Reprint requests: Thomas A. E. Platts-Mills, MD, PhD, Allergy Division, University of Virginia Health System, PO Box 801355, Charlottesville, VA 22908.
In 2009, we reported a novel form of delayed anaphylaxis to red meat that is related to serum IgE antibodies to the oligosaccharide galactose-α-1,3-galactose (alpha-gal). Most of these patients had tolerated meat for many years previously. The implication is that some exposure in adult life had stimulated the production of these IgE antibodies.
Objectives
We sought to investigate possible causes of this IgE antibody response, focusing on evidence related to tick bites, which are common in the region where these reactions occur.
Methods
Serum assays were carried out with biotinylated proteins and extracts bound to a streptavidin ImmunoCAP.
Results
Prospective studies on IgE antibodies in 3 subjects after tick bites showed an increase in levels of IgE to alpha-gal of 20-fold or greater. Other evidence included (1) a strong correlation between histories of tick bites and levels of IgE to alpha-gal (χ2 = 26.8, P < .001), (2) evidence that these IgE antibodies are common in areas where the tick Amblyomma americanum is common, and (3) a significant correlation between IgE antibodies to alpha-gal and IgE antibodies to proteins derived from A americanum (rs = 0.75, P < .001).
Conclusion
The results presented here provide evidence that tick bites are a cause, possibly the only cause, of IgE specific for alpha-gal in this area of the United States. Both the number of subjects becoming sensitized and the titer of IgE antibodies to alpha-gal are striking. Here we report the first example of a response to an ectoparasite giving rise to an important form of food allergy.
Shortly thereafter, it became clear that a significant number of patients were experiencing severe hypersensitivity reactions during their first infusion of this mAb. Surprisingly, those reports appeared to be restricted to an area of the Southeast, including Tennessee, North Carolina, Arkansas, Virginia, and the southern half of Missouri.
In 2007, a study was published by the oncology groups at the University of North Carolina and Vanderbilt, suggesting that the prevalence of severe reactions to cetuximab was as high as 20%.
At that time, a detailed investigation of serum antibodies established that these reactions were occurring in patients who had preexisting IgE antibodies specific for the glycosylation on the Fab fragment of the mAb.
This analysis was, in part, made possible because Dr Zhou and his colleagues, working at ImClone (New York, NY), had published the full glycosylation of cetuximab.
Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion.
After establishing the assay for IgE antibodies to cetuximab (ie, alpha-gal), we screened large numbers of sera. The results established that these IgE antibodies were regionally distributed in a pattern consistent with the cases of cetuximab-related anaphylaxis. More importantly, the results led to the realization that IgE to alpha-gal was also associated with a novel form of food allergy.
Those patients reported that they had generalized urticaria or frank anaphylaxis starting 3 to 6 hours after eating beef, pork, or lamb; however, they reported tolerating chicken, turkey, or fish without difficulty.
Thus, the specificity of IgE antibodies present in their serum, which was known to be specific for a carbohydrate common to mammals but absent from poultry and fish, matched their symptoms.
In most cases, these patients were adults who had consumed red meat for many years before having the delayed reactions. This history implies that some new exposure had triggered the production of IgE antibodies to alpha-gal.
Initially, we investigated the possible role of a regionally important inhalant or fungal allergen, but those results were uniformly negative. The next possibility was that a regional helminth could have induced IgE antibodies to the oligosaccharide. However, both from studying the known distribution of helminths in the United States and from sharing sera with Dr Amy Klion at the National Institutes of Health, we excluded Ascaris species and found little evidence for any other helminth.
By contrast, the known distribution of the immediate reactions to cetuximab was similar to the areas with high prevalence of Rocky Mountain spotted fever.
We were also aware of patients who thought that their reactions to red meat started after receiving multiple tick bites. Finally, we were informed that Dr van Nunen in Sydney, Australia, and Dr Deutsch in Georgia (personal communication, 2010) had reported to their local allergy meetings about patients who had become allergic to meat after experiencing multiple tick bites.
We report here the evidence that tick bites in the United States can induce IgE antibodies to alpha-gal. The evidence comes from (1) prospective studies of the response to tick bites in 3 subjects, (2) epidemiologic evidence that these IgE antibodies are present in areas where tick bites are common, (3) correlation between IgE antibodies to tick proteins and IgE antibodies to alpha-gal, and (4) evidence for an expanding range of the lone star tick, Amblyomma americanum.
Methods
Patients, control subjects, and random populations
Clinic populations in Virginia
Patients presenting to clinic in Charlottesville for evaluation of recurrent anaphylaxis or severe urticarial reactions (n = 121), asthma (n = 56) or control subjects (n = 40) were enrolled and provided serum. Of these, the most recently enrolled 125 subjects responded to a full questionnaire, including questions about tick bites (see Fig E1 in this article’s Online Repository at www.jacionline.org). Patients who specifically presented with urticarial or anaphylactic reactions were enrolled as subjects and had their serum tested for IgE antibodies to alpha-gal. Other patients were enrolled sequentially upon their agreeing to the informed consent process.
Random populations in the southeastern United States where tick bites are common
The Tennessee control cohort was obtained as a random control population in a cancer-screening clinic at Vanderbilt University.
The North Carolina cohort was collected randomly from hospital patients at the University of North Carolina as part of a study on the genetics of this anaphylactic response (unpublished data). The Virginia patients included 70 with acute asthma and 66 control subjects presenting to the emergency department at the University of Virginia.
As part of a prospective study on asthma among children in the Norrbotten area of northern Sweden, sera were obtained from 963 subjects at age 18 years.
From these sera, all those with positive assay results for IgE antibodies to cat epithelium and dander of class 2 or higher (n = 150) were assayed for IgE to alpha-gal. Northern California was included because reactions to cetuximab are rare, and although some ticks are present there, A americanum is not.
and (3) patients with asthma (n = 102) and control subjects (n = 41) enrolled in studies on asthma at the University of California San Francisco.
Populations from Kenya and the Esmeraldas Province of Ecuador (ie, tropical areas where tick bites are common)
In a previous study in Kenya, we reported that IgE antibodies to cat were common among schoolchildren in the village of Kabati (n = 131) and less common in the small town of Thika (n = 123). In the village, helminth infections, tick bites, and other ectoparasites are extremely common and less so in the town.
Sera from children in the Esmeraldas Province of northern Ecuador (n = 295) were collected as part of ongoing studies on the relevance of helminth infections to asthma and allergic disease.
Approval for these studies was obtained locally in the area where subjects were enrolled and from the University of Virginia Human Investigation Committee. Further details regarding the individual cohorts can be found in Appendix E1 (available in this article’s Online Repository at www.jacionline.org) and in the indicated references.
Allergen source and preparation
Adult, pathogen-free A americanum and Dermacentor variabilis ticks were purchased from the Oklahoma State Tick Rearing Facility (Stillwater, Okla) and were maintained at 4°C until preparation of whole-body extracts. The ticks were frozen with liquid nitrogen and crushed with a mortar and pestle. Proteins from the resultant powder were extracted overnight in borate-buffered saline, pH 8.0, with added protease inhibitors (Sigma Aldrich, St Louis, Mo). After removal of the solid pellet, the protein solution was defatted with ethyl ether (Sigma Aldrich), and after centrifugation at 3000 rpm for 5 minutes, the sample was separated into 3 layers, the bottom of which (the aqueous layer) was collected for analysis.
Total and specific IgE antibodies were measured by using either commercially available ImmunoCAP (Phadia US, Portage, Mich) or a modification of the assay with streptavidin on the solid phase.
The assays were performed with the ImmunoCAP 250 instrument, and the results were expressed as international units per milliliter, with the international unit both for specific and total IgE being approximately 2.4 ng. For specific assays, the standard cutoff point for a positive reaction was 0.35 IU/mL. The streptavidin CAP technique was used to measure IgE antibodies to alpha-gal and tick proteins (A americanum and D variabilis), with approximately 5 μg of biotinylated antigen added to each CAP before adding 40 μL of undiluted serum. This assay is now available commercially. Selected sera were tested with commercially available assays for IgE antibodies to dust mite (Dermatophagoides pteronyssinus; d1), dog epithelium (e2), ryegrass pollen (g5), beef (f27), Trichophyton rubrum (m205), common silver birch pollen (t3), and German cockroach (i6).
Statistical analyses
We compared quantitative measures of IgE antibodies by using Spearman rank correlation and compared qualitative measures of tick bite severity and the presence of IgE antibodies by using the χ2 test for trend. A 2-sided P value of less than .05 was considered to indicate statistical significance. Statistical analyses were performed with SPSS software, version 18.0 (SPSS, Inc, Chicago, Ill), and GraphPad Prism, version 4 (GraphPad Software, Inc, La Jolla, Calif).
Results
Rapid development of IgE antibodies to α-gal after tick bites in 3 subjects
Over the last 3 years, we have had the opportunity to follow serum IgE responses prospectively in 3 subjects whose sera were available from before they experienced multiple tick bites. In each case IgE antibody levels to alpha-gal increased more than 20-fold after tick bites, and there was a parallel, although not identical, increase in total IgE levels (Fig 1). Furthermore, 2 of these subjects (nos. 1 and 3 in Fig 1 and Table I) experienced an episode of generalized urticaria 3 to 4 hours after eating red meat, something that had not occurred before the tick bites. In these 2 cases the IgE antibodies to alpha-gal represented 30% or more of the total IgE. By contrast, in subject 2 IgE levels to α-gal increased from less than 0.35 to 8 IU/mL, but this accounted for less than 1% of the total IgE, and this subject has yet to report any allergic symptoms after eating mammalian meat. IgE antibodies to several other allergens were measured in serial samples from each of these subjects (Table I). The results show that none of the 3 subjects had new specificities of IgE antibodies, other than those that could be explained by IgE antibodies to alpha-gal (eg, cat, dog, beef, and milk). Although IgE antibodies to inhalants in sera from subject 2 did increase, these increases were much less than the increase in IgE levels to alpha-gal. In cases 1 and 2 the ticks responsible for the bites were identified as A americanum.
Fig 1Time course of IgE antibodies to alpha-gal and total IgE levels collected as sequential samples after episodes of multiple tick bites (red arrows) in 3 subjects. The time intervals between samples vary considerably, and the specific times can be seen in Table I. Subjects 1 and 3 experienced episodes of generalized urticaria starting 3 to 4 hours after eating mutton and beef, respectively (indicated by red asterisk). In each case this was the first such episode.
∗ Values for serum IgE antibodies and total IgE after episodes of multiple tick bites. Values are shown for 5 allergens unrelated to alpha-gal: dust mite, cockroach, ryegrass, silver birch, and the fungus T rubrum. Values for IgE antibodies that are related to the oligosaccharide alpha-gal are shown in shaded areas.
‡ The timing of episodes of multiple tick bites are shown as vertical black lines. In each case the bites occurred within 10 days of the subsequent blood draw.
§ Subjects 1 and 3 experienced generalized hives 3 to 4 hours after eating red meat. In each case this was their first such episode and occurred when the IgE antibody titer to alpha-gal was high (subject 1: November 20, 2007; subject 3: October 10, 2010).
§ Subjects 1 and 3 experienced generalized hives 3 to 4 hours after eating red meat. In each case this was their first such episode and occurred when the IgE antibody titer to alpha-gal was high (subject 1: November 20, 2007; subject 3: October 10, 2010).
June 1996
June 1999
August 2008
August 2009
March 2010
September 2010
October 2010
November 2010
Dust mite
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
Cockroach
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
Rye grass
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
Silver birch
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
Trichophyton species
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
Dog epithelium
0.35
0.35
3.36
0.78
0.35
0.67
9.3
4.66
Beef
0.35
0.35
4.15
1.00
0.35
0.85
12.0
5.9
Alpha-gal
0.35
0.35
7.29
1.24
0.35
1.18
16.2
11
Total IgE
7.01
10.1
17.1
6.95
3.25
9.05
34.9
20.5
∗ Values for serum IgE antibodies and total IgE after episodes of multiple tick bites. Values are shown for 5 allergens unrelated to alpha-gal: dust mite, cockroach, ryegrass, silver birch, and the fungus T rubrum. Values for IgE antibodies that are related to the oligosaccharide alpha-gal are shown in shaded areas.
† Subject 2 was allergic to mite, grass, and birch before tick bites.
‡ The timing of episodes of multiple tick bites are shown as vertical black lines. In each case the bites occurred within 10 days of the subsequent blood draw.
§ Subjects 1 and 3 experienced generalized hives 3 to 4 hours after eating red meat. In each case this was their first such episode and occurred when the IgE antibody titer to alpha-gal was high (subject 1: November 20, 2007; subject 3: October 10, 2010).
Relationship between histories of tick bites and serum IgE antibody levels to alpha-gal on a local and regional level
From questionnaire results, it was clear that a large proportion (>90%) of the subjects with serum IgE antibodies to alpha-gal had a history of tick bites. However, tick bites are now common in central Virginia. Among 125 subjects, including patients with delayed anaphylaxis, as well as patients with asthma and control subjects, a positive response to questions, including “Have you ever had local reactions to tick bites last for weeks or longer?” correlated highly significantly with the presence of IgE antibodies to alpha-gal (P < .001, Fig 2). We have now assayed sera or received results on more than 300 subjects who presented with anaphylaxis or urticaria and about whom we knew where they lived at the time of the first reaction. Although this is by no means a random sample, the results are interesting. Plotting positive cases on a map of the United States showed a striking similarity to the known distribution of A americanum.
Fig 2Prevalence of IgE antibodies to alpha-gal among 125 patients with or without histories of tick bites. Histories of bites were analyzed by length of time that reactions persisted at the site. χ2 Analysis on 3 bite categories versus assays for IgE antibodies of greater than 0.35 IU/mL was performed.
Although ticks are ubiquitous in temperate and tropical regions, there are major regional differences in species and also regions or populations where tick bites are rare or absent (Table II).
These include large cities and areas where the climate prevents survival of ticks or their natural hosts (eg, deer and small mammals). In keeping with the absence of ticks in the inland areas of northern Sweden, assays of sera from Norrbotten were negative for IgE antibodies to alpha-gal (Table II).
In contrast, we have assayed sera from Africa and Ecuador, which show a significant number of positive sera among rural populations. The positive results from Africa and Ecuador can only be interpreted as providing evidence that ticks could be the cause of these IgE antibodies because other ectoparasites or helminths could be relevant (Table II). Of note, the generally negative data from Norrbotten, Boston, and northern California argue strongly that eating food containing this oligosaccharide (eg, beef or milk) does not induce sensitization to alpha-gal.
Table IIPrevalence of IgE antibodies to alpha-gal in different populations related to local prevalence of tick bites
∗ Values are the number of sera at greater than 0.35 IU/mL, and the values in parentheses are the number of sera at greater than 3.5 IU/mL. Also shown is the percentage of positive sera.
Percentage
Virginia clinic populations
Patients presenting with delayed anaphylaxis to red meat
† Patients presenting to the clinic with a history of recurrent anaphylaxis, angioedema, or generalized urticaria occurring 3 to 5 hours after eating mammalian meat.
‖ Tick bites of several species are known to be common in each of these areas, but there are also multiple helminths and other ectoparasites that could be relevant.
∗ Values are the number of sera at greater than 0.35 IU/mL, and the values in parentheses are the number of sera at greater than 3.5 IU/mL. Also shown is the percentage of positive sera.
† Patients presenting to the clinic with a history of recurrent anaphylaxis, angioedema, or generalized urticaria occurring 3 to 5 hours after eating mammalian meat.
‡ Patients evaluated in the University of Virginia Allergic Disease clinic for asthma, including 30 with severe or moderately severe asthma.
§ Three studies in which enrollment was unrelated to histories that could be related to IgE antibody responses to alpha-gal.
‖ Tick bites of several species are known to be common in each of these areas, but there are also multiple helminths and other ectoparasites that could be relevant.
Studies from both Europe and Australia have reported the presence of IgE antibodies to tick salivary proteins in sera from patients who experienced anaphylactic reactions to tick bites.
Using whole-body extracts of A americanum or D variabilis, we assayed sera with or without IgE antibodies to alpha-gal for IgE antibodies specific for tick proteins. The results show a strong correlation between IgE antibodies to A americanum and IgE antibodies to alpha-gal (Fig 3). The titers of IgE antibodies to tick proteins were lower than the IgE antibodies to alpha-gal. When sera were absorbed using a solid phase coated with alpha-gal, half of the sera retained more than 80% of the IgE to ticks (see Table E1 in this article’s Online Repository at www.jacionline.org). Four of the 12 sera had over 80% of the IgE that bound the tick extract removed by absorption with alpha-gal–coated beads. Performing the same experiments with blank sepharose beads or as a mock experiment caused less than a 1% change in alpha-gal or A americanum IgE binding (data not shown). The results for D variabilis did not show a significant relationship with IgE antibodies to alpha-gal (see Fig E2 in this article’s Online Repository at www.jacionline.org). Thus, our evidence suggests that bites from A americanum ticks can induce both IgE antibodies to tick proteins and IgE antibodies to alpha-gal. The sera from the inland town of Kiruna in the Norrbotten province of Sweden were uniformly negative for IgE antibodies to ticks.
Fig 3Correlation between IgE antibodies to alpha-gal and IgE antibodies to proteins derived from the tick A americanum in 125 sera obtained from patients living in Virginia.
The results presented here strongly suggest that tick bites are a cause, if not the only significant cause, of IgE antibody responses to alpha-gal in the southeastern United States. This evidence includes following the response prospectively in 3 cases, a strong correlation with histories of tick bites, epidemiologic evidence that these antibodies are not found in regions where tick bites are rare, and correlation with IgE antibodies specific for tick proteins. As recently as January 2006, we were totally unaware of this phenomenon. Our present results in Virginia, North Carolina, and Tennessee suggest that as many as 20% of the population in these states have serum IgE antibodies to alpha-gal. In 2009-2010, delayed anaphylaxis to red meat is a common reason for presentation to the clinics of this predominantly rural or suburban area of central Virginia. Nevertheless, we do not mean to suggest that 15% of the population in these states has an allergy to mammalian meat. We are aware of many subjects with IgE to alpha-gal (and beef) who do not have reactions after eating red meat. In addition to the high prevalence, the titers of IgE antibodies to alpha-gal can be very high. In many cases, patients have greater than 100 IU/mL IgE antibodies to alpha-gal, often in conjunction with high total IgE levels. Indeed, our results suggest that tick bites can be an important cause of high total serum IgE levels. That the saliva of ticks is a potent immunogen is well known
Amblyomma americanum: Physiochemical isolation of a protein derived from the tick salivary gland that is capable of inducing immune resistance in guinea pigs.
; however, it has not previously been reported that ticks could induce IgE antibodies to a cross-reactive oligosaccharide.
Our original observation was that the distribution of anaphylactic reactions to cetuximab (Fig 4, A) was similar to the maximum prevalence of Rocky Mountain spotted fever (Fig 4, B). The major vectors of Rocky Mountain spotted fever in this region are the ticks D variabilis and A americanum, and the geographic range of A americanum has been expanding over the last 30 years, probably in parallel with the massive increase in the deer population (Fig 4, C and D).
Many persons are not aware that in 1950 deer had been hunted close to extinction in the southeastern United States. Indeed, at about that time, the Virginia state game commission “reintroduced” deer to sections of the Blue Ridge Mountains. Over the last 20 years, the deer population has expanded to become a major pest in suburban areas. This further increase might have been made possible by the introduction of leash laws for dogs. Our focus has been on A americanum because (1) 2 of our cases involved this species, (2) the cases match the known distribution of this tick (Fig 4, Fig 3 this species has a well-deserved reputation for being “aggressive.”
Both adult and larval forms of this tick are very willing to bite humans, as well as other mammals. The larval ticks are about 1 mm in diameter and are generally not seen or appear as small black dots. This creates a confusion that is important in taking histories. The term used for larval ticks is “seed ticks”; however, the term “chiggers” is better known, and it is not easy to identify either seed ticks or chiggers correctly. Chiggers are correctly the larval forms of trombiculid mites, which can also bite humans but are less common than seed ticks in the region in question.
Fig 4A and B, Comparison of the states where anaphylactic reactions to cetuximab were reported in 2006 (Fig 4, A) and the reported prevalence of Rocky Mountain spotted fever (Fig 4, B). C, Distribution of known cases of patients with delayed anaphylaxis to red meat whose serum contained IgE antibodies to alpha-gal. Markers indicate the number of cases: dots indicate single cases, smaller black stars indicate 5 to 24 cases within a state, and larger black stars indicate states with 25 or more cases. Data in Fig 4, A, are from Bristol-Myers Squibb, and data in Fig 4, B, are from the Centers for Disease Control and Prevention Web site. Numbers of cases in states with stars: Arkansas, 65; Georgia, 14; Maryland, 8; Mississippi, 5; Missouri, 30; North Carolina, 35; Oklahoma, 25; Tennessee, 40; and Virginia, greater than 200. D, Known distribution of the tick A americanum (data from the Centers for Disease Control and Prevention Web site).
Our evidence about the regional distribution of these reactions does not favor a role for Ixodes scapularis. Indeed, the Lyme disease literature has very few mentions of multiple bites or severe pruritic reactions to bites. This might well be because persons who consistently report itching in association with tick bites are less likely to experience an episode of Lyme disease than those who do not react against tick bites of I scapularis.
In the same study from Block Island, where Lyme disease is highly endemic, it was found that the frequency of itch increased as the number of reported tick bites increased, suggesting that tick-related itch was associated with an acquired cutaneous hypersensitivity response.
Of note, the larval forms of the ticks that carry Borrelia burgdorferi are not known to bite humans. In other areas of the world, different ticks have been associated with allergic reactions in humans. In Australia, bites of Ixodes holocyclus can precede subsequent allergic reactions to beef.
In Europe the pigeon tick, Argas reflexus, is known to cause sensitization and anaphylactic reactions to subsequent bites from ticks of the same species.
At present, it seems likely that several different ticks can cause an IgE antibody response to alpha-gal; however, the behavior of the ticks (eg, propensity to bite humans) might also be important.
The severity of the pruritic symptoms that our patients report has been impressive. Although many different patterns of tick bites have been reported, 3 forms stand out. The first pattern is a few bites from adult ticks that persist for weeks or months, remaining pruritic. The most severe case of this kind was a report of 2 tick bites removed surgically 6 months after the original contact. The second pattern is repeated bites, often around the ankles, in subjects who work outside or hunt regularly. In a few cases the local reactions to the ticks have been so severe as to preclude further work outside. The third pattern is multiple bites from larval ticks, generally 10 or more, but often hundreds, which are again severely pruritic but generally do not last more than a few weeks.
We have seen high or very high (>100 IU/mL) titers of IgE antibodies to alpha-gal in patients with histories of each of these types. Surprisingly, despite the severity of pruritic reactions locally and the presence of IgE antibodies to tick proteins, we are not aware of any cases of anaphylactic reactions to subsequent tick bites among patients we have seen. There is, however, 1 case report of anaphylaxis to a tick bite in the United States.
The number of cases of delayed anaphylaxis diagnosed in central Virginia and the surrounding areas has increased dramatically, suggesting that we might be looking at an epidemic. However, there were some cases of this condition seen, if not correctly diagnosed, 20 years ago; in fact, 2 patients have recently come to see us and reported that they had told us the same story 15 and 18 years previously! Equally, we know that IgE antibodies to alpha-gal were present in this area in 1988 because sera from an emergency department study at that time have recently been assayed for IgE to alpha-gal.
If the incidence has increased, there are several possible causes related to ticks. There is good evidence that A americanum has increased its range and that its primary host has increased dramatically in numbers.
It is also possible that some factor about the tick has changed, such as a minor variation in the composition of saliva or the presence of a symbiotic organism. This species is known to carry multiple species of rickettsiae, including Rickettsia amblyommii and the organism responsible for human ehrlichiosis, Ehrlichia chaffeensis.
Although the results of absorption experiments provide clear evidence for antibodies that can be absorbed by alpha-gal and those that cannot, we do not know the target for the IgE antibodies that bind to tick-derived proteins (Fig 3 and see Table E1). Future studies will focus on identifying the possible causes of the IgE response to ticks, which include a symbiotic organism, bystander polyclonal IgE induced in the alpha-gal response or a concomitant sensitization to tick salivary protein(s).
Taken together, the evidence pointing toward tick bites as the primary cause of IgE antibody responses to alpha-gal creates a compelling argument. In addition to the 3 cases reported in detail here, there is epidemiologic evidence that (1) these IgE antibodies are found in areas where tick bites are common, (2) the responses correlate with reports of tick bites, and (3) IgE antibodies to alpha-gal correlate with the presence of IgE antibodies to tick proteins. Furthermore, the currently known distribution of delayed anaphylactic reactions to red meat is similar to the known distribution of A americanum (Fig 4, C and D). Predictions are that populations of both deer and ticks will continue to increase their range, particularly in suburban areas. Thus the number of Americans exposed to these ticks and their larval forms will, in all probability, continue to increase.
Key messages
•
IgE antibodies to the oligosaccharide alpha-gal are related to 2 forms of anaphylaxis that are most common in the southeastern United States (eg, anaphylaxis during cetuximab infusion and anaphylaxis after eating mammalian meat).
•
Tick bites are directly related to the presence of these IgE antibodies as assessed prospectively, by history of tick bites, and by analyses of the areas where these IgE antibodies are found.
•
This IgE antibody response provides a model of an ectoparasite-specific response that has significant clinical consequences.
We are grateful to the many physicians who have shared data with us, including Tina Hatley-Merritt, Jane Purser, Carol Wiggins, Michael Land, Marty Fiser, Dan Atkins, Phil Lieberman, Luis Matos, Saju Eapen, Dane McBride, Joey Lane, Gary Rakes, Eddie Shields, and Peter Bressler. We also thank Amy Klion at the National Institutes of Health for discussion about IgE responses to parasites and for sharing sera of patients with helminth infestation. We would also like to thank Dr Raymond Mullins for thoughtful conversations regarding the experience of meat allergy and ticks in Australia. We are grateful to Dr Robert Lane, who identified the seed ticks that bit subject 1. We are also grateful to Walter Oliveira, who has established the assay for IgE to alpha-gal in the hospital laboratory at the University of Virginia.
Appendix E1. Further description of cohorts contained in Table II
Virginia, Allergy Clinic
Patients seen at the University of Virginia Allergy and Asthma Clinic with the complaints of anaphylaxis, angioedema, generalized urticaria, or asthma were enrolled. Additionally, 40 subjects seen in the clinic without these complaints (eg, immune deficiency and contact dermatitis) were enrolled as control subjects.
Tennessee
Sera (n = 107) from Vanderbilt University Medical Center included subjects with cancer who had received cetuximab, as well as subjects who were enrolled as healthy volunteers at a yearly cancer-screening event.
North Carolina
Sera from 75 hospitalized patients were chosen in May 2009 from the general medicine and surgical inpatient census at University of North Carolina Hospital. None of the patients were known to have a cancer diagnosis, and none had been exposed to cetuximab.
Virginia, Emergency Department
Sixty-six randomly selected patients with acute asthma and 70 age- and sex-matched control subjects between the ages of 18 and 50 years were recruited in the emergency department of the University of Virginia Health Sciences Center in Charlottesville. The exclusion criterion for the control subjects was acute breathlessness at the time of the study.
Boston Women’s Study
Sera screened were samples from a metropolitan Boston prospective birth cohort study designed to examine relationships between exposure to indoor allergens and the development of allergic sensitization and asthma. All mothers who delivered at a large Boston hospital were approached for screening within 24 to 48 hours after delivery if they lived within Route 128 (encircling the metropolitan area), were 18 years old or older, and were able to speak English or Spanish.
Norrbotten
Sera samples were from the research program Obstructive Lung Disease in Northern Sweden. In 1996 and 2006, all children in the first-grade and second-grade classes (age 7 and 8 years) in 2 municipalities in northern Sweden were invited to participate in skin prick tests and a questionnaire study. The participation rate for skin prick tests in 1996 was 88%, and 90% of the children participated in 2006, with a similar participation rate among girls and boys and in the 2 areas. All children with any positive skin prick test responses in 4 schools in Luleå were invited to undergo a second skin prick test, and sera were taken at age 18 years for analyses of specific IgE antibodies.
Stanford University
These 49 sera samples were from subjects with cancer of the head and neck who had presented at the Stanford University Medical Center in Stanford, California.
Travis Air Force Base
These sera were obtained from 60 adult patients presenting with wheeze and 57 control subjects evaluated for a nonrespiratory complaint at Travis Air Force Hospital, Fairfield, California, within 2 weeks of the peak grass pollen counts.
University of California, San Francisco
Sera were from subjects with asthma (n = 102) or control subjects (n = 41) enrolled in studies on asthma at the University of California San Francisco.
Kenya
Sera were collected from 2 cohorts. The rural cohort was from the Mukerengu Primary School located in Kabati, an area approximately 80 km north of Nairobi. The major sources of income in Kabati are farming, raising livestock, and working for large pineapple and coffee farms. Most of the children live in thatched-roof homes. Three of 4 classes from the fourth grade were randomly selected. The more urban cohort was from the Kenyatta Primary School located in Thika, a small industrial town 50 km north of Nairobi and only 30 km from Kabati. The many factories in Thika include pineapple processing, battery manufacturing, and bread making. Most of the children from the school live in housing built by the companies in the town: 1-room dirt-floor brick houses arranged as 8 units around a courtyard (1 family per room).
Ecuador
The sera from this cohort were collected from schoolchildren aged 5 to 18 years in adjacent districts in the provinces of Pichincha and Esmeraldas. The area is subtropical/tropical rain forest at altitudes of 50 to 1000 m above sea level. Economic activities in the area are centered largely on agriculture and cattle. The study area consists of small (15-100 houses) and homogeneous (in terms of economic means, lifestyle, and living conditions) communities interconnected by dirt roads. General features of housing are wood or breeze-block walls, corrugated iron roofs, and uncovered wooden or cement floors. Cooking is with propane or wood.
Results are shown for the correlation between IgE antibodies to alpha-gal and IgE antibodies to A americanum(A) and D variabilis(B). Data are shown for the 61 sera for which assay results were available for both ticks.
Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion.
Amblyomma americanum: Physiochemical isolation of a protein derived from the tick salivary gland that is capable of inducing immune resistance in guinea pigs.
Supported by National Institutes of Health grant RO1 AI-20565, AI-AADCRC-U19-070364, K08 AI085190, R21 AI087985 , and the Wellcome Trust 072405/Z/03/Z (to P. J. C.).
Disclosure of potential conflict of interest: S. P. Commins is a volunteer committee member for the American Association of Allergy, Asthma & Immunology (AAAAI) and the American College of Allergy, Asthma & Immunology and is an author for Up-to-Date. M. S. Perzanowski receives research support from the National Institutes of Health (NIH) and is a member of the AAAAI. J. V. Fahy receives research support from the NIH . P. J. Cooper receives research support from Wellcome Trust . T. A. E. Platts-Mills receives research support from Phadia and the NIH and has a patent on an assay. The rest of the authors have declared that they have no conflict of interest.
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