Escherichia coli (STEC) in food safety enforcement as well as clinical care primarily relates to the current inability of an accurate risk assessment of individual strains due to the large variety in serotype and genetic content associated with (severe) disease. In order to classify the clinical and/or epidemic potential of a STEC isolate at an early stage it is crucial to identify virulence characteristics of putative pathogens from genomic information, which is referred to as ‘predictive hazard identification’. This study aimed at identifying associations between virulence factors, phylogenetic groups, isolation sources and seropathotypes. Most non-O157 STEC in the Netherlands belong to phylogroup B1 and are characterized by the presence of ehxA, iha and stx2, but absence of eae. The large variability in the number of virulence factors present among serogroups and seropathotypes demonstrated that this was merely indicative for the virulence potential. While all the virulence gene associations have been worked out, it appeared that there is no specific pattern that would unambiguously enable hazard identification for an STEC strain. However, the strong correlations between virulence factors indicate that these arrays are not a random collection but are rather specific sets. Especially the presence of eae was strongly correlated to the presence of many of the other virulence genes, including all non-LEE encoded effectors. Different stx-subtypes were associated with different virulence profiles. The factors ehxA and ureC were significantly associated with HUS-associated strains (HAS) and not correlated to the presence of eae. This indicates their candidacy as important pathogenicity markers next to eae and stx2a.The complexity regarding Shiga toxin-producing  Escherichia coli (STEC) are potential lethal zoonotic pathogens with a clinical spectrum including diarrhea, hemorrhagic colitis, and the hemolytic uremic syndrome (HUS) [Shiga toxin producing me (HUS) . STEC isme (HUS) –4. Some me (HUS) . This obme (HUS) . Serotypme (HUS)  an alterex post facto determinant of virulence potential, the dynamic nature of STEC virulence in time and place exposes a limitation of SPT classification as a predictive indicator of microbial risk [The EFSA network recently concluded that the seropathotype classification does not define pathogenic STEC nor does it provide an exhaustive list of pathogenic serotypes . This reial risk .E. coli strains belonged to four main phylogenetic groups, A, B1, B2, and D [E. coli strains belong mainly to group B2 and group D. STEC strains were found to fall into phylogenetic groups A, B1, and D [Phylogenetic analyses have shown that most 2, and D . Whereas1, and D . HoweverThe serogroups most frequently associated with severe human disease in the EU, in particular HUS, are O157, O26, O111, O103, and O145 . Over thrpoS genotype. The results are discussed in relation to the epidemiology of STEC in the Netherlands.The goal of this study was to investigate the distribution of known virulence factors among clinical, food and animal STEC isolates from the Netherlands. More specific, the research aimed at identifying associations between virulence factors and phylogenetic groups, isolation sources, seropathotypes, serogroups, intimin presence/absence, type of Shiga toxin, and the A set of 209 STEC non-O157 isolates  was obtained from the collection of the Food and Consumer Product Safety Authority  and the National Institute for Public Health and The Environment . The animal isolates  were isolated from 2002 to 2009 during national surveys and were maintained in Microbank vials . The strains from meat  were isolated during national surveys by the Food and Consumer Product Safety Authority in the Netherlands 2005–2010. The clinical human isolates were strains isolated from patients with STEC symptoms and sent in by hospitals for confirmation in the period 2006 to 2010. All clinical human isolates were maintained at room temperature in Mueller-Hinton agar.Isolates were propagated on blood agar or nutrient agar (Oxoid) and DNA was extracted using Chelex-100  resin-based technique. One colony of each isolate was transferred into 300 μl 10% Chelex-100 solution, which was subsequently heated for 5 min at 56°C to resuspend the cells. The tubes were briefly cooled at room temperature and mixed for 15 seconds before heating for 15 min at 98–99°C for lysis of the bacteria. After cooling at room temperature, the lysates were centrifuged for 5 min. at 13,000 rpm and up to 200 μl of the supernatant was transferred to a clean tube and stored at -20°C.Isolates were grouped according to the Karmali seropathotype classification . RankingadfO, astA, ckf, efaI, ent/espL2, etpD, iha, iha_homologue, saa, stx2a, stx2b, stx2c, stx2d, stx2dact, stx2e, stx2f, stx2g, subA, toxB and ureC were performed on a Thermo Hybaid PCR Express Thermal Cycler  using iQ Supermix  and 0.2 μM of each primer at an annealing temperature as indicated in eae, ehxA, stx1, stx2 and terB were performed on an iQ5 Thermal Cycler using iQ Supermix  0.2 μM of each forward and reverse primer and 0.4 μM of the probe at the temperature as indicated in PCR was used to screen isolates for the presence of 40 virulence markers and determination of the phylogenetic group. Primers and probes used in this study are displayed in (nle) and ent/espL2 were performed as described in Coombes et al. [nleA, the forward primer of each primer set was fluorescently labelled with FAM, VIC, NED, or PET. Amplicons were generated essentially as described and pooled in five sets, resulting in distinctive combinations of fragment size and fluorescent label. Fluorescently labelled fragments were analyzed on a capillary sequencer  in the presence of an internal marker . The GeneScan 600 LIZ size standard was used for pooled amplicons smaller than 600 bp . The GeneScan 1200 LIZ size standard was used for pooled PCR products between 600 and 1,200 bp (nleC and nleG). Fragments larger than 1,200 bp  were analyzed by agarose gel electrophoresis. Raw data were analyzed using BioNumerics 6.1 (Applied Maths) to determine fragment sizes.PCR amplification and sequencing of the rpoS gene was performed as described earlier [PCRs for the non-LEE encoded effectors s et al. . Except  earlier , but forchuA, TspE4.C2 and yjaA was carried out in a multiplex format using the Qiagen multiplex PCR mix and 0.2 μM of each primer at an annealing temperature of 60°C  between groups and associations between markers were evaluated using the Chi-square test with a significance level of 0.05 (IBM SPSS Statistics version 19).Isolate characteristics and the PCR results of all strains used can be accessed in eae-negative (169/209 = 80.9%) (stx1(78%) was observed  in the top-EU serotypes  isolated in the Netherlands while the prevalence of stx1 was relatively high among these serogroups. The genes stx2 (more specific: stx2b and stx2d) and subA occurred at significant higher frequency (P<0.05) among the most frequently isolated clinical serogroups in the Netherlands  compared to the top 4 EU serogroups . The following genes occurred at significant lower frequency among the top 4 Dutch serogroups compared to the top 4 EU serogroups (in random order): eae, stx2a, etpD, toxB, adfO, cfk, efa. ureC, terB, ehxA, ent/espL2, nleB, nleE, nleG23, nleG62, nleG52, nleB2, nleH11, nleG, nleF, nleH12, nleA, nleG21 and nleG9. In addition, the top Dutch serogroups were significantly stronger associated with phylogroup A (12/45 versus 0/20) (P = 0.021) and showed significantly less total number of virulence genes (median of 6 versus 11) (P = 0.002).Overall, the vast majority of STEC strains included in this study were = 80.9%) . A relatobserved . InteresThe number of virulence genes present in the top 4 EU serogroups showed considerable variation: O26 (n = 7) average 20.7 (14–25), O111 (n = 5) average 17.7 (7–23), O103 (n = 3) average 17.3 (7–24), O145 (n = 5) average 16.4 (7–19), and O121 (n = 2) 5. Some isolates classified as Karmali SPT-C-E or modified SPT-D and E contained relatively high numbers of virulence genes: O84:H- , O5:H- , O165: H- , O6:H25 ; O80: H- , O55:H7  and O177: H- .stx2a, stx2c and stx2f showed higher number of virulence genes compared to isolate with the other stx-subtypes present (stx2a and stx2f were significantly (Chi-square P<0.05) associated with eae  positive associations with : adfO, astA, cfk, eae, nleB2, nleD, and nleF. Negative associations were observed with ehxA, iha, nleG21, stx1, subA and terB  of the STEC isolates included in this study was characterized as phylogenetic group (phylotype) B1, followed by A (20.1%), D (9.1%) and B2 (7.7%). The distribution of phylotypes was not significantly different among animal, meat and human isolates . However, a trend was observed with relatively more A and B1 isolates among non-human isolates (90.0%) compared to human isolates (79.1%). In contrast, relatively more B2 and D isolate were observed among human isolates (20.9%) compared to non-human isolates (10.0%). A significant association between phylogenetic group and HAS was observed , with no HAS among phylogenetic group A (n = 42) and B2 (n = 16). In contrast, 85.2% of the HAS belonged to phylogenetic group B1, and 14.8% to phylogenetic group D.eae gene was more likely to be associated with B2 and D isolates compared to A and B1 isolates. Shiga toxin subtype 2f showed a significant association with phylogenetic group B2. Isolates with stx2e and stx2g were significantly associated with phylogenetic group A. Similarly, stx2f, adfO, and nleB2 were more likely to be associated with B2 and D isolates. In contrast, stx2, iha, and ehxA were more likely to be associated with A and B1 isolates.No difference was observed in the number of virulence genes present in isolates of different phylogenetic groups (P = 0.515). However, some genes differed significantly in frequency between different phylogenetic groups . The eaenle-genes.The total number of markers present decreased progressively from the Karmali SPT-B to SPT-E . SPT-B seae, stx2f and ckf. Highly significant (P<0.01) associations with isolates of non-human origin were observed for stx2dact and iha. Other targets occurring in marginally significantly (0.01<P<0.05) higher frequency among isolates of human origin compared to isolates of non-human origin included ent/espL2, nleA, nleG9, efa1, adfO, and nleH1–2  and human isolates (mean 7.0) (P = 0.142). Irrespective of serogroup and seropathotype, some genetic targets were found at a significantly different frequency among isolates of human and non-human origin . Highly  nleH1–2 .nleG5–2, efa1, ent/espL2, ehxA, toxB, adfO, nleG2–3, nleE, cfk, ureC, nleA, nleG, nleB, eae, stx2a, and terB .In total, 20/40 virulence factors showed a significant higher association with HAS  compared to non-HAS  . Highly and terB . Marginaeae-positive  compared to eae-negative isolates  (P<0.001). All eae-negative strains lacked all 15 tested nle-genes, whereas the eae-positive strains on average showed 7.7 nle-genes (median 8.5). The virulence factors stx2a, stx2f, ureC, terB, toxB, etpD, adfO, and cfk showed a significant stronger association with eae-positive strains compared to eae-negative strains : O76:H19 (n = 6), O128:H2 (n = 4) and O174:H21 (n = 4). The virulence factors iha_homologue, saa, stx2b, and subA showed a significant higher frequency among eae-negative strains. When comparing eae-negative non-human with clinical human STEC isolates, the factors stx2dact (OR 4.9) and iha (OR 1.9) occurred in a significant higher frequency among eae-negative strains.A significant higher number of virulence markers was observed among  strains . SeveralrpoS gene revealed that 7/80 (8.8%) non-human isolates and 31/129 (24.0%) of the clinical isolates were characterized by mutations, including deletions, insertions and single nucleotide polymorphisms in the open reading frame (ORF). Surprisingly, the mutation found in the animal/meat isolates was nearly the same for all, i.e. A967G (N323D) (6/7). This mutation was also found in one human isolate. The majority of the rpoS mutated strains were phylogroup B1 (60.5%), followed by A (28.9%), D (10.5%) and B2 (none). With respect to the distribution of rpoS genotypes over seropathotypes, only one (1/15 = 6.7%) SPT-B isolate (O26:H11) was found with a mutated rpoS. Mutations were identified in 18/69 (26.1%) SPT-C isolates, 12/94 (12.8%) SPT-D isolates, and 6/31 (19.4%) SPT-E isolates. No relation was observed with the virulence profile.Sequencing the The complexity regarding STEC in food safety enforcement as well as clinical care primarily relates to the current inability of designating individual strains as pathogens due to the large variety in serotype and genetic content associated with (severe) disease. Subsequently, pathogenicity can neither be excluded nor confirmed for a given STEC isolate based on the seropathotype concept or analysis of the public health surveillance data . To claset al. [eae-negative (and therefore “non-virulent”) could not be confirmed in the present study: i.e. 23.8% of the A isolates were eae-positive compared to only 12.9% of the B1 isolates. However, only phylogenetic group B1 and D contained HUS-associated strains (HAS). Possibly, these associations differ with respect to isolation sources and geographical regions. In contrast to intimin, the typical STEC virulence factors stx2 and ehxA were significantly associated with A and B1 isolates.Consistent with previous studies ,17, phylet al.  that STEnle-genes from SPT-A to SPT-E strains [nle-genes as compared to the modified EFSA classification [Earlier studies showed a clear progressive decline in the number of  strains ,22. In tfication  and the eae and other virulence markers, the disease mechanism employed by LEE-positive strains seems (unlike LEE-negative trains) strongly related to other virulence factors like terB, toxB, etpD, adfO, ckf, efa1 (in random order) and almost all nle-factors. Primarily the isolates belonging to the EU top-5 serogroups possess this array of correlated virulence genes.STEC containing the LEE-island are characterized by their ability to express the attaching and effacing (A/E) phenotype, leading to substantial cytoskeletal rearrangements within the enterocyte . Given teae-negative . With the present study, the percentage of eae-negative human isolates was almost twice as high (80.9%). It has been postulated that in the absence of LEE-island mechanisms are emerging by which LEE-negative STEC interact with the host mucosa and induce disease [saa), the iron-regulated gene homolog adhesion (iha) and the subtilase cytotoxin (subAB) have been reported as alternative adhesins [Although the LEE-island is considered a hallmark virulence factor for STEC pathogenesis, it appears not to be essential since sporadic cases and small outbreaks (including HC and HUS) have been caused by LEE-negative STEC ,24. Althadhesins –27. In tstx2a was positively associated with an array of additional virulence factors (incl. eae) while stx2b, stx2d, stx2e and stx2g showed very little positive association with additional (known) virulence factors. Recently, the emergence of stx2f-STEC in the Netherlands was described, which are generally associated with more mild disease [ehxA and terB, both showing a significant association with HAS in the current study and in general with EHEC/HUS [Specific sets of virulence factors were also associated with different Stx-subtypes. Especially  disease . This mi disease , but alsEHEC/HUS ,31. ThesEHEC/HUS .adfO, cfk, eae, efa1, nle-genes, stx2a, terB, toxB, and ureC; in alphabetic order). This coincides with the observation that most disease caused by these serogroups is relatively mild [In the Netherlands, the EU top-5 serogroups  represent approximately half of all clinical STEC isolates . The othely mild .et al. [eae-positive STEC strains. This is also supported by recent comparative genomics which revealed the absence of all known phage-encoded non-LEE effector genes eae-negative STEC [et al. [eae, ent/espL2, nleB, nleE, and nleH1–2 is a strong signature of a human-pathogenic EHEC. A Belgian study presented the combined presence of efa, nleE and stx2 as a high-risk virulence profile [et al. [eae, nle and subA genes as a high risk profile. However, all these markers were strongly correlated in the present study with eae, questioning the added value of using the nle-genes as an additional markers. Consequently, all eae-negative virulent STEC strains, including HAS [et al. [eae-negative STEC. The factors ehxA and ureC were, independently from eae, associated with HAS. This indicates their candidacy as important pathogenicity markers next to eae and stx2a. The ureC was earlier identified as a suitable marker for pathogenicity [E. coli O157:H7 in ligated pig intestine [ureC was strongly correlated to intimin [ehxA) is also is known for its association with HUS [In line with the results described here, Ju et al.  demonstrive STEC . Several [et al.  conclude profile . Bosilev [et al.  reportedding HAS  would be [et al.  that addgenicity . Mutationtestine  and strantestine . However intimin ,37. Probwith HUS ,31.E. coli induce the rpoS-controlled general stress response [rpoS variants (i.e. deviating from E. coli O157:H7 Sakai strain as wild-type (WT) reference) was three times higher among human clinical isolates compared to animal and food isolates. A similar skewed distribution of WT and variants was demonstrated for STEC O157 isolates [et al. [rpoS variants is strengthened by the current results on non-O157. There is evidence that a WT functional rpoS is advantageous for bovine colonization [rpoS negatively regulates the expression of the LEE-island [rpoS variants as these are characterized by increased nutrient scavenging abilities at the expense of stress-resistance [E. coli could be avoided by the establishment of an STEC specific metabolic niche [Stationary-phase and almost any environmental stress that slows the growth rate of response . In thisnization ,42. In cE-island  and negaE-island . Non-bovsistance . Hereby,ic niche .ehxA, iha and stx2, but absence of eae. Especially eae was strongly correlated to many of the other virulence genes, including all non-LEE encoded effectors. Different stx-subtypes were associated with different virulence profiles. The factors ehxA and ureC were independently from eae associated with HUS-associated strains (HAS). This indicates their candidacy as important pathogenicity markers next to eae and stx2a. However, since some serogroups are only represented by a limited number of isolates definitive conclusions on association of virulence factors with these groups require a more specific strain set.The large variability in the number of virulence factors present among serogroups and seropathotypes indicated that this was merely indicative for the virulence potential. While all the virulence gene associations have been compared, it appeared that there is no specific pattern that would unambiguously enable hazard identification for an STEC strain. However, the strong correlations between virulence factors indicate that these arrays are not a random collection but are rather specific sets. Most non-O157 STEC in the Netherlands are phylogroup B1 and characterized by the presence of S1 Table(XLSX)Click here for additional data file.


Raptors may contract highly pathogenic avian influenza virus H5N1 by hunting or scavenging infected prey. However, natural H5N1 infection in raptors is rarely reported. Therefore, we tested raptors found dead during an H5N1 outbreak in wild waterbirds in Mecklenburg-Western Pomerania, Germany, in 2006 for H5N1-associated disease. We tested 624 raptors of nine species—common buzzard (385), Eurasian sparrowhawk (111), common kestrel (38), undetermined species of buzzard (36), white-tailed sea eagle (19), undetermined species of raptor (12), northern goshawk (10), peregrine falcon (6), red kite (3), rough-legged buzzard (3), and western marsh-harrier (1)—for H5N1 infection in tracheal or combined tracheal/cloacal swabs of all birds, and on major tissues of all white-tailed sea eagles. H5N1 infection was detected in two species: common buzzard  and peregrine falcon . In all necropsied birds , H5N1 was found most consistently and at the highest concentration in the brain, and the main H5N1-associated lesion was marked non-suppurative encephalitis. Other H5N1-associated lesions occurred in air sac, lung, oviduct, heart, pancreas, coelomic ganglion, and adrenal gland. Our results show that the main cause of death in H5N1-positive raptors was encephalitis. Our results imply that H5N1 outbreaks in wild waterbirds are more likely to lead to exposure to and mortality from H5N1 in raptors that hunt or scavenge medium-sized birds, such as common buzzards and peregrine falcons, than in raptors that hunt small birds and do not scavenge, such as Eurasian sparrowhawks and common kestrels. Anser indicus), great black-headed gulls (Larus ichthyaetus), and brown-headed gulls  [Cygnus cygnus) and mute swans (Cygnus olor), Canada geese (Branta canadensis), tufted ducks (Aythya fuligula), greater black-backed gulls (Larus marinus), herring gulls (Larus argentatus), but also in raptors such as northern goshawks (Accipiter gentilis) and common buzzards (Buteo buteo) [Highly pathogenic avian influenza virus causes high mortality in poultry, but is rarely reported to cause clinical disease in wild waterbirds. However, since 2000 there have been several reported outbreaks of highly pathogenic avian influenza virus of the subtype H5N1 causing mortality in wild waterbirds. A major outbreak occurred in 2005 in western China, causing mortality of more than thousand birds, including bar-headed geese (o buteo) ,4. By hio buteo) . Additioo buteo) .Spizaetus nipalensis) [Falco cherrug) [Since several raptor species selectively hunt moribund or otherwise conspicuous prey, including waterbirds, or scavenge on their carcasses, they may be assumed to have a high rate of exposure to H5N1 during outbreaks of this virus. However, there are only rare case reports of natural H5N1 infection in raptors: common buzzard ,5,6, moualensis) ,8, and scherrug) , with liOf the total of 9 356 dead wild birds from Mecklenburg-Western Pomerania examined during the 2006 H5N1 outbreak, 624 were raptors  and other raptor species. For white-tailed sea eagles, all 19 individuals underwent necropsy, regardless whether their swabs tested positive or negative for H5N1, and tissue samples of lung, liver, spleen, kidney, pancreas and brain were collected for virology and stored at −70 °C until analysis. Duplicate samples of the same tissues were collected for histopathology and immunohistochemistry and fixed in 10% neutral-buffered formalin until analysis. For other raptor species, only individuals with H5N1-positive swabs and either freshly dead or intact and expected to be in a mild state of autolysis underwent necropsy: six common buzzards (B1 to B6) and two peregrine falcons  (F1 and F2). During this necropsy, tissue samples of nasal turbinate, air sac, trachea, bronchus, lung, heart, liver, spleen, kidney, adrenal gland, pancreas, stomach, jejunum, colon, cerebrum, cerebellum, reproductive tract and cloacal bursa  were collected for virology and stored at −70 °C until analysis. Duplicate samples of the same tissues were collected for histopathology and immunohistochemistry and fixed in 10% neutral-buffered formalin until analysis. In addition, separate pharyngeal and cloacal swabs for virology were placed in Hank’s balanced salt solution containing 0.5% lactalbumin, 10% glycerol, 200 U/mL penicillin, 200 μg/mL streptomycin, 100 U/mL polymyxin B sulfate, 250 μg/mL gentamycin, and 50 U/mL nystatin , and stored at −70 °C until analysis.Both selection of carcasses for necropsy and level of sampling of necropsied carcasses differed between white-tailed sea eagles  for histopathological examination by light microscopy. For immunohistochemical detection of influenza virus antigen, duplicate sections of the same tissue samples were stained with a primary antibody against the influenza A nucleoprotein as described previously . PeroxidOut of 624 individual raptors belonging to 9 different species, H5N1 was detected by RT-PCR in the swabs of only 2 species: common buzzard 12 of 385) and peregrine falcon (2 of 6)  in the abdominal air sac (F1 and F2), and multifocal hemorrhage in thorax or upper respiratory tract, suggestive of trauma (F1 and F2). Freeze-thaw artifacts and autolytic changes often hampered gross examination.The nutritional state of the white-tailed sea eagles ranged from poor to good. All common buzzards and peregrine falcons were in poor nutritional state, lacking fat in the subcutis or body cavity, and showing mild to advanced atrophy of the breast muscles. Externally, no lesions were found. Internally, there were lesions in several organs: multifocal hemorrhage in the pancreas (B2 and B3); increased redness in the cerebrum ; enlargement of the spleen  and kidneys ; wet lungs ; many nematodes (most likely Serratospiculum sp. infection). Freeze-thaw artifacts and mild autolytic changes were present in most tissues, but did not prevent histopathological assessment.By histopathology, lesions were seen in the brain, air sac, oviduct, coelomic ganglion, heart, pancreas and adrenal gland of common buzzards and peregrine falcons Figure . In the By immunohistochemistry, influenza virus antigen expression was visible as diffuse red staining mainly in the cytoplasm and occasionally in the nucleus of affected cells. Virus antigen expression was found in multiple tissues Figure  and was Our study shows that, during the 2006 H5N1 outbreak in wild waterbirds in Mecklenburg- Western Pomerania, Germany, both common buzzards and peregrine falcons were exposed to and suffered fatal disease from H5N1 infection, while seven other raptor species examined showed no evidence of H5N1 infection. In both species, H5N1 showed a distinct neurotropism associated with marked encephalitis, comparable to that observed in most other susceptible wild bird species.Aythya fuligula) and Eurasian pochards (A. ferina) showed clinical signs of disease, while the remaining four species, all dabbling ducks belonging to the genus Anas, were clinically unaffected [The differences in diet and hunting or scavenging behavior among the nine raptor species was likely an important factor in their exposure to H5N1 in the 2006 outbreak in Germany. This outbreak affected mainly swans, geese, gulls, and diving ducks, all medium-sized birds  and the affected .Falco sparverius) [The tissue tropism of H5N1 and the character of the associated lesions in these common buzzards and peregrine falcons correspond with those seen previously in other avian species  . The detrverius) ,23. The rverius) . In contThe discrepancy between the results in the immunohistochemistry, RT-PCR and virus titration can most likely be attributed to the extended storage time in the −20 °C freezer and to the difference in sensitivity of the tests. Also, autolysis before storage and freezing artifacts hampered the necropsy and data analyses.Although it is likely that these common buzzards and peregrine falcons became infected with H5N1 because of feeding on H5N1-contaminated carcasses, as has been shown experimentally in gulls , it is nIn conclusion, our study implies that the position of raptors at the top of the food pyramid puts them at risk of acquiring infectious pathogens such as H5N1 by eating sick or dead prey. Because of their broad geographic range and migratory nature, they may contribute to long-distance movement of such pathogens; however, this contribution is likely much smaller than that of more numerous species of migratory birds. Our data suggest that some raptor species, such as common buzzards and peregrine falcons, are more likely to eat sick or dead wild waterbirds and subsequently are more exposed to H5N1 infection than other species, such as Eurasian sparrowhawks and common kestrels. Whether there is species-specific resistance to disease from H5N1 infection in some raptor species, such as white-tailed sea eagles, remains to be determined.