Climate predictors and climate change projections for avian haemosporidian prevalence in Mexico.

Long-term, inter-annual and seasonal variation in temperature and precipitation influence the distribution and prevalence of intraerythrocytic haemosporidian parasites. We characterized the climatic niche behind the prevalence of the three main haemosporidian genera (Haemoproteus, Plasmodium and Leucocytozoon) in central-eastern Mexico, to understand their main climate drivers. Then, we projected the influence of climate change over prevalence distribution in the region. Using the MaxEnt modelling algorithm, we assessed the relative contribution of bioclimatic predictor variables to identify those most influential to haemosporidian prevalence in different avian communities within the region. Two contrasting climate change scenarios for 2070 were used to create distribution models to explain spatial turnover in prevalence caused by climate change. We assigned our study sites into polygonal operational climatic units (OCUs) and used the general haemosporidian prevalence for each OCU to indirectly measure environmental suitability for these parasites. A high statistical association between global prevalence and the bioclimatic variables 'mean diurnal temperature range' and 'annual temperature range' was found. Climate change projections for 2070 showed a significant modification of the current distribution of suitable climate areas for haemosporidians in the study region.

New PCR primers targeting the cytochrome b gene reveal diversity of Leucocytozoon lineages in an individual host.

Avian haemosporidian parasites have received considerable attention in ecology and evolution as a result of their wide distribution and ease of detection. However, conventional PCR-based detection methods may sometimes underestimate haemosporidian mixed infections, which are frequent in natural populations. This underestimation is due to differences in PCR sensitivity for detection of lineages within the mixed infections. Therefore, we designed new primers to amplify sequences that were not detected by the conventional primers and examined if our primers were useful for accurate detection of mixed infections. Blood samples were collected from 32 wild birds captured in Hokkaido, and 16 of these were positive for Leucocytozoon using the conventional primers, while 15 were positive using our primers. All positively amplified samples were sequenced, and we found that the conventional primers detected 16% (5/32) of multiple infections and none of them was a novel lineage, whereas our primers detected 44% (14/32) of multiple infections and ten of them were novel lineages. A phylogenetic analysis showed that the new primers can detect a wide range of Leucocytozoon lineages compared with that detected by the conventional primers. The results indicate that our primers are particularly suitable for revealing unique strains from multiple infections. Highly variable multiple infections in the same population of birds at the same location were found for the first time. We revealed a higher diversity of Leucocytozoon lineages in nature than expected, which would provide more information to better understand parasite diversity and host-vector interactions in wildlife.

Avian haemosporidia in native and invasive sparrows at an Afrotropical region.

Bio-invasions are a major threat to biodiversity and ecosystems globally and may contribute to the proliferation of emerging infectious diseases. We examined the prevalence and phylogenetic diversity of avian haemosporidian parasites infecting the non-native house sparrows (Passer domesticus) and the native southern grey-headed sparrows (Passer diffusus). Blood samples from 104 sparrows (74 house sparrows and 30 southern grey-headed sparrows) mist-netted inside and around the Kruger National Park were used. Genomic DNA was extracted from each blood sample and subjected to nested PCR analyses, Sanger sequencing and phylogenetic analyses. Overall, 35.57% (37/104) of the birds sampled were infected with at least one haemosporidian parasites. Southern grey-headed sparrows had a higher parasite prevalence (60%) than house sparrows (24.3%). A total of 16 parasite lineages were identified, of which eight were novel lineages. Whereas Haemoproteus spp. showed the highest lineage diversity, Leucocytozoon spp. were the most prevalent parasites, albeit with significant differences between sparrow species. A single Plasmodium sp. infection was recorded in a southern grey-headed sparrow. In support of the enemy release hypothesis, we found that prevalence on non-native house sparrows was lower than prevalence recorded in their region of origin and also that they were infected only by indigenous parasites lineages.

Diversity, distribution, and methodological considerations of haemosporidian infections among Galliformes in Alaska.

Using samples spanning 10-degrees of latitude in Alaska, we provide the first comparative assessment of avian haemosporidia distribution of Arctic Alaska with subarctic host populations for four species of grouse and three species of ptarmigan (Galliformes). We found a high overall prevalence for at least one haemospordian genus (88%; N = 351/400), with spruce grouse (Canachites canadensis) showing the highest prevalence (100%; N = 54/54). Haemoproteus and Plasmodium lineages were only observed within grouse, while Leucocytozoon species were found within both grouse and ptarmigan. Further, different Leucocytozoon lineages were obtained from blood and tissue samples from the same individual, potentially due to the differential timing and duration of blood and tissue stages. Using different primer sets, we were able to identify different Leucocytozoon lineages within 55% (N = 44/80) of sequenced individuals, thereby detecting coinfections that may have otherwise gone undetected. The commonly used Haemoproteus/Plasmodium primers amplified Leucocytozoon for 90% (N = 103/115) of the products sequenced, highlighting the potential value of alternate primers to identify intra-genus coinfections and the importance of obtaining sequence information rather than relying solely on PCR amplification to assess parasite diversity. Overall, this dataset provides baseline information on parasite lineage distributions to assess the range expansion associated with climate change into Arctic regions and underscores methodological considerations for future studies.

Avian haemosporidian parasites in captive and free-ranging, wild birds from zoological institutions in Switzerland: Molecular characterization and clinical importance.

Avian haemosporidian parasites are widespread and infect birds from a broad variety of avian families with diverse consequences ranging from subclinical infections to severe and fatal disease. This study aimed to determine the occurrence and diversity of avian haemosporidia including associated clinical signs and pathomorphological lesions in captive and free-ranging, wild birds from two zoos and the near environment in Switzerland. Blood samples from 475 birds, including 230 captive and 245 free-ranging, wild individuals belonging to 42 different avian species from 15 orders were examined for the presence of avian haemosporidian DNA by a one-step multiplex PCR designed to simultaneously detect and discriminate the genera Plasmodium, Haemoproteus and Leucocytozoon by targeting mitochondrial genome sequences. Positive samples were additionally tested using a nested PCR targeting the cytochrome b gene of Plasmodium and Haemoproteus. The obtained amplicons were bidirectionally sequenced. This study revealed haemosporidian DNA in 42 samples, belonging to ten host species. The most commonly detected lineage was Plasmodium relictum SGS1, which was identified in 29 birds (Phoenicopterus roseus: n = 24, Alectoris graeca: n = 1, Lamprotornis superbus: n = 1, Somateria mollissima: n = 1, Spheniscus demersus: n = 1, Tetrao urogallus crassirostris: n = 1), followed by Haemoproteus sp. STRURA03 in six avian hosts (Bubo bubo: n = 5, Bubo scandiacus = 1), Plasmodium relictum GRW11 in four individuals (Phoenicopterus roseus: n = 3, Spheniscus demersus: n = 1) and Plasmodium elongatum GRW06 in one Alectura lathami lathami. A Phalacrocorax carbo was infected with Plasmodium relictum, but the exact lineage could not be determined. One mixed infection with P. relictum and Haemoproteus sp. was detected in a Bubo scandiacus. Only five individuals (Spheniscus demersus: n = 2, Somateria mollissima: n = 1, Bubo scandiacus: n = 1, Alectoris graeca: n = 1) showed clinical and pathomorphological evidence of a haemosporidian infection.

The first detection of avian haemosporidia from Culicoides biting midges in Japan, with notes on potential vector species and the transmission cycle.

BACKGROUND: Biting midges (Ceratopogonidae) are capable of transmitting a variety of pathogens including viruses, trypanosomes and haemosporidia. The majority of Haemoproteus parasites are transmitted by biting midges predominantly of the genus Culicoides and are known to cause significant physical and reproductive impacts on both wild and domestic birds. In Japan, Haemoproteus had been detected from various avian hosts, but not from arthropod vectors. In this study, we investigated the prevalence of avian haemosporidia at an educational forest in central Japan in attempt to reveal possible vector species of Haemoproteus, which would help to better understand the transmission cycle of Haemoproteus within Japan and to develop preventative measures for captive and domestic birds. METHODS: Biting midges were caught using UV light traps from 2016 to 2018. The collected samples were morphologically identified, and haemosporidian parasites were detected using PCR-based methods. The detected lineages were phylogenetically analyzed and compared with lineages previously detected from birds. Bloodmeal analyses were also carried out for part of the blood-fed individuals. RESULTS: Six Haemoproteus lineages were detected from 17 of 1042 female Culicoides (1.63%), including three species (C. sigaensis, C. arakawae, and C. pictimargo) in which Haemoproteus was detected for the first time. All detected lineages were placed in the subgenus Parahaemoproteus clade and were previously detected from crows of central Japan, strongly suggesting that parasites of these genetic lineages are transmitted between Culicoides and crows. Two Plasmodium lineages were also detected but are thought to be transmitted between Culex mosquitoes and birds of the educational forest based on previous detections. No amplifications were seen in bloodmeal analysis, possibly due to insufficient amount of blood, denaturation via digestion, or insufficient detectability of the used protocol. CONCLUSION: Haemoproteus DNA was detected from Culicoides for the first time in Japan, suggesting that transmission is possible within the country. These findings highlight the necessity to investigate Culicoides populations and Haemoproteus infections dynamics in Japan. However, vector competence could not be confirmed in this study and further studies are anticipated.

Insights on the taxonomy of Haemoproteus parasites infecting cracid birds.

The Haemosporida order is a well-supported clade of heteroxenous parasites transmitted by dipteran insects and frequently found parasitizing wild birds. These parasites have already been reported in all zoogeographic regions of the world, except for Antarctica. One of the potential hosts of haemosporidians is the Cracidae family, which includes approximately 50 species, 22 of which are present in Brazil, classified within nine genera. Data on haemosporidian infecting individuals of the Cracidae family is scarce, with only three Haemoproteus species being recorded in this group of birds. We found Haemoproteus spp. infection in all Penelope obscura bronzina analyzed. Among the parasites found, we observed two lineages of Haemoproteus (PENOBS02 and PENOBS03), which were characterized by morphological, molecular and phylogenetic approaches. The morphological data on cracid haemosporidian parasites, together with our phylogenetic results, allows discussions on the taxonomy of the Haemoproteus parasites that infect birds of the Cracidae family.

First report of haemosporidia and associated risk factors in red junglefowl (Gallus gallus) in China.

BACKGROUND: Avian haemosporidia infect both domestic and wild birds, causing anemia, acute tissue degeneration, and depopulation in wild birds. Poultry and wild birds have been reported as common reservoirs of haemosporidia, but limited information is available for red junglefowl (Gallus gallus) in China. The present study investigated the prevalence and molecular characterization of haemosporidia in red junglefowl. METHODS: Blood samples were collected from 234 red junglefowl from Jinghong City of Yunnan Province, and genomic DNA was extracted from these samples. The prevalence of haemosporidia was determined by nested PCR targeting the mitochondrial cytochrome b (cytb) gene. Molecular characterization was investigated based on phylogenetic analysis of cytb sequences, and associated risk factors were analyzed using the Chi-square (χ2) test. RESULTS: The overall prevalence of haemosporidia was 74.8% (175/234), and three species were identified, namely Haemoproteus enucleator, Leucocytozoon californicus, and Plasmodium juxtanucleare. The prevalence of haemosporidia in adult fowl (81.1%, 107/132) was significantly higher (χ2 = 6.32, df = 1, P = 0.012) than that in juveniles (66.7%, 68/102). Three novel haemosporidian lineages were revealed. CONCLUSIONS: This study examined the prevalence and identified species of avian haemosporidians in red junglefowl, providing new information on the molecular epidemiology and geographical distribution of haemosporidian parasites. Our results indicated high prevalence and diverse species distribution of these haemosporidians in red junglefowl. To the best of our knowledge, this is the first record of haemosporidian infection in red junglefowl in China.

A widespread survey of avian haemosporidia in deceased wild birds of Japan: the hidden value of personally collected samples.

Widespread surveys of avian haemosporidia (Plasmodium, Haemoproteus, and Leucocytozoon) in wild birds have substantially advanced information on the haemosporidian fauna of Japan. However, many areas and bird species remain insufficiently investigated. Bird carcasses collected for personal specimen collection seldom reach academic audience particularly in the veterinary field. The presence of avian haemosporidia was investigated in these personally collected bird carcasses, in order to better understand the avian haemosporidian fauna in Japan. Bird carcasses were donated through personal contact upon approval of the study. Tissue samples were collected from the birds and examined for haemosporidian parasites using nested-PCR targeting the cytochrome b gene. One hundred and forty-three birds of 85 species were donated, including 34 species and two subspecies that were molecularly or collectively investigated for the first time in Japan. Avian haemosporidian DNA was detected from 37 of the 134 tested birds (27.61%). In 8 bird species, avian haemosporidia was detected for the first time. Twenty-nine lineages were detected, including 8 novel and 9 known lineages detected in Japan for the first time. Furthermore, 16 lineages were detected from novel host species. While information that could be drawn was limited and risk management of zoonotic diseases needs re-consideration, these findings expanded information on the host range and distribution of several lineages. Collectively, this method of investigation using personally collected bird samples can provide important additions to more fully understand the avian haemosporidian fauna of Japan, as well as other areas with limited investigations.

First records of prevalence and diversity of avian haemosporidia in snipe species (genus Gallinago) of Japan.

Migratory birds are important carriers of pathogens such as viruses, bacteria and protozoa. Avian haemosporidia have been detected from many wild birds of Japan, but the infection status of migratory birds and transmission area are still largely unknown. Gallinago snipes are long-distance migratory shorebirds, and five species migrate to or through Japan, including Latham's snipe which is near threatened. Haemosporidian parasites in four snipe species were investigated to understand the role of migratory birds in the transmission of avian haemosporidia. Namely, this study aimed: i) to investigate differences in parasite prevalence and related factors explaining infection likelihood among these migratory species, ii) to explore the diversity in haemosporidian lineages and possible transmission areas, and iii) to assess the possibility of morphological effects of infection. Blood samples were collected from snipes caught in central and southwest Japan during migration. Parasites cytb gene DNA were detected via PCR-based testing, and detected lineages were phylogenetically analyzed. Additionally, factors related to prevalence and morphological effects of infection were statistically tested. 383 birds from four Gallinago snipe species were caught, showing higher overall prevalence of avian haemosporidia (17.8 %) than reported in other wader species in previous studies. This high infection rate is presumably due to increased contact with vector insects, resultant of environmental preferences. The prevalence of Plasmodium spp. Was higher in Swinhoe's snipes, while Haemoproteus spp. Was higher in Latham's snipes. These differences are thought to be related to ecological factors including habitat use, distribution and migratory route. Six lineages detected from juveniles indicate transmission between the breeding and sampling area. Contrary to expectations, a direct link between morphological features and haemosporidian parasite infection were not detected. These findings provide valuable information for conservation of this endangered migratory bird group. Further studies linking biological and parasitological research are anticipated to contribute to conservational actions.

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

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.

PREVALENCE AND PHYLOGENETIC ANALYSIS OF AVIAN HAEMOSPORIDIA IN WILD BIRDS IN THE REPUBLIC OF KOREA.

Avian blood parasites, including Plasmodium spp. and Haemoproteus spp., are found worldwide but only limited information of their occurrence is available in the Republic of Korea (ROK). We determined the prevalence of Plasmodium and Haemoproteus and their phylogenetic characteristics in wild birds in ROK. Blood samples were collected from 118 wild birds of 27 species in the Chonbuk Province, ROK. While 43 (36%) were positive for avian haemosporidia on microscopic examination of blood smears, 53 (45%) were positive by PCR targeting the cytochrome b gene. By direct sequencing of PCR amplicons, 47 (89%) were identified as Haemoproteus spp. and 6 (11%) as Plasmodium spp. Phylogenetic analysis using the cytochrome b gene revealed that resident and migrant birds have very similar genetic lineages of both parasites in ROK, suggesting the possibility that migrant birds may act as a mediator for the parasite among Asian countries.

Prevalence of avian haemosporidia among injured wild birds in Tokyo and environs, Japan.

Avian haemosporidia have been reported in various birds of Japan, which is part of the East Asian-Australian flyway and is an important stopover site for migratory birds potentially carrying new pathogens from other areas. We investigated the prevalence of avian malaria in injured wild birds, rescued in Tokyo and surrounding areas. We also evaluated the effects of migration by examining the prevalence of avian malaria for each migratory status. 475 birds of 80 species were sampled from four facilities. All samples were examined for haemosporidian infection via nested polymerase chain reaction (PCR) of the cytochrome b (cytb) gene. 100 birds (21.1%) of 43 species were PCR positive for avian haemosporidia. Prevalence in wintering birds, migratory breeders, and resident birds was 46.0%, 19.3%, 17.3% respectively. There was a bias in wintering birds due to Eurasian coot (Fulica atra) and Anseriformes. In wintering birds, lineages which are likely to be transmitted by Culiseta sp. in Northern Japan and lineages from resident species of Northern Japan or continental Asia were found, suggesting that wintering birds are mainly infected at their breeding sites. Meanwhile, there were numerous lineages found from resident and migratory breeders, suggesting that they are transmitted in Japan, some possibly unique to Japan. Although there are limits in studying rescued birds, rehabilitation facilities make sampling of difficult-to-catch migratory species possible and also allow for long-term monitoring within areas.