indicating that the light parasitaemia is not always indication of improved health during avian malaria. Application of traditional histological and ISH methods in parallel simplifies investigation of exoerythrocytic development and is recommended in avian malaria research.

Establishment of a quantitative real-time PCR for the detection of Pneumocystis carinii f. sp. suis in bronchoalveolar lavage samples from pigs.

Respiratory diseases in pigs are mostly polymicrobial, and the involved pathogens can vary from farm to farm. The impact of Pneumocystis carinii (P. c.) f. sp. suis on respiratory disorders has not been comprehensively appraised because tests were limited to lung tissue samples and, for this reason, it was not possible to detect the fungus in living animals. In the present study, bronchoalveolar lavage fluid (BALF) from 12 pigs and oral fluid samples from 9 pigs were tested for the presence of Pneumocystis by quantitative real-time PCR. The results from these 2 clinical specimens were compared with Pneumocystis quantities in lung tissue samples. Pneumocystis quantities in BALF correlated significantly to those in lung tissue. BALF has proved to be an adequate specimen for detection of various respiratory pathogens in pigs, and the collection procedure directly on farms is also well established. In contrast to the BALF results, all oral fluid samples were negative. Thus, specimens from the lower respiratory tract should generally be preferred for the detection of Pneumocystis Additionally, under farm conditions, oral fluid is mainly collected in the form of collective samples per pen. In the present study, oral swab sampling of individual pigs was intended but failed in 3 of 12 pigs because they did not salivate sufficiently. As a conclusion, only BALF can be recommended as a useful tool for Pneumocystis herd monitoring in vivo.

In situ hybridization and sequence analysis reveal an association of Plasmodium spp. with mortalities in wild passerine birds in Austria.

Native European passerine birds are frequently clinically inapparent carriers of haemosporidian parasites of the genus Plasmodium. Clinical disease and death are only exceptionally reported. In the present study, tissue samples of 233 wild passerine birds found dead in Eastern Austria were examined by in situ hybridization (ISH) and partial cytochrome B gene sequence analysis for the presence, abundance and taxonomic assignment of Plasmodium spp. In 34 cases (14.6%), ISH yielded a positive result with large numbers of developmental stages in different cell types of the spleen, liver, brain and lung. The abundance of the tissue stages, which was comparable to fatal cases of avian malaria in penguins, suggested a major contribution to the cause of death. Genetic analysis revealed infections with representatives of three different valid species of Plasmodium, Plasmodium elongatum, Plasmodium lutzi and Plasmodium vaughani. Genetically identical parasite lineages had been found in a previous study in penguins kept in the Vienna zoo, providing evidence for the role of wild birds as reservoir hosts. Further, this study provides evidence that several species of Plasmodium are able to abundantly proliferate in endemic wild birds ultimately resulting in mortalities.

Finding your way through Pneumocystis sequences in the NCBI gene database.

Pneumocystis sequences can be downloaded from GenBank for purposes as primer/probe design or phylogenetic studies. Due to changes in nomenclature and assignment, available sequences are presented with a variety of inhomogeneous information, which renders practical utilization difficult. The aim of this study was the descriptive evaluation of different parameters of 532 Pneumocystis sequences of mitochondrial and ribosomal origin downloaded from GenBank with regard to completeness and information content. Pneumocystis sequences were characterized by up to four different names. Official changes in nomenclature have only been partly implemented and the usage of the "forma specialis", a special feature of Pneumocystis, has only been established fragmentary in the database. Hints for a mitochondrial or ribosomal genomic origin could be found, but can easily be overlooked, which renders the download of wrong reference material possible. The specification of the host was either not available or variable regarding the used language and the localization of this information in the title or several subtitles, which limits their applicability in phylogenetic studies. Declaration of products and geographic origin was incomplete. The print version of this manuscript is completed by an online database which contains detailed information to every accession number included in the meta-analysis.

Detection of Pneumocystis infections by in situ hybridization in lung samples of Austrian pigs with interstitial pneumonia.

Pneumocystis carinii f. sp. suis is a fungus multiplying in the respiratory tract of pigs which occasionally is associated with interstitial pneumonia. Identification of Pneumocystis in tissue samples is considered difficult and there are only scarce data on its occurrence in European pigs. This investigation presents an in situ hybridization (ISH) procedure for identification of Pneumocystis spp. in paraffin wax embedded tissue samples and its application for labeling the agent in lung samples of pigs with interstitial pneumonia. Thirty-two out of 100 lung samples from pigs on Austrian farms were identified as positive, five of them with multiple, 12 with moderate and 15 with few organisms but Grocott's methenamine silver staining demonstrated that only 20 cases were unequivocally positive for Pneumocystis carinii. In addition to interstitial pneumonia Pneumocystis-positive pigs were more frequently affected with granulomatous pneumonia than Pneumocystis-negative pigs. Frequently concurrent infections with different viral or bacterial lung pathogens were noted but there was no positive correlation between Pneumocystis- and PCV-2-infections. With other infections, no clear-cut differences between Pneumocystis-positive and Pneumocystis-negative animals were found. This study shows that Pneumocystis infections occur frequently in Austrian pigs with interstitial pneumonia. It remains to be shown which are the factors triggering severe multiplication and whether infection with Pneumocystis alone is able to induce lung disease in pigs.