Preserving avian blood and DNA sampled in the wild: a survey of personal experiences

Collecting and storing biological material from wild animals in a way that does not deteriorate data quality for analyses using DNA is instrumental for research in ecology and evolution. Our aims were to collect methods commonly used by researchers for the field collection and long-term storage of blood samples and DNA extracts from wild birds and gather reports on their effectiveness. Personal experiences were collected with an online survey targeted specifically at researchers sampling wild birds. Many researchers experienced problems with blood sample storage but not with DNA extract storage. Storage issues generated problems with obtaining adequate DNA quality and sufficient DNA quantity for the targeted molecular analyses, but were not related to season of blood sampling, access to equipment, transporting samples, temperature and method of blood storage. Final DNA quality and quantity were also not affected by storage time before DNA extraction or the methods used to extract DNA. We discuss practical aspects of field collection and storage and provide some general recommendations, with a list of pros and cons of different preservation methods of avian blood samples and DNA extracts.

Description and Molecular Characterization of Two Species of Avian Blood Parasites, with Remarks on Circadian Rhythms of Avian Haematozoa Infections

Avian blood parasites are remarkably diverse and frequently occur in co-infections, which predominate in wildlife. This makes wildlife pathogen research challenging, particularly if they belong to closely related groups, resulting in diagnostic problems and poor knowledge about such infections as well as the patterns of their co-occurrence and interactions. This is particularly true due to the periodicity (circadian rhythms) of parasitemia, which means that different parasitemia and parasite stages might be found throughout the day. We analysed blood samples from a Eurasian blackbird (Turdus merula) and a Song thrush (Turdus philomelos). This study aimed to describe a new avian Lankesterella species and molecularly characterize and redescribe Splendidofilaria mavis, a common avian filarioid nematode. Additionally, it was possible to investigate the circadian rhythms of the avian blood parasites belonging to Plasmodium, Haemoproteus, Leucocytozoon, and Trypanosoma, which occurred in co-infection in the same avian host individuals. Different circadian rhythms were seen in different parasites, with Plasmodium sp. peaks occurring at midday, Leucocytozoon spp. peaks mainly during the evening and night, and Trypanosoma spp. and microfilariae peaks at midnight. No periodicity was seen in Haemoproteus and Lankesterella species infections. The time of parasitemia peaks most likely coincides with the time of vectors’ activity, and this should be beneficial for transmission. Knowledge about the circadian rhythms is needed for better understanding patterns in host-parasite interactions and disease transmission.

Effect of RNA preservation methods on RNA quantity and quality of field collected avian whole blood

A limitation of comparative transcriptomic studies of wild avian populations continues to be sample acquisition and preservation to achieve resulting high-quality RNA (i.e., ribonucleic acids that transfers, translates, and regulates the genetic code from DNA into proteins). Field sampling of wild bird samples provides challenges as RNA degradation progresses quickly and because cryopreservation is often not feasible at remote locations. We collected blood samples from songbirds, as avian blood is nucleated and provides sufficient transcriptionally active material in a small and non-lethal sample, to compare the efficacy of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at differing concentrations along with a dry ice-based flash freezing method (Isopropanol 99% and dry ice mixture, -109°C). Each blood sample was divided among five different preservation treatments (dry ice-based flash freezing, RNAlater with 1:5 or 1:10 dilution, or DNA/RNA Shield with 1:2 or 1:3 dilution). A new protocol was optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of small passerines. We quantified quality measures, RNA integrity numbers (RINe), rRNA ratios, and total RNA concentrations. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations, provide sample protection from RNA degradation. We suggest caution against using dry ice-based flash-freezing alone for samples preservation as these samples resulted in lower quality measures then samples in preservation buffer. Total RNA concentration was generally not affected by preservation treatment and may vary due to differences in initial samples volumes and carryover across processing steps.

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.

Haemosporidian infection does not alter aerobic performance in the Pink-sided Junco (Junco hyemalis mearnsi)

ABSTRACTAvian haemosporidia are blood parasites that can have dramatic fitness consequences on their hosts, including largescale population declines when introduced to naïve hosts. Yet the physiological effects that accompany haemosporidian infection and underlie these fitness decrements are poorly characterized in most wild birds. Because haemosporidia destroy host red blood cells and consume host hemoglobin, they are predicted to have detrimental impacts on avian blood-oxygen transport and, as a result, reduce aerobic performance. However, the documented effects of infection on avian hematological traits vary across species and no effects have been demonstrated on avian aerobic performance to date. Here we quantified the physiological effects of haemosporidian infections on wild ‘Pink-sided’ Juncos (Junco hyemalis mearnsi) breeding in northwestern Wyoming, USA. We assayed hematological traits (hemoglobin concentration and hematocrit) and aerobic performance (resting and summit metabolic rates, thermogenic endurance, and aerobic scope), then screened individuals for haemosporidian infection post-hoc (n = 106 adult juncos). We found that infection status did not correlate with any of the physiological indices that we measured, suggesting there is little cost of haemosporidian infection on either junco aerobic performance or energy budgets. Our results highlight the need for more studies of haemosporidia infections in a broader range of species and in a wider array of environmental contexts.

A Rapid and Simple Assay Correlates In Vitro NetB Activity with Clostridium perfringens Pathogenicity in Chickens

Necrotic enteritis is an important enteric disease in poultry, caused by NetB-producing Clostridium (C.) perfringens strains. As no straight-forward method to assess the NetB activity of C. perfringens was available, we aimed to develop an easy, high-throughput method to measure the NetB activity produced by C. perfringens. First, the appearance of C. perfringens on different avian blood agar plates was assessed. Based on the size of the haemolysis surrounding the C. perfringens colonies, NetB-positive strains could phenotypically be discriminated from NetB-negative strains on both chicken and duck blood agar. Additionally, strains producing the consensus NetB protein induced more pronounced haemolysis on chicken blood agar as compared to the weak outer haemolysis induced by A168T NetB-variant-producing C. perfringens strains. Next, a 96-well plate-based haemolysis assay to screen NetB activity in the C. perfringens culture supernatants was developed. Using this assay, a positive correlation between the in vitro NetB activity and virulence of the C. perfringens strains was shown. The developed activity assay allows us to screen novel C. perfringens isolates for their in vitro NetB activity, which could give valuable information on their disease-inducing potential, or identify molecules and (bacterial) metabolites that affect NetB expression and activity.

Sex and nest type influence avian blood parasite prevalence in a high-elevation bird community

Background The prevalence of avian haemosporidian parasites and the factors influencing infection in the Colorado Rocky Mountains are largely unknown. With climate change expected to promote the expansion of vector and avian blood parasite distributions, baseline knowledge and continued monitoring of the prevalence and diversity of these parasites is needed. Methods Using an occupancy modeling framework, we conducted a survey of haemosporidian parasite species infecting an avian community in the Colorado Rocky Mountains in order to estimate the prevalence and diversity of blood parasites and to investigate species-level and individual-level characteristics that may influence infection. Results We estimated the prevalence and diversity of avian Haemosporidia across 24 bird species, detecting 39 parasite haplotypes. We found that open-cup nesters have higher Haemoproteus prevalence than cavity or ground nesters. Additionally, we found that male Ruby-crowned Kinglets, White-crowned Sparrows, and Wilson’s Warblers have higher Haemoproteus prevalence compared to other host species. Plasmodium prevalence was relatively low (5%), consistent with the idea that competent vectors may be rare at high altitudes. Conclusions Our study presents baseline knowledge of haemosporidian parasite presence, prevalence, and diversity among avian species in the Colorado Rocky Mountains and adds to our knowledge of host–parasite relationships of blood parasites and their avian hosts.

Sex and nest type influence avian blood parasite prevalence in a high elevation bird community

Background - Prevalence of avian haemosporidian parasites and the factors influencing infection in the Colorado Rocky Mountains are largely unknown. With climate change expected to promote the expansion of vector and avian blood parasite distributions, baseline knowledge and continued monitoring of the prevalence and diversity of these parasites is needed. Methods - Using an occupancy modeling framework, we conducted a survey of haemosporidian parasite species infecting an avian community in the Colorado Rocky Mountains in order to estimate prevalence and diversity of blood parasites and to investigate species-level and individual-level characteristics that may influence infection. Results - We estimated prevalence and diversity of avian haemosporidia across 24 bird species, detecting 39 parasite haplotypes. We found that open cup nesters have higher Haemoproteus prevalence than cavity or ground nesters. Additionally, we found that male Ruby-crowned Kinglets, White-crowned Sparrows, and Wilson’s Warblers have higher Haemoproteus prevalence compared to other host species. Conclusions - Our study presents baseline knowledge of haemosporidian parasite presence, prevalence, and diversity among avian species in the Colorado Rocky Mountains and adds to our knowledge of host-parasite relationships of blood parasites and their avian hosts.