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.
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.
The antimicrobial resistance (AMR) of E. coli, Salmonella spp. and thermophilic Campylobacter spp. isolated from wild birds in the Austrian-Czech border region, predominantly within the vicinity of Vienna and Brno, was determined. Bacteria were isolated from cloacal swabs taken from 53 avian species belonging to 14 orders, the majority being feral pigeons and various songbirds. Consequently, 1978 E. coli, 24 Salmonella and 99 Campylobacter isolates were tested by microdilution method against a panel of 14 (E. coli and Salmonella spp.) or ten antimicrobial substances (Campylobacter spp.). The AMR varied greatly between Austrian and Czech isolates, between different bird and bacterial species and between the different antimicrobials without consistent trends being recognizable. In 331 from 664 birds, differences in the AMR profile between the two or three E. coli isolates from the same bird were observed. Concerning E. coli, the least effective antimicrobial was cephalotin (90.8% of isolates resistant); for Campylobacter it was trimethoprim-sulfamethoxazole (71.4% of isolates resistant). None of the antimicrobials was effective against all E. coli or Campylobacter isolates. In contrast, more than half of the antimicrobials were effective against all Salmonella isolates, but 71.4% of the Czech Salmonella isolates and all of the Austrian Salmonella isolates were multi-resistant (resistant to three or more antimicrobial substances); streptomycin being the least effective antimicrobial substance (90.5% of isolates resistant). It is concluded that AMR in zoonotic bacteria from wild birds in the investigated densely populated areas is widespread and diverse, arguing for certain care at the wildlifehuman interface, but AMR to clinically important and critical antimicrobials is relatively low.
Objective Female reproductive tract disorders are common conditions of backyard poultry with an increasing demand for individual veterinary care. However, only limited case reports are available on diagnostic workup and outcomes of individual cases. This study aims at giving an overview of usually presented reproductive tract disorders, comparing diagnostic imaging findings with final diagnoses, and summarizing the outcome of the respective diseases. Material and methods The digital medical records archive of the University for Veterinary Medicine in Vienna was searched for chickens that were finally diagnosed with diseases of the reproductive tract, including all patients from May 1st, 2009 to May 31st, 2019. Information such as patient age, medical history, results of diagnostic imaging, final confirmed diagnosis, outcome, surgical protocol and necropsy findings was extracted. Results Finally confirmed reproductive tract diseases were found in 57 of 315 female chickens. The most common conditions were egg-related coelomitis along with salpingitis or impacted salpinx (25/57), followed by ovarian or oviductal neoplasia (17/57). Clinical findings were unspecific in the majority of cases, but most conspicuous were chickens presented with a distended coelomic cavity and apathy. Coelomic ultrasonography as well as computed tomography proved to be valuable tools for distinguishing between the respective conditions. However, ultrasonography alone mainly failed (10/11) to differentiate between ovary or oviductal neoplasia and egg-related coelomitis with salpingitis or impacted salpinx, respectively. Computed tomography was perceived as a superior tool for final diagnosis. In total 6/6 CT-scans correctly made a definitive diagnosis. Nevertheless, accurate diagnosis was only possible after celiotomy with the necessity of consecutive surgery for most of the reported cases. As an overall outcome 34 of 57 patients were either euthanised or died, whereas only 23 chickens could be successfully treated and discharged. Several hens were reported to be doing well at home, according to regular check-up procedures within 4 years post-surgery. Conclusion and clinical relevance Diagnostic work-up and treatment of hens with reproductive tract diseases can be challenging. Clinical presentation and diagnostic imaging provide important information, still celiotomy is often required for final diagnosis. The condition of the chickens is usually serious. Within our study, 40 % of the hens could be treated successfully. Therefore, a realistic assessment of each individual case and clarification for the owners are important.
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