Dataset Information

A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays.

ABSTRACT:

Background

Accurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and considerable experience, while the widely used semi-quantitative qPCR methods are mostly employed with ideal, laboratory-based golden samples and standard curves, which may limit the comparison of parasitemia from different laboratories.

Methods

Here we present a digital droplet PCR (ddPCR) protocol for absolute quantification of avian haemosporidians in raptors. Novel primers were designed that target a conserved fragment of a rRNA region of the mitochondrial genome of the parasites. Sensitivity and repeatability were evaluated compared to qPCR and other assays.

Results

This ddPCR assay enables accurate quantification of haemosporidian parasites belonging to Plasmodium, Haemoproteus and Leucocytozoon with minimum infection quantities of 10^-5 (i.e. one parasite copy in 10⁵ host genomes) without the use of standard curves. Quantities assessed by ddPCR were more accurate than qPCR using the same primers through reduction of non-specific amplification in low-intensity samples. The ddPCR technique was more consistent among technical duplicates and reactions, especially when infection intensities were low, and this technique demonstrated equal sensitivity with high correspondence (R²?=?0.97) compared to the widely used qPCR assay. Both ddPCR and qPCR identified more positive samples than the standard nested PCR protocol for the cytb gene used for barcoding avian haemosporidians.

Conclusions

We developed a novel ddPCR assay enabling accurate quantification of avian haemosporidians without golden samples or standard curves. This assay can be used as a robust method for investigating infection patterns in different host-parasite assemblages and can facilitate the comparison of results from different laboratories.

SUBMITTER: Huang X

PROVIDER: S-EPMC7368712 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Publications

A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays.

Huang Xi X Huang Di D Liang Yuge Y Zhang Linlin L Yang Guocheng G Liu Boye B Peng Yangyang Y Deng Wenhong W Dong Lu L

Parasites & vectors 20200717 1

<h4>Background</h4>Accurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and considerable experience, while the widely used semi-quantitative qPCR methods are mostly employed with ideal, laboratory-based golden samples and standard curves, which may limit the comparison of parasit ...[more]

PMID: 32680557

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Project description:BackgroundThe 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.MethodsBlood 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.ResultsOf 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.ConclusionThe 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.

Project description:BackgroundVarious haemosporidian parasites infect raptors, especially captive hosts who may be more exposed. Diagnosis of threatening factors such as infectious diseases indirectly has a significant role in protecting endangered or threatened species that may boost the mortality or extinction resulting from declined reproduction. Few investigations have been performed in captive hosts to detect the prevalence of haemosporidian parasites and define genetic diversity in west Asia. For the first time, the current study was designed to determine the prevalence and genetic diversity of haemosporidian parasites in captive raptors by molecular methods in two rehabilitation facilities in North and North-east Iran and to define phylogenetic relationships of detected lineages circulating in raptors.ResultsMolecular characterization of the haemosporidian parasite was accomplished by PCR-based method and DNA sequencing in 62 captive raptors. The overall prevalence was ~ 36% with higher infection of Haemoproteus spp. than Leucocytozoon spp. Plasmodium infection was not detected in any host. Results showed that 22 individuals (of 10 species) were infected with unique lineages. Genus Haemoproteus was detected in 26.66% of examined individuals (of eight species) and Leucocytozoon was found in 10% of individuals (of four species). The molecular analysis could detect ten lineages (nine Haemoproteus spp. and one Leucocytozoon spp.) which were categorizes as new and six lineages which have been previously detected in the other investigations.ConclusionsThe Bayesian phylogenetic analysis derived from obtained data in the present study and published lineages in previous investigations indicated the probable host specificity of Haemoproteus and Leucocytozoon parasites in several sub-clades at hosts' order and genus level. As monitoring the parasite loads of captive birds when admitted reduce the risk of infecting hosts in captivity at those locations, we designed this study to determine infection prevalence and genetic diversity of blood parasites in raptors examined in Iran. These results allow mapping of haemosporidian distribution and shed light on the depth of their diversity in Iran to protect species by identification of risk in rehabilitation facilities.

Project description:BackgroundPlasmodium species feature only four to eight nuclear ribosomal units on different chromosomes, which are assumed to evolve independently according to a birth-and-death model, in which new variants originate by duplication and others are deleted throughout time. Moreover, distinct ribosomal units were shown to be expressed during different developmental stages in the vertebrate and mosquito hosts. Here, the 18S rDNA sequences of 32 species of avian haemosporidian parasites are reported and compared to those of simian and rodent Plasmodium species.MethodsAlmost the entire 18S rDNAs of avian haemosporidians belonging to the genera Plasmodium (7), Haemoproteus (9), and Leucocytozoon (16) were obtained by PCR, molecular cloning, and sequencing ten clones each. Phylogenetic trees were calculated and sequence patterns were analysed and compared to those of simian and rodent malaria species. A section of the mitochondrial CytB was also sequenced.ResultsSequence patterns in most avian Plasmodium species were similar to those in the mammalian parasites with most species featuring two distinct 18S rDNA sequence clusters. Distinct 18S variants were also found in Haemoproteus tartakovskyi and the three Leucocytozoon species, whereas the other species featured sets of similar haplotypes. The 18S rDNA GC-contents of the Leucocytozoon toddi complex and the subgenus Parahaemoproteus were extremely high with 49.3% and 44.9%, respectively. The 18S sequences of several species from all three genera showed chimeric features, thus indicating recombination.ConclusionGene duplication events leading to two diverged main sequence clusters happened independently in at least six out of seven avian Plasmodium species, thus supporting evolution according to a birth-and-death model like proposed for the ribosomal units of simian and rodent Plasmodium species. Patterns were similar in the 18S rDNAs of the Leucocytozoon toddi complex and Haemoproteus tartakovskyi. However, the 18S rDNAs of the other species seem to evolve in concerted fashion like in most eukaryotes, but the presence of chimeric variants indicates that the ribosomal units rather evolve in a semi-concerted manner. The new data may provide a basis for studies testing whether differential expression of distinct 18S rDNA also occurs in avian Plasmodium species and related haemosporidian parasites.

Dataset Information

A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays.

Background

Methods

Results

Conclusions

Publications

A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays.

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