Second International Diagnostic Accuracy Study for the Serological Detection of West Nile Virus Infection

Background

In recent decades, sporadic cases and outbreaks in humans of West Nile virus (WNV) infection have increased. Serological diagnosis of WNV infection can be performed by enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) neutralization test (NT) and by hemagglutination-inhibition assay. The aim of this study is to collect updated information regarding the performance accuracy of WNV serological diagnostics.

Methodology/Principal findings

In 2011, the European Network for the Diagnostics of Imported Viral Diseases-Collaborative Laboratory Response Network (ENIVD-CLRN) organized the second external quality assurance (EQA) study for the serological diagnosis of WNV infection. A serum panel of 13 samples (included sera reactive against WNV, plus specificity and negative controls) was sent to 48 laboratories involved in WNV diagnostics. Forty-seven of 48 laboratories from 30 countries participated in the study. Eight laboratories achieved 100% of concurrent and correct results. The main obstacle in other laboratories to achieving similar performances was the cross-reactivity of antibodies amongst heterologous flaviviruses. No differences were observed in performances of in-house and commercial test used by the laboratories. IFA was significantly more specific compared to ELISA in detecting IgG antibodies. The overall analytical sensitivity and specificity of diagnostic tests for IgM detection were 50% and 95%, respectively. In comparison, the overall sensitivity and specificity of diagnostic tests for IgG detection were 86% and 69%, respectively.

Conclusions/Significance

This EQA study demonstrates that there is still need to improve serological tests for WNV diagnosis. The low sensitivity of IgM detection suggests that there is a risk of overlooking WNV acute infections, whereas the low specificity for IgG detection demonstrates a high level of cross-reactivity with heterologous flaviviruses.     

Mosquito‐borne West Nile virus (WNV) surveillance in the Upper Rhine Valley,Germany

West Nile virus (WNV) could be introduced into Germany via migratory birds originating from Africa or southern Europe and subsequently transmitted to indigenous birds, humans, or horses by mosquitoes. Neither the virus itself nor antibodies against WNV have yet to be found in mosquitoes and horses, whereas antibodies have been detected in migrating birds and in humans that were in close contact with birds. At present, the West Nile virus itself has yet to be detected in Germany. This investigation was conducted primarily in major bird breeding, resting, and roosting habitats (hotspots) in the Upper Rhine Valley. Adult mosquitoes were trapped using CO₂‐baited Encephalitis Vector Surveillance (EVS)‐traps and were tested for WNV by the VecTest WNV Antigen Assay. In 2007 and 2008, a total of 11,073 host‐seeking adult female mosquitoes (13 species) were tested, and all tests were negative for WNV. Statistical calculations could be performed only where sufficient numbers of mosquitoes were trapped. For these sites, WNV infection among mosquitoes could be ruled out with 80% certainty. For the evaluation of the WNV situation in Germany, the results of this investigation are a further indication that the virus has not yet arrived.     

An Integrative Eco-Epidemiological Analysis of West Nile Virus Transmission

West Nile disease, caused by the West Nile virus (WNV), is a mosquito-borne zoonotic disease affecting humans and horses that involves wild birds as amplifying hosts. The mechanisms of WNV transmission remain unclear in Europe where the occurrence of outbreaks has dramatically increased in recent years. We used a dataset on the competence, distribution, abundance, diversity and dispersal of wild bird hosts and mosquito vectors to test alternative hypotheses concerning the transmission of WNV in Southern France. We modelled the successive processes of introduction, amplification, dispersal and spillover of WNV to incidental hosts based on host–vector contact rates on various land cover types and over four seasons. We evaluated the relative importance of the mechanisms tested using two independent serological datasets of WNV antibodies collected in wild birds and horses. We found that the same transmission processes (seasonal virus introduction by migratory birds, Culex modestus mosquitoes as amplifying vectors, heterogeneity in avian host competence, absence of ‘dilution effect’) best explain the spatial variations in WNV seroprevalence in the two serological datasets. Our results provide new insights on the pathways of WNV introduction, amplification and spillover and the contribution of bird and mosquito species to WNV transmission in Southern France.     

Exposure of Eurasian magpies and turtle doves to West Nile virus during a major human outbreak, Greece, 2011

A major number of West Nile virus (WNV) infections in humans occurred in 2010 in northern Greece, with 262 laboratory confirmed cases. In 2011, fewer cases were reported, but the pattern was more dispersed throughout the Greek mainland. Isolated strains were similar to lineage 2 strains detected in previous years in Austria and Hungary from birds of prey. We conducted a serological surveillance study on hunter-harvested wild birds, to determine possible exposure of avian species during the current outbreak. Serum samples from a total of 113 Eurasian magpies and 85 turtle doves (abundant resident and migratory avian species, respectively, with potential roles in WNV epidemiology) were tested. These birds were hunter-harvested during 2011 from various prefectures both affected and not affected by the WNV outbreak in Greece. Sera were tested for the presence of WNV IgG antibodies by indirect immunofluorescence assay (IFA). Verification of positive results by a micro-virus neutralization test (VNT) was also performed. A total of 23 out of 113 (20.4%) Eurasian magpies and 6/85 (7.1%) turtle doves were found positive. Results showed association of human cases with wild birds’ exposure to the virus; no avian sera were found positive in prefectures not affected by the WNV outbreak. In contrast, positive avian sera were found in every prefecture that human WNV cases occurred in 2011. High seroprevalence in Eurasian magpies suggests high activity of WNV in the areas. Findings of past exposure of migratory birds like turtle doves to WNV upon their arrival in resting areas in Greece suggest various avian species with similar migration traits as target species for viral isolation studies, as they can be considered candidates for the introduction of WNV lineage 2 in Greece from Central Europe.     

West Nile Virus and California Breeding Bird Declines

Since it was first detected in 1999, West Nile virus (WNV) quickly spread, becoming the dominant vector-borne disease in North America. Sometimes fatal to humans, WNV is even more widespread among birds, with hundreds of species known to be susceptible to WNV infection in North America alone. However, despite considerable mortality and local declines observed in American crows (Corvus brachyrhynchos), there has been little evidence of a large regional association between WNV susceptibility and population declines of any species. Here we demonstrate a correlation between susceptibility to WNV measured by large-scale testing of dead birds and two indices of overall population change among bird species following the spread of WNV throughout California. This result was due primarily to declines in four species of Corvidae, including all species in this family except common ravens (Corvus corax). Our results support the hypothesis that susceptibility to WNV may have negative population consequences to most corvids on regional levels. They also provide confirmation that dead animal surveillance programs can provide important data indicating populations most likely to suffer detrimental impacts due to WNV.     

Population Seroprevalence Study after a West Nile Virus Lineage 2 Epidemic,Greece, 2010

Introduction

During summer 2010, 262 human cases including 35 deaths from West Nile virus (WNV) infection were reported from Central Macedonia, Greece. Evidence from mosquitoes, birds and blood donors demonstrated that the epidemic was caused by WNV lineage 2, which until recently was considered of low virulence. We conducted a household seroprevalence study to estimate the spread of infection in the population during the epidemic, ascertain the relationship of infection to clinical disease, and identify risk factors for infection.

Methods

We used a two-stage cluster design to select a random sample of residents aged ≥18 years in the outbreak epicentre. We collected demographic, medical, and risk factor data using standard questionnaires and environmental checklists, and tested serum samples for presence of WNV IgG and IgM antibodies using ELISA.

Results

Overall, 723 individuals participated in the study, and 644 blood samples were available. Weighted seropositivity for IgG antibodies was 5.8% (95% CI: 3.8–8.6; n=41). We estimated that about 1 in 130 (1:141 to 1:124) infected individuals developed WNV neuroinvasive disease, and approximately 18% had clinical manifestations attributable to their infection. Risk factors for infection reflected high exposure to mosquitoes; rural residents were particularly at risk (prevalence ratio: 8.2, 95% CI: 1.1–58.7).

Discussion

This study adds to the evidence that WNV lineage 2 strains can cause significant illness, demonstrating ratios of infection to clinical disease similar to those found previously for WNV lineage 1.     

Comparing Competitive Fitness of West Nile Virus Strains in Avian and Mosquito Hosts

Enzootic transmission of West Nile virus (WNV; Flaviviridae, Flavivirus) involves various species of birds and ornithophilic mosquitoes. Single nucleotide substitutions in the WNV genome may impact viral fitness necessary for WNV adaptation and evolution as previously shown for the WN02 genotype. In an effort to study phenotypic change, we developed an in vivo fitness competition model in two biologically relevant hosts for WNV. The House Finch (HOFI; Haemorhous mexicanus) and Culex tarsalis mosquitoes represent moderately susceptible hosts for WNV, are highly abundant in Western North America and frequently are infected with WNV in nature. Herein, we inoculated HOFIs and Cx. tarsalis competitively (dually) and singly with infectious-clone derived viruses of the founding California isolate COAV997-2003 (COAV997-IC), the founding North American isolate NY99 (NY99-IC), and a 2004 field isolate from California (CA-04), and compared the replicative capacities (fitness) of these viruses to a genetically marked virus of COAV997 (COAV997-5nt) by measuring RNA copy numbers. COAV997 and COAV997-5nt exhibited neutral fitness in HOFIs and Cx. tarsalis, and the temperature-sensitive phenotype of COAV997 did not affect replication in HOFIs as none of the infected birds became febrile. The NY99 and CA-04 isolates demonstrated elevated fitness in HOFIs compared to COAV997-5nt, whereas all viruses replicated to similar titers and RNA copies in Cx. tarsalis, and the only fitness differences were related to infection rates. Our data demonstrated that competitive replication allows for the sensitive comparison of fitness differences among two genetically closely related viruses using relevant hosts of WNV while eliminating host-to-host differences. In conclusion, our approach may be helpful in understanding the extent of phenotypic change in fitness associated with genetic changes in WNV.     

Using Avian Surveillance in Ecuador to Assess the Imminence of West Nile Virus Incursion to Galápagos

Infectious disease emergence represents a global threat to human, agricultural animal and wildlife health. West Nile virus (WNV) first emerged in the Americas in 1999 following its introduction to New York from the Old World. This flavivirus rapidly spread across much of North America, causing human, equine and avian mortalities and population declines of multiple wild bird species. It has now spread to Central and South America, and there is concern that the virus will reach the Galápagos Islands, a UNESCO World Heritage Site famous for its unique biodiversity, with potentially catastrophic results. Here, we use wild bird surveillance to examine the current WNV status in the Galapagos Islands and around the Ecuadorian city of Guayaquil (the main air and sea port serving Galápagos). We conducted serosurveys of wild birds on three Galápagos Islands (Baltra, San Cristobal and Santa Cruz) with direct transport links to the South American continent. In addition, dead birds killed by car collisions on Santa Cruz were tested for WNV infection. On mainland Ecuador, serosurveys of wild birds were conducted at three sites around Guayaquil. No evidence of WNV seropositivity or infection was detected. Although wider testing is recommended on the mainland, the study highlights a limit of WNV spread within South America. Our results indicate the continued absence of WNV on Galápagos and suggest the current likelihood of human-mediated transport of WNV to Galápagos to be low. The risk of emergence will almost certainly increase over time, however, and stringent biosecurity and surveillance measures should be put in place to minimise the risk of the introduction of WNV (and other alien pathogens) to Galápagos.     

Vector competence of northern and southern European Culex pipiens pipiens mosquitoes for West Nile virus across a gradient of temperatures

In Europe, West Nile virus (WNV) outbreaks have been limited to southern and central European countries. However, competent mosquito vectors and susceptible bird hosts are present in northern Europe. Differences in temperature and vector competence of mosquito populations may explain the absence of WNV outbreaks in northern Europe. The aim of the present study was to directly compare vector competence of northern and southern European Culex pipiens (Cx. p.) pipiens mosquitoes for WNV across a gradient of temperatures. WNV infection and transmission rates were determined for two Cx. p. pipiens populations originating from The Netherlands and Italy, respectively. Mosquitoes were orally exposed by providing an infectious bloodmeal, or by injecting WNV (lineage 2) in the thorax, followed by 14‐day incubation at 18, 23, or 28 °C. No differences in infection or transmission rates were found between the Cx. p. pipiens populations with both infection methods, but WNV transmission rates were significantly higher at temperatures above 18 °C. The absence of WNV outbreaks in northern Europe cannot be explained by differences in vector competence between Cx. p. pipiens populations originating from northern and southern Europe. This study suggests that low temperature is a key limiting factor for WNV transmission.     

Avian diversity and West Nile virus: testing associations between biodiversity and infectious disease risk

The emergence of several high profile infectious diseases in recent years has focused attention on our need to understand the ecological factors contributing to the spread of infectious diseases. West Nile virus (WNV) is a mosquito-borne zoonotic disease that was first detected in the United States in 1999. The factors accounting for variation in the prevalence of WNV are poorly understood, but recentideas suggesting links between high biodiversity and reduced vector-borne disease risk may help account for distribution patterns of this disease. Since wild birds are the primary reservoir hosts for WNV, we tested associations between passerine (Passeriform) bird diversity, non-passerine (all other orders) bird diversity and virus infection rates in mosquitoes and humans to examine the extent to which bird diversity is associated with WNV infection risk. We found t h at non-passerine species richness (number of non-passerine species) was significantly negatively correlated with both mosquito and human infection rates, whereas there was no significant association between passerine species richness and any measure of infection risk. Our findings suggest that non-passerine diversity may play a role in dampening WNV amplification rates in mosquitoes, minimizing human disease risk.     

Ticks (Acari: Ixodidae) parasitizing migrating and local breeding birds in Finland

Ticks are globally renowned vectors for numerous zoonoses, and birds have been identified as important hosts for several species of hard ticks (Acari: Ixodidae) and tick-borne pathogens. Many European bird species overwinter in Africa and Western Asia, consequently migrating back to breeding grounds in Europe in the spring. During these spring migrations, birds may transport exotic tick species (and associated pathogens) to areas outside their typical distribution ranges. In Finland, very few studies have been conducted regarding ticks parasitizing migrating or local birds, and existing data are outdated, likely not reflecting the current situation. Consequently, in 2018, we asked volunteer bird ringers to collect ticks from migrating and local birds, to update current knowledge on ticks found parasitizing birds in Finland. In total 430 ticks were collected from 193 birds belonging to 32 species, caught for ringing between 2018 and 2020. Furthermore, four Ixodes uriae were collected from two roosting islets of sea birds in 2016 and 2020. Ticks collected on birds consisted of: Ixodes ricinus (n?=?421), Ixodes arboricola (4), Ixodes lividus (2) and Hyalomma marginatum (3). Ixodes ricinus loads (nymphs and larvae) were highest on thrushes (Passeriformes: Turdidae) and European robins (Erithacus rubecula). The only clearly imported exotic tick species was H. marginatum. This study forms the second report of both I. uriae and I. arboricola from Finland, and possibly the northernmost observation of I. arboricola from Europe. The importation of exotic tick species by migrating birds seems a rare occurrence, as over 97% of all ticks collected from birds arriving in Finland during their spring migrations were I. ricinus, a species native to and abundant in Finland.

     

Quantitative Risk Assessment of the Pathways by Which West Nile Virus Could Reach Hawaii

The introduction of West Nile virus (WNV) to Hawaii could have severe impacts on human health, wildlife health and, as a result, Hawaiis tourism-based economy. To provide guidance for management agencies seeking to prevent the introduction of WNV, we performed a quantitative assessment of the pathways by which WNV could reach Hawaii from North America. We estimated the rate of infectious individuals reaching Hawaii by the following means (1) humans on aircraft, (2) wind-transported mosquitoes, (3) human-transported mosquitoes, (4) human-transported birds or other vertebrates, and (5) migratory birds. We found that pathways 3 and 4 represented the highest risk. We estimated that each year, 7–70 WNV infectious mosquitoes will reach Hawaii by airplane when WNV becomes well established in the Western U.S. Exemptions in current quarantine regulations will also result in the import of birds that will be infectious with WNV for 0.3–2.2 bird-days each year. We propose actions that would substantially reduce the risk of WNV reaching Hawaii by these means, including reinstating aircraft disinsection in cargo holds and altering bird quarantine rules. Finally, research is urgently needed to determine whether a migratory bird can survive the migration from North America to Hawaii with a viremic WNV infection.     

Use of Wild Bird Surveillance,Human Case Data and GIS Spatial Analysis for Predicting Spatial Distributions of West Nile Virus in Greece

West Nile Virus (WNV) is the causative agent of a vector-borne, zoonotic disease with a worldwide distribution. Recent expansion and introduction of WNV into new areas, including southern Europe, has been associated with severe disease in humans and equids, and has increased concerns regarding the need to prevent and control future WNV outbreaks. Since 2010, 524 confirmed human cases of the disease have been reported in Greece with greater than 10% mortality. Infected mosquitoes, wild birds, equids, and chickens have been detected and associated with human disease. The aim of our study was to establish a monitoring system with wild birds and reported human cases data using Geographical Information System (GIS). Potential distribution of WNV was modelled by combining wild bird serological surveillance data with environmental factors (e.g. elevation, slope, land use, vegetation density, temperature, precipitation indices, and population density). Local factors including areas of low altitude and proximity to water were important predictors of appearance of both human and wild bird cases (Odds Ratio = 1,001 95%CI = 0,723–1,386). Using GIS analysis, the identified risk factors were applied across Greece identifying the northern part of Greece (Macedonia, Thrace) western Greece and a number of Greek islands as being at highest risk of future outbreaks. The results of the analysis were evaluated and confirmed using the 161 reported human cases of the 2012 outbreak predicting correctly (Odds = 130/31 = 4,194 95%CI = 2,841–6,189) and more areas were identified for potential dispersion in the following years. Our approach verified that WNV risk can be modelled in a fast cost-effective way indicating high risk areas where prevention measures should be implemented in order to reduce the disease incidence.     

Naturally Induced Humoral Immunity to West Nile Virus Infection in Raptors

West Nile virus (WNV) infection can be fatal to many bird species, including numerous raptors, though population- and ecosystem-level impacts following introduction of the virus to North America have been difficult to document. Raptors occupy a diverse array of habitats worldwide and are important to ecosystems for their role as opportunistic predators. We documented initial (primary) WNV infection and then regularly measured WNV-specific neutralizing antibody titers in 16 resident raptors of seven species, plus one turkey vulture. Most individuals were initially infected and seroconverted between July and September of 2003, though three birds remained seronegative until summer 2006. Many of these birds became clinically ill upon primary infection, with clinical signs ranging from loss of appetite to moderate neurological disease. Naturally induced WNV neutralizing antibody titers remained essentially unchanged in some birds, while eight individuals experienced secondary rises in titer presumably due to additional exposures at 1, 2, or 3 years following primary infection. No birds experienced clinical signs surrounding or following the time of secondary exposure, and therefore antibodies were considered protective. Results of this study have implications for transmission dynamics of WNV and health of raptor populations, as well as the interpretation of serologic data from free-ranging and captive birds. Antibodies in raptors surviving WNV may persist for multiple years and protect against potential adverse effects of subsequent exposures.     

Apoptosis in mosquito midgut epithelia associated with West Nile virus infection

The mosquito Culex pipiens pipiens is a documented vector of West Nile virus (WNV, Flaviviridae, Flavivirus). Our laboratory colony of C. p. pipiens, however, was repeatedly refractory to experimental transmission of WNV. Our goal was to identify if a cellular process was inhibiting virus infection of the midgut. We examined midguts of mosquitoes fed control and WNV-infected blood meals. Three days after feeding, epithelial cells from abdominal midguts of mosquitoes fed on WNV fluoresced under an FITC filter following Acridine Orange staining, indicating apoptosis in this region. Epithelial cells from experimental samples examined by TEM exhibited ultrastructural changes consistent with apoptosis, including shrinkage and detachment from neighbors, heterochromatin condensation, nuclear degranulation, and engulfment of apoptotic bodies by adjacent cells. Virions were present in cytoplasm and within cytoplasmic vacuoles of apoptotic cells. No apoptosis was detected by TEM in control samples. In parallel, we used Vero cell plaque assays to quantify infection after 7 and 10 day extrinsic incubation periods and found that none of the mosquitoes (0/55 and 0/10) which imbibed infective blood were infected. We propose that programmed cell death limits the number of WNV-infected epithelial cells and inhibits disseminated viral infections from the mosquito midgut.     

Migrating Birds as Dispersal Vehicles for West Nile Virus

Whereas migrating birds have been implicated in the spread of West Nile virus (WNV), there is no direct evidence of birds actively migrating while infectious. The role of birds in WNV dispersal is difficult to assess in the field. However, this role can be evaluated experimentally because birds in migratory disposition display increased locomotor activity or restlessness under captive conditions. We tested the following hypotheses: (1) migrating passerine birds continue to exhibit migratory activity while infectious with WNV and (2) the migratory state of the individual affects the magnitude of viremia. We examined the migratory activity of two neoarctic-neotropical passerine migrants, Swainson’s thrush (Catharus ustulatus) and gray catbird (Dumetella carolinensis), during acute WNV infection. All gray catbirds and six of nine Swainson’s thrushes exhibited migratory activity while infectious. Moreover, migratory status did not appear to influence viremia titers, as might be expected if individuals were immunosuppressed during migration. Therefore, we demonstrate that migrating passerine birds are potential dispersal vehicles for WNV.     

Evaluation of vector competence for West Nile virus in Italian Stegomyia albopicta (=Aedes albopictus) mosquitoes

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle that involves wild birds as reservoir hosts. The virus is responsible for outbreaks of viral encephalitis in humans and horses. In Europe, Culex pipiens (Diptera: Culicidae) is considered to be the main vector of WNV, but other species such as Stegomyia albopicta (=Aedes albopictus) (Diptera: Culicidae) may also act as competent vectors of this virus. Since 2008 human cases of WNV disease have been reported in northeast Italy. In 2011, new areas of southern Italy became involved and a first outbreak of WNV lineage 1 occurred on the island of Sardinia. On the assumption that a potential involvement of St. albopicta in WNV transmission cannot be excluded, and in order to evaluate the competence of this species for the virus, an experimental infection of an St. albopicta laboratory colony, established from mosquitoes collected in Sardinia, was carried out. The results were compared with those obtained in a colony of the main vector Cx. pipiens. The study showed St. albopicta collected on Sardinia to be susceptible to WNV infection, which suggests this Italian mosquito species is able to act as a possible secondary vector, particularly in urban areas where the species reaches high levels of seasonal abundance.     

West Nile Virus Infection in American Robins: New Insights on Dose Response

West Nile virus (WNV) is a vector-borne pathogen that was first detected in the United States in 1999. The natural transmission cycle of WNV involves mosquito vectors and avian hosts, which vary in their competency to transmit the virus. American robins are an abundant backyard species in the United States and appear to have an important role in the amplification and dissemination of WNV. In this study we examine the response of American robins to infection with various WNV doses within the range of those administered by some natural mosquito vectors. Thirty American robins were assigned a WNV dosage treatment and needle inoculated with 10^0.95 PFU, 10^1.26 PFU, 10^2.15 PFU, or 10^3.15 PFU. Serum samples were tested for the presence of infectious WNV and/or antibodies, while oral swabs were tested for the presence of WNV RNA. Five of the 30 (17%) robins had neutralizing antibodies to WNV prior to the experiment and none developed viremia or shed WNV RNA. The proportion of WNV-seronegative birds that became viremic after WNV inoculation increased in a dose dependent manner. At the lowest dose, only 40% (2/5) of the inoculated birds developed productive infections while at the highest dose, 100% (7/7) of the birds became viremic. Oral shedding of WNV RNA followed a similar trend where robins inoculated with the lower two doses were less likely to shed viral RNA (25%) than robins inoculated with one of the higher doses (92%). Viremia titers and morbidity did not increase in a dose dependent manner; only two birds succumbed to infection and, interestingly, both were inoculated with the lowest dose of WNV. It is clear that the disease ecology of WNV is a complex interplay of hosts, vectors, and viral dose delivered.     

Seasonal dynamics in mosquito abundance and temperature do not influence avian malaria prevalence in the Himalayan foothills

We examined seasonal prevalence in avian haemosporidians (Plasmodium and Haemoproteus) in migrant and resident birds in western Himalaya, India. We investigated how infection with haemosporidians in avian hosts is associated with temporal changes in temperature and mosquito abundance along with host abundance and life‐history traits (body mass). Using molecular methods for parasite detection and sequencing partial cytochrome b gene, 12 Plasmodium and 27 Haemoproteus lineages were isolated. Our 1‐year study from December 2008 to December 2009 in tropical Himalayan foothills revealed a lack of seasonal variation in Plasmodium spp. prevalence in birds despite a strong correlation between mosquito abundance and temperature. The probability of infection with Plasmodium decreased with increase in temperature. Total parasite prevalence and specifically Plasmodium prevalence showed an increase with average avian body mass. In addition, total prevalence exhibited a U‐shaped relationship with avian host abundance. There was no difference in prevalence of Plasmodium spp. or Haemoproteus spp. across altitudes; parasite prevalence in high‐altitude locations was mainly driven by the seasonal migrants. One Haemoproteus lineage showed cross‐species infections between migrant and resident birds. This is the first molecular study in the tropical Himalayan bird community that emphasizes the importance of studying seasonal variation in parasite prevalence. Our study provides a basis for further evolutionary study on the epidemiology of avian malaria and spread of disease across Himalayan bird communities, which may not have been exposed to vectors and parasites throughout the year, with consequential implications to the risk of infection to naïve resident birds in high altitude.