All across Africa: highly individual migration routes of Eleonora's falcon

Eleonora's falcon (Falco eleonorae) is a rare raptor species that delays its breeding period until late summer to feed its young with passerines at the peak of autumn migration. Since the 1950s, this slender winged falcon has been believed to migrate along a historical route via the Red Sea to its main wintering area in Madagascar. In our study, we used satellite telemetry to investigate the real migration route of Eleonora's falcons and found that the species displayed a highly individual migration pattern. Furthermore, juvenile falcons migrated via West Africa to Madagascar and two juveniles could be tracked during spring migration and to their summering areas in East and West Africa. As juveniles migrated independently of adults, we discuss inherited navigation strategies forming part of a complex navigation system. We propose the idea of an orientation mechanism that naive falcons could apply during their long-distance migration towards their faraway wintering area located in the open ocean.     

Nesting ecology of solitary‐nesting Amur Falcons (Falco amurensis) in central Mongolia

Amur Falcons (Falco amurensis) are a migratory species that face a variety of threats across their range, but little is known about their breeding ecology. These falcons breed in forest habitats in Eastern and Central Asia using nests constructed by corvids, including Eurasian Magpies (Pica pica). We monitored nests of 21 pairs of Amur Falcons at Hustai National Park in central Mongolia in 2017. Our objectives were to describe their basic nesting ecology, estimate nest survival by modeling the daily survival rate (DSR), examine nest selection by modeling it as a function of nest and site covariates, and use a spatial simulation to test hypotheses concerning intra‐ and interspecific avoidance. Clutch sizes averaged 4.1 eggs (N = 21 nests), and incubation and nestling periods averaged 25.7 and 26.1 d, respectively. The daily survival rate was 0.98, with young in 12 nests surviving to fledging. Nest structures were more likely to be selected as percent cover of nest bowls increased, usually in the form of a dome of sticks with multiple side entrances. Closed nests likely provide increased protection from predators. In contrast to congeneric Red‐footed Falcons (F. vespertinus) that nest in large colonies, Amur Falcons nested no farther from or closer to nests of either conspecifics or congeners than expected by chance. One factor likely contributing to this difference is that Red‐footed Falcons often use the nests of colonial‐nesting Rooks (Corvus frugilegus), whereas Amur Falcons typically use the nests of non‐colonial Eurasian Magpies. The ongoing loss of deciduous trees like white birch (Betula platyphylla) across the breeding range of Amur Falcons, probably due to climate change and increased grazing pressure, is likely to reduce the availability of nesting habitat for Eurasian Magpies which, in turn, will likely reduce availability of nests for Amur Falcons and other small falcons.     

Predator versus prey: on aerial hunting and escape strategies in birds

Predator and prey attack-escape performance is likely to bethe outcome of an evolutionary arms race. Predatory birds aretypically larger than their prey, suggesting different flightperformances. We analyze three idealized attack-escape situationsbetween predatory and prey birds: climbing flight escape, horizontalspeeding, and turning and escape by diving. Generally a smallerbird will outclimb a larger predator and hence outclimbing shouldbe a common escape strategy. However, some predators such asthe Eleonora's falcon (Falco elenorae) has a very high rateof climb for its size. Prey species with an equal or highercapacity to climb fast, such as the swift Apus apus, usuallyadopt climbing escape when attacked by Eleonora's falcons.To analyze the outcome of the turning gambit between predatorand prey we use a Howland diagram, where the relative lineartop speeds and minimum turning radii of prey and predator definethe escape and danger zones. Applied to the Eleonora's falconand some potential prey species, this analysis indicates thatthe falcon usually wins against the example prey species; thatis, the prey will be captured. Level maneuvering hunting isthe most common strategy seen in Eleonora's falcons. To avoidcapture via use of this strategy by a predator, the prey shouldbe able to initiate tight turns at high linear speed, whichis facilitated by a low wing loading (weight per unit of wingarea). High diving speed is favored by large size. If close enough to safe cover, a prey might still opt for a verticaldive to escape in spite of lower terminal diving speed thanthat of the predator. On the basis of aerodynamic considerationswe discuss escape flight strategies in birds in relation tomorphological adaptations.     

Autumn migration and wintering areas of Peregrine Falcons Falco peregrinus nesting on the Kola Peninsula, northern Russia

Four female Peregrine Falcons Falco peregrinus breeding on the Kola Peninsula, Russia, were fitted with satellite-received transmitters in 1994. Their breeding home ranges averaged 1175 (sd = ±714) km², and overlapped considerably. All left their breeding grounds in September and migrated generally south-west along the Baltic Sea. The mean travel rate for three falcons was 190 km/day. Two Falcons wintered on the coasts of France and in southern Spain, which were, respectively, 2909 and 4262 km from their breeding sites. Data on migration routes suggested that Falcons took a near-direct route to the wintering areas. No prolonged stopovers were apparent. The 90% minimum convex polygon winter range of a bird that migrated to Spain encompassed 213 km² ( n  = 54). The area of the 50% minimum convex polygon was 21.5 km² ( n  = 29). Data from this study agree with others from North America that show that Falcons breeding in a single area do not necessarily follow the same migratory path southward and do not necessarily use the same wintering grounds.     

Translocation of threatened New Zealand falcons to vineyards increases nest attendance, brooding and feeding rates

Anthropogenic landscapes can be rich in resources, and may in some cases provide potential habitat for species whose natural habitat has declined. We used remote videography to assess whether reintroducing individuals of the threatened New Zealand falcon Falco novaeseelandiae into a highly modified agricultural habitat affected the feeding rates of breeding falcons or related breeding behavior such as nest attendance and brooding rates. Over 2,800 recording hours of footage were used to compare the behavior of falcons living in six natural nests (in unmanaged, hilly terrain between 4 km and 20 km from the nearest vineyard), with that of four breeding falcon pairs that had been transported into vineyards and nested within 500 m of the nearest vineyard. Falcons in vineyard nests had higher feeding rates, higher nest attendance, and higher brooding rates. As chick age increased, parents in vineyard nests fed chicks a greater amount of total prey and larger prey items on average than did parents in hill nests. Parents with larger broods brought in larger prey items and a greater total sum of prey biomass. Nevertheless, chicks in nests containing siblings received less daily biomass per individual than single chicks. Some of these results can be attributed to the supplementary feeding of falcons in vineyards. However, even after removing supplementary food from our analysis, falcons in vineyards still fed larger prey items to chicks than did parents in hill nests, suggesting that the anthropogenic habitat may be a viable source of quality food. Although agricultural regions globally are rarely associated with raptor conservation, these results suggest that translocating New Zealand falcons into vineyards has potential for the conservation of this species.     

Sooty Falcon Falco concolor reproduction and population dynamics on the islands in the Sea of Oman

Knowledge of demographic parameters affecting population dynamics is critical to the formulation of effective conservation strategies. Sooty Falcon Falco concolor is a little‐studied, Near‐threatened species; estimates of global population size and trend for this species are uncertain. They lay eggs during mid‐summer and sometimes nest in colonies. This unusual breeding ecology suggests that demographic parameters driving their population growth rate may differ from those of most other falcons. We studied Sooty Falcon reproduction at breeding aggregations on Fahal Island and the Daymaniyat islands in the Sea of Oman during 2007–2014, modelled population growth and identified important life history parameters using elasticity analysis. The mean (± se) clutch and brood size was 2.83 ± 0.06 and 2.11 ± 0.07, respectively. Overall, 11.7% of nests failed between the egg and nestling stages, and the failure rate differed significantly between Fahal and the Daymaniyats, and across years. The mean proportion of eggs that hatched annually was 0.66 ± 0.02, and broods were significantly smaller on the Daymaniyats than on Fahal. Falcons on Fahal Island had a higher rate of hatching, a higher rate of nests that produced at least one chick, and produced more chicks per nest than on the Daymaniyats. We suggest that Fahal's proximity to the mainland gives breeding Sooty Falcons access to a more plentiful and stable source of food, especially during the period between arrival from the wintering grounds and the onset of the autumn migration of prey birds, resulting in the better reproductive rates for falcons on Fahal Island, relative to those on the Daymaniyat Islands. The annual asymptotic population growth rate (λ) was 0.87 (95% confidence interval (CI) 0.75–0.99), suggesting a declining population, although Sooty Falcons enjoyed a slightly higher population growth rate on Fahal than on the Daymaniyats. Because our study population is on the edge of the breeding range and is isolated from other breeding areas, measures to improve reproductive success of Sooty Falcons breeding on the islands in the Sea of Oman could be important for conservation of Sooty Falcons in Oman.     

The Sooty Falcon Falco concolor on the Red Sea coast of Saudi Arabia: distribution, numbers and conservation

Ground and aerial surveys of breeding Sooty Falcons Falco concolor were conducted along the western coast of Saudi Arabia and its offshore islands from August to October 1991 and 1992. An estimated population of 260–381 pairs was found. The known world breeding population is still below 1000 pairs. Most rocky islands were occupied except the largest islands of the Farasan archipelago. Among the many low islets surrounded with mangrove, only one small group near Al Lith harboured breeding Sooty Falcons. The distribution pattern, density and breeding success of the falcons seemed to be strongly influenced by food availability (autumn migration of small and medium-size birds) and the deterring presence of terrestrial predators. Human disturbance and trapping are still of local importance.     

Trapping of Saker Falcon Falco cherrug and Peregrine Falcon Falco peregrinus in Saudi Arabia: Implications for biodiversity conservation

The numbers of Falco cherrug and Falco peregrinus trapped during their migration over the Kingdom of Saudi Arabia (KSA) were investigated from published reports and through interviews with well-known trappers and dealers over several years (1989–2013). The number of trapped individuals increased for both species over a 23 year period, which is probably related to an enhanced trapping effort. Time series analysis suggests that the number of Saker Falcons being trapped is likely to be stable with annual fluctuations in the coming ten-year period, whereas the number of trapped Peregrine Falcons will probably decline with a small fluctuation initially. Using the population viability analysis suggests a high extinction rate for the Saker Falcon population migrating through KSA during the coming 10 and 20 years; whereas Peregrine Falcons probably take more than 100 years to reach the extinction threshold. However, the increase in the trapping period, especially in the spring, that has been observed during the last five years could increase the number of falcons trapped in the future. As both falcon species are migratory, implementing conservation actions across all range states is important to ensure a favourable conservation status for the Saker and Peregrine Falcons. Both species will benefit through the implementation of the Global Action Plan (GAP), developed by the Saker Falcon Task Force.     

Induction of epitope-specific neutralizing antibodies against West Nile virus

Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies.     

Visual configuration of two species of Falconidae with different foraging ecologies

Significant interspecific differences in avian vision occur, even in congeneric species, and these have been correlated with differences in the perceptual challenges associated with foraging. Although diurnal raptors are assumed to be mainly visually guided in their foraging, they differ markedly in their foraging tactics and this may result in different visual demands. Among the Falconidae (Falconiformes), most falcons forage mainly on the wing for highly mobile prey, whereas caracaras forage on the ground for carrion and insects. We assessed whether Saker Falcon Falco cherrug and Southern Caracara Caracara plancus differ in their visual abilities by determining the visual fields and foveal characteristics of both species. Using an ophthalmoscopic reflex technique, we found a higher degree of binocular overlap in the caracaras than in the falcons. The high binocular overlap (47°) of the Southern Caracara may facilitate object manipulation (e.g. moving rocks) when foraging. We used an ultra‐high resolution spectral‐domain optical coherence tomography to determine foveal characteristics. We found two foveas (depressions in the retina where high visual resolution is expected) in the falcons (one central and one temporal) but only a central fovea in the caracaras. The presence of a shallower temporal fovea in Saker Falcons may help to fixate visually upon a highly mobile prey item during pursuit. We conclude that these differences in visual field configurations and foveal characteristics reflect different foraging demands, suggesting that the extraction of visual information is finely tuned to the demands of their foraging tactics.     

Serologic evidence of West Nile virus infection in black bears (Ursus americanus) from New Jersey

Serum samples obtained from 51 free-ranging black bears (Ursus americanus) in northwestern New Jersey in February and March 2002 were analyzed for neutralizing antibodies to West Nile virus (WNV) and St. Louis encephalitis virus. Three (6%) of the black bears tested positive for WNV-neutralizing antibodies. One additional sample was positive for flavivirus-neutralizing antibodies but could not be differentiated for a specific virus type. This is the first report of WNV infection in black bears.     

Serologic evidence of West Nile virus infection in patients with exanthema in Hungary

The presence of WNV in Europe has been well known for decades, although the first human infections and avian outbreaks were diagnosed in Hungary only in 2003. An annual average of 6-8 cases of the neuroinvasive form of WNV infection has been detected in the region since then, but a higher number (17) of WNV associated neuroinvasive disease occurred in 2008. In 2004, a surveillance system was established for monitoring WNV-associated meningo-encephalitis cases in Hungary, but a milder type of illness (with fever, rash and/or influenza like symptoms) is not followed. Fifty-two sera of 45 patients with mild clinical symptoms (fever, exanthema) were tested for anti-WNV antibodies in 2008 in a retrospective study by immunofluorescence test and ELISA. Seven patients had antibodies against WNV, serologic evidence of recent WNV infection was found in 4 out of the 7 patients. Infections could be acquired predominantly in August and in September, which seems to be a risk period for WNV in Hungary. The possibility of a recent WNV infection should be taken into consideration in the occurrence of fever and rush at late summer. Differential diagnosis of exanthematous patients should include WNV serology tests and should be done routinely.     

Mosquito saliva causes enhancement of West Nile virus infection in mice

West Nile virus (WNV) is transmitted to vertebrate hosts primarily by infected Culex mosquitoes. Transmission of arboviruses by the bite of infected mosquitoes can potentiate infection in hosts compared to viral infection by needle inoculation. Here we examined the effect of mosquito transmission on WNV infection and systematically investigated multiple factors that differ between mosquito infection and needle inoculation of WNV. We found that mice infected with WNV through the bite of a single infected Culex tarsalis mosquito exhibited 5- to 10-fold-higher viremia and tissue titers at 24 and 48 h postinoculation and faster neuroinvasion than mice given a median mosquito-inoculated dose of WNV (10(5) PFU) by needle. Mosquito-induced enhancement was not due to differences in inoculation location, because additional intravenous inoculation of WNV did not enhance viremia or tissue titers. Inoculation of WNV into a location where uninfected mosquitoes had fed resulted in enhanced viremia and tissue titers in mice similar to those in mice infected by a single infected mosquito bite, suggesting that differences in where virus is deposited in the skin and in the virus particle itself were not responsible for the enhanced early infection in mosquito-infected mice. In addition, inoculation of mice with WNV mixed with salivary gland extract (SGE) led to higher viremia, demonstrating that mosquito saliva is the major cause of mosquito-induced enhancement. Enhanced viremia was not observed when SGE was inoculated at a distal site, suggesting that SGE enhances WNV replication by exerting a local effect. Furthermore, enhancement of WNV infection still occurred in mice with antibodies against mosquito saliva. In conclusion, saliva from C. tarsalis is responsible for enhancement of early WNV infection in vertebrate hosts.     

Efficacy of wildlife rehabilitation centers in surveillance and monitoring of pathogen activity: a case study with West Nile virus

Surveillance is critical for identifying and monitoring pathogen activity in wildlife populations, but often is cost- and time-prohibitive and logistically challenging. We tested the hypothesis that wildlife rehabilitation centers are useful for monitoring pathogen activity using West Nile virus (WNV) as a case study. We hypothesized that birds submitted to wildlife rehabilitation centers would have a similar prevalence of antibody to WNV as free-ranging birds. From 2008 to 2010, we collected sera from peridomestic birds submitted to the Wildlife Care Clinic (WCC), a wildlife rehabilitation center in central Iowa, and tested them for antibodies to WNV. We also collected and tested sera from free-ranging peridomestic birds in the area from which approximately 50% of WCC submissions historically originated. Prevalences of WNV antibodies in free-ranging birds and in peridomestic WCC birds were 2.3% (44/1,936) and 2.8% (2/72), respectively. However, none of the birds submitted to the WCC from the area where we captured free-ranging birds had antibodies (0/29). Our results indicate that rehabilitation facilities are not likely to be useful for monitoring WNV activity at small spatial scales or over short-time periods due to the low endemic prevalence of WNV, and low and variable submission rates. However, at larger spatial scales (ca. nine Iowa counties) WNV antibody prevalence in peridomestic birds submitted to the WCC was similar to that of free-ranging birds. Although limitations to using rehabilitation birds to monitor WNV must be considered, testing these birds could be useful for monitoring WNV activity regionally, especially with many states limiting surveillance due to budgetary constraints.     

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.     

Gyr Falcons, ptarmigan and microtine rodents in northern Sweden

A Gyr Falcon Falco rusticolus population in Northern Sweden (66°N, 17°E) was monitored from 1996 to 2002 in relation to its predator–prey interactions with its main and alternative prey species. Ptarmigan species Lagopus spp., and especially Rock Ptarmigan L. mutus , were the Gyr Falcons' most important prey and constituted more than 90% of the prey biomass. A 21-fold difference in ptarmigan abundance was found across Falcon breeding territories. However, this great variation in prey availability corresponded to only about a 10% shift in Gyr Falcon diet across territories, suggesting that the Falcons were reluctant or unable to compensate for declining ptarmigan availability by using alternative prey categories. Gyr Falcons did not respond functionally to microtine rodent abundance. Their diets were unaffected by a peak in the microtine rodent population cycle when Norwegian Lemmings Lemmus lemmus occurred in high numbers in the study area. Gyr Falcons responded numerically to their prey in two ways. First, there was a reproductive response with a significant relationship between the number of chicks fledged and the number of ptarmigan in the breeding territories. Secondly, although the Gyr Falcons did not utilize microtines as prey, there was a relationship between the microtine rodent abundance and the number of pairs that attempted to breed each year. This could be a result of an indirect community interaction, assuming that other predators switched from ptarmigan to microtines as prey, which could have had a positive effect on the breeding performance of the Gyr Falcons. The Gyr Falcons acted as true specialist predators, and their narrow food niche probably reflected a general lack of suitable alternative prey in the study area.     

A Therapeutic Antibody against West Nile Virus Neutralizes Infection by Blocking Fusion within Endosomes

Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West Nile virus (WNV) that neutralizes infection even after virus has spread to the central nervous system. Herein, we define its mechanism of inhibition. E16 blocks infection primarily at a post-attachment step as antibody-opsonized WNV enters permissive cells but cannot escape from endocytic compartments. These cellular experiments suggest that E16 blocks the acid-catalyzed fusion step that is required for nucleocapsid entry into the cytoplasm. Indeed, E16 directly inhibits fusion of WNV with liposomes. Additionally, low-pH exposure of E16–WNV complexes in the absence of target membranes did not fully inactivate infectious virus, further suggesting that E16 prevents a structural transition required for fusion. Thus, a strongly neutralizing anti–WNV MAb with therapeutic potential is potently inhibitory because it blocks viral fusion and thereby promotes clearance by delivering virus to the lysosome for destruction.     

Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines development for WNV and other viruses of the JEV serocomplex.     

Effect of Serum Heat-Inactivation and Dilution on Detection of Anti-WNV Antibodies in Mice by West Nile Virus E-protein Microsphere Immunoassay

Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.