Entomologic and serologic evidence of zoonotic transmission of Babesia microti, Eastern Switzerland.We evaluated human risk for infection with Babesia microti Babesia mi·cro·ti
A species of Babesia that causes babesiosis in humans, usually transmitted by the northern deer tick.
Babesia microti at a site in eastern Switzerland where several B. microti-infected nymphal nymph
1. Greek & Roman Mythology Any of numerous minor deities represented as beautiful maidens inhabiting and sometimes personifying features of nature such as trees, waters, and mountains.
2. lxodes ricinus ticks had been found. DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. from pooled nymphal ticks amplified by polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is was highly homologous to published B. microti sequences. More ticks carried babesial infection in the lower portion of the rectangular 0.7-ha grid than in the upper (11% vs. 0.8%). In addition, we measured seroprevalence seroprevalence Immunology The proportion of a population that is seropositive–ie, has been exposed to a particular pathogen or immunogen; the seropositivity of a population is calculated as the number of individuals who produce a particular antibody divided of immunoglobulin (Ig) G antibodies against B. microti antigen in nearby residents. Serum from 1.5% of the 396 human residents of the region reacted to B. microti antigen ([greater than or equal to] 1:64), as determined by indirect immunofluorescence assay (IgG). These observations constitute the first report demonstrating B. microti in a human-biting vector, associated with evidence of human exposure to this agent in a European site.
A malaria-like syndrome due to Babesia microti infection has been recognized in parts of the northeastern United States for more than three decades (1,2). This protozoon protozoon
pl. protozoa [Gr.] any member of the Protozoa. pathogen was first isolated more than half a century earlier from a Portuguese vole vole, name for a large number of mouselike rodents, related to the lemmings. Most range in length from 3 1-2 to 7 in. (9–18 cm) and have rounded bodies with gray or brown coats, blunt muzzles, small ears concealed in the long fur, and short tails. (3); the pathogen has since been detected in small mammals and ticks throughout Eurasia (3,4).
Despite its broad geographic distribution, B. microti has not been implicated im·pli·cate
tr.v. im·pli·cat·ed, im·pli·cat·ing, im·pli·cates
1. To involve or connect intimately or incriminatingly: evidence that implicates others in the plot.
2. as a cause of human illness in Europe. A host-specific, rodent-feeding tick, Ixodes trianguliceps, is widely regarded as the main enzootic en·zo·ot·ic
Prevalent among or restricted to animals of a specific geographic area. Used of a disease.
An enzootic disease.
peculiar to or present constantly in a location. See also endemic. vector on that continent. I. ricinus, the most common human-biting tick of Europe, transmits the Lyme borreliosis Lyme borreliosis
Another name for Lyme disease.
Mentioned in: Lyme Disease spirochete spirochete
Any of an order (Spirochaetales) of spiral-shaped bacteria. Some are serious pathogens for humans, causing such diseases as syphilis, yaws, and relapsing fever. Spirochetes are gram-negative (see gram stain) and motile. , tick-borne encephalitis virus tick-borne encephalitis virus
An arbovirus of the genus Flavivirus that occurs in two subtypes, Central European and Eastern, causing two forms of encephalitis; it is transmitted by ticks. , the agent of human granulocytic ehrlichiosis human granulocytic ehrlichiosis: see ehrlichiosis. , and B. divergens, but I. ricinus was believed to be infected only occasionally with B. microti (5). This vector-pathogen association may account for the absence of human disease due to B. microti (3,6). However, subadult I. ricinus ticks feed abundantly on the reservoirs of B. microti, such as voles and mice, and appear to be competent vectors for B. microti (7). In fact, recent studies indicate that Swiss residents may have concurrent infection with the Lyme disease spirochete and B. microti (8) and that the human population of certain parts of Germany is exposed to B. microti (9).
Human exposure to B. microti may occur more often in Europe than has been recognized. Accordingly, we assessed the potential of zoonotic Zoonotic
A disease which can be spread from animals to humans.
Mentioned in: Zoonosis transmission in eastern Switzerland, where other I. ricinus--transmitted infections are present. In particular, we determined how frequently B. microti parasites infect I. ricinus ticks locally, how infection in ticks is spatially distributed, and how frequently the sera of nearby residents react to B. microti antigen.
Tick Collection and B. microti Detection in Ticks To assess local prevalence of B. microti in host-seeking nymphal I. ricinus ticks, we developed a tick-sampling procedure with high spatial resolution (Figure 1). The roughly rectangular, 0.7-ha field site, Ruetiwis (9[degrees]38' E, 46[degrees]59' N), is located on a steep southwesterly south·west·er·ly
1. Situated toward the southwest.
2. Coming or being from the southwest.
south·west slope near Seewis in the lower Praettigau Valley of eastern Switzerland at an approximate mean altitude of 850 m above sea level. The site is characterized by abandoned pastures that are partly overgrown overgrown
said of a part that has not been kept trimmed.
overgrown hooves put unusual stresses on bones and tendons and allow for distortion of the wall and sole. by young stands of deciduous deciduous /de·cid·u·ous/ (de-sid´u-us) falling off or shed at maturity, as the teeth of the first dentition.
1. and coniferous trees and bushes, as well as by mature mixed forest. All ticks were collected by flagging in July 1997.
[FIGURE 1 OMITTED]
To ensure the quality of tick-&rived DNA, pools of 2-11 nymphal ticks were transferred to 0.5-mL microcentrifuge tubes containing 50 [micro]L guanidium thiocyanate thiocyanate /thio·cy·a·nate/ (-si´ah-nat) a salt analogous in composition to a cyanate, but containing sulfur instead of oxygen. solution and stored at room temperature until further processing. Before homogenization homogenization (həmŏj'ənəzā`shən), process in which a mixture is made uniform throughout. Generally this procedure involves reducing the size of the particles of one component of the mixture and dispersing them evenly with a glass pestle pestle /pes·tle/ (pes´'l) an implement for pounding drugs in a mortar.
A club-shaped, hand-held tool for grinding or mashing substances in a mortar. , the solution containing the ticks was incubated at 60[degrees]C for 2 h. DNA was extracted from the tick homogenate homogenate /ho·mog·e·nate/ (ho-moj´in-at) material obtained by homogenization.
material obtained by homogenization. by the phenol-chloroform method. The resulting DNA pellet was suspended in 30 [micro]L DNAse-free [H.sub.2]O.
To determine whether B. microti-specific DNA was present, the extracted DNA was subjected to polymerase chain reaction (PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) ) with the primer pair Bab-1 (5'-ttagtataagcttttatacagc-3') and Bab-4 (5'-ataggtcagaaacttgaatgataca-3') (10), which targets a 250-bp fragment of the 18s rRNA gene of B. microti. After denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures. for 2 min at 94[degrees]C, 40 cycles were performed, with 45 sec at 94[degrees]C, 45 sec at 55[degrees]C, and 45 sec at 72[degrees]C, followed by a 7-min final extension. Amplification products were separated on 2% agarose agarose
more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments. gel in Tris-borate-EDTA buffer, stained with ethidium bromide, and visualized under UV light. To differentiate the sequence of interest from a frequently observed, slightly smaller fragment, purified PCR products were digested with XhoI (Life Technologies, Invitrogen Corp., Gaithersburg, MD). The resulting two fragments specific for B. microti migrate in one band on the 2% agarose gel. The corresponding sequence of B. divergens lacks the restriction site.
To verify the taxonomic status of the Babesia Babesia /Ba·be·sia/ (bah-be´ze-ah) a genus of protozoa found as parasites in red blood cells and transmitted by ticks; its numerous species include B. bige´mina, B. bo´vis, and B. spp. detected in these ticks, we conducted a phylogenetic phy·lo·ge·net·ic
1. Of or relating to phylogeny or phylogenetics.
2. Relating to or based on evolutionary development or history. analysis on a representative sample from which DNA had been amplified (Bab-1/Bab-4). For this purpose we used the primer pair PIRO A and PIRO B, which also targets 18s rDNA but is less specific for B. microti; the resulting sequence is longer (400 bp) than the Bab-1/Bab-4 amplicon (11). After PCR amplification, the respective band was excised from the agarose gel, purified with spin columns (Qiagen Inc., Valencia, CA), and sent to the University of Maine "UMO" redirects here, but this abbreviation is also used informally to mean the Mozilla Add-ons website, formerly Mozilla Update
Should not be confused with Université du Maine, in Le Mans, France
The University of Maine sequencing facility for sequence analysis. The resulting sequence was aligned against other Babesia spp. listed in GenBank by using Clustal X and consecutive adjustment visually. Phylogenetic analysis was performed by both maximum parsimony (Swofford D. Phylogenetic analysis using parsimony par·si·mo·ny
1. Unusual or excessive frugality; extreme economy or stinginess.
2. Adoption of the simplest assumption in the formulation of a theory or in the interpretation of data, especially in accordance with the rule of , PAUP PAUP Phylogenetic Analysis Using Parsimony *4b61; Sinauer Associates, Inc., Sunderland, MA) and neighbor-joining analyses (12) with Toxoplasma gondii (GenBank accession no. X68523) as outgroup. Robustness of the nodes was assessed by bootstrap See boot.
(operating system, compiler) bootstrap - To load and initialise the operating system on a computer. Normally abbreviated to "boot". From the curious expression "to pull oneself up by one's bootstraps", one of the legendary feats of Baron von Munchhausen. analysis with 500 bootstrap replicates.
To accurately estimate the local prevalence of B. microti infection in I. ricinus ticks, we developed a maximum likelihood method for point estimation as well as a method for calculating confidence intervals. Briefly, the maximum likelihood estimate (MLE MLE Maximum Likelihood Estimation
MLE Managed Learning Environment
MLE Maximum Likelihood Estimate
MLE Medical Laboratory Evaluation (Medical Laboratory Proficiency Testing Program, Washington, DC) ) of the prevalence p is based on the likelihood
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII ASCII or American Standard Code for Information Interchange, a set of codes used to represent letters, numbers, a few symbols, and control characters. Originally designed for teletype operations, it has found wide application in computers. ]
where [beta] = Log(p / 1-p), N- is the number of ticks in negative pools, J is the set of pool sizes with at least one positive pool, and [n.sub.j] is the number of positive pools of size j. Test-inversion bootstrap confidence intervals (13) were calculated for the prevalence estimate.
To determine whether infected ticks were clustered in the study site, we tested the null hypothesis [[beta].sub.1] = [[beta].sub.2] = 0 by the model [p.sub.k] = Exp ([[beta].sub.0] + [[beta].sub.1][x.sub.1k] + [[beta].sub.2][x.sub.2k]), where [x.sub.1k] = 1 if the [x.sub.1k] = 0 pool is from section C and otherwise, and [x.sub.2k] = 1 if the pool is from section D and [x.sub.1k] = 0 otherwise. It the large sample confidence interval around the MLEs [[beta].sub.1], [[beta].sub.2] or both does not include 0, evidence for clustering is positive.
n. pl. se·rol·o·gies
1. The science that deals with the properties and reactions of serums, especially blood serum.
To determine whether humans may be exposed to B. microti in the study area, we recruited 400 blood donors living within 10 km of the field site for a serologic survey of tick-borne zoonoses Zoonoses
Infections of humans caused by the transmission of disease agents that naturally live in animals. People become infected when they unwittingly intrude into the life cycle of the disease agent and become unnatural hosts. . Volunteers were recruited for this cross-sectional seroprevalence study during blood drives from December 1997 to May 1998 in towns within a 10-kin radius of the study site. This protocol was approved by the Human Subjects Committee of the Harvard School of Public Health The Harvard School of Public Health is (colloquially, HSPH) is one of the professional graduate schools of Harvard University. Located in Longwood Area of the Boston, Massachusetts neighborhood of Mission Hill, next to Harvard Medical School and Cambridge, Massachusetts, (protocol number 9712THEE). The sera of participants who gave their written informed consent were tested by indirect immunofluorescence assay (IFA Immunofluorescent assay (IFA)
A blood test sometimes used to confirm ELISA results instead of using the Western blotting. In an IFA test, HIV antigen is mixed with a fluorescent compound and then with a sample of the patient's blood. ) as described (14). Antigen slides were prepared from erythrocytes Erythrocytes
Red blood cells.
Mentioned in: Bartonellosis
n.pl red blood cells. of B. microti-infected hamsters (the GI strain, originally derived from a Nantucket Island patient). Sera were first screened by IFA at 1:64 dilution. A panel of sera included all samples reactive in the screening test for which enough serum was available, including samples with borderline reactivity and representative controls. This panel was coded and blindly retested for B. microti IFA at the University of Connecticut The University of Connecticut is the State of Connecticut's land-grant university. It was founded in 1881 and serves more than 27,000 students on its six campuses, including more than 9,000 graduate students in multiple programs.
UConn's main campus is in Storrs, Connecticut. laboratories, which specialize in Babesia serology Serology
The division of biological science concerned with antigen-antibody reactions in serum. It properly encompasses any of these reactions, but is often used in a limited sense to denote laboratory diagnostic tests, especially for syphilis. . An IFA titer [greater than or equal to] 1:64 was considered reactive. All reactive sera were titrated ti·trate
tr. & intr.v. ti·trat·ed, ti·trat·ing, ti·trates
To determine the concentration of (a solution) by titration or perform the operation of titration. to endpoint.
To verify the identity of the amplified DNA, a phylogenetic analysis was performed. The sequence amplified from our I. ricinus ticks, which was deposited in GenBank (accession no. AF494286), differed by 1 bp from the North American B. microti sequence (GenBank accession no. AF231348) and was identical with that of B. microti from Slovenia (GenBank accession no. AF373332). Accordingly, our sequence clearly clustered with the European and the American strain of B. microti (Figure 2), with concordant results from both maximum parsimony and neighbor-joining analyses. Therefore, the piroplasms detected in I. ricinus ticks from Switzerland must be considered B. microti.
[FIGURE 2 OMITTED]
We then determined the prevalence of B. microti infection in nymphal I. ricinus ticks from the study site, on the basis of PCR amplification of B. microti in DNA tick pools. Overall, we analyzed 408 ticks in 64 pools. We detected B. microti-specific DNA in 14 pools (Table 1). Thus, B. microti infection appears to be common in human-biting ticks at this central European study site.
To determine whether the prevalence of B. microti is distributed homogeneously within the study site, prevalence of infection in ticks was estimated for selected segments of the sampling grid. The overall prevalence of B. microti infection in ticks, as estimated by MLE, was close to 4%. More detailed spatial analysis, however, indicates that the distribution of babesial infection at the site is heterogeneous (Table 2). In the lower portion of the site (section D), >10% of all ticks were infected, while in the upper portion (B-C), prevalence was <1%. Prevalence of infection in ticks was similar in sections B and C (p=0.161), but greater in section D than in sections B and C (p=0.003). Thus, B. microti transmission is focal at this site.
To determine how frequently humans are exposed to B. microti infection in the region around the study site, sera of local residents were screened by IFA for the presence of antibodies to B. microti antigen. Most sera (80%) were collected from December through March, when I. ricinus is not active. Five (1.5%) of 396 human sera were immunoglobulin (Ig) G-reactive against B. microti antigen at a titer [greater than or equal to] 1:64. All the samples positive on initial testing and one with a borderline result were confirmed on retesting to be reactive at a titer >1:64, while none of the samples with a negative screening result reacted (Table 3). B. microti-specific IgM seroreactivity was not found in any of the sera, a finding compatible with the time of the year when most of the sera had been collected. These results indicate that residents of our central European study site are exposed to bites of ticks infected with B. microti.
B. microti-infected nymphal I. ricinus ticks are present at this Swiss study site, and human residents of the area appear to be exposed to this agent. However, the presence of B. microti in I. ricinus ticks has been reported only rarely (5).
A phylogenetic analysis clearly demonstrated that the piroplasms found in our study site belong to B. microti, rather than to other Babesia or Theileria species (Figure 2). The detection of B. microti DNA in host-seeking nymphal I. ricinus ticks may, however, simply reflect "spill-over" from enzootic transmission by the accepted maintenance vector for B. microti in Eurasia, the tick I. trianguliceps (15). This tick, which does not bite humans, infests small rodents that also are abundantly parasitized by I. ricinus (3,16). The prevalence of infection in ticks may be underestimated as only one genome copy is present per parasite in unfed ticks before sporogony sporogony /spo·rog·o·ny/ (spo-rog´ah-ne) sporulation involving multiple fission of a sporont, resulting in the formation of sporocysts and sporozoites.sporogon´ic
n. . Therefore, low parasite loads may escape detection, thus increasing the specificity of the assay.
In our study, maximizing specificity was desirable because infection of I. ricinus with B. microti was a priori assumed to be rare, and underestimation of prevalence therefore is conservative. Regardless, the proportion of ticks that appear to be infected by B. microti is similar to that in coastal New England (S. Telford, unpub. data). Recently, Duh duh
Used to express disdain for something deemed stupid or obvious, especially a self-evident remark.
[Imitative of an utterance attributed to slow-witted people.] et al. reported a similarly high prevalence of B. microti infection in nymphal I. ricinus ticks collected in Slovenia (7 of 69 ticks tested by PCR) (17). In combination with our findings, this report suggests that B. microti infection in I. ricinus ticks is far more common than traditionally thought. In addition, vector competence of I. ricinus for B. microti has been demonstrated experimentally (7,18). The frequent infection of I. ricinus with this piroplasm pir·o·plasm
[New Latin Piroplasma, genus name : Latin pirum, pear + Greek plasma, image; see plasma.]
Noun 1. therefore implies zoonotic relevance of this vector-pathogen association in Switzerland and possibly in other parts of Europe.
B. microti transmission is clustered in the study site. Similar to tick-borne encephalitis virus, B. microti seems to be maintained in small focal areas. The risk of human infection therefore is spatially highly variable and conditional on tick density. Preliminary analysis of tick infection data from the study site over a 3-year period (Foppa, unpub. data) suggests that B. microti is locally maintained, especially in the lower portion of that site, while the exact location of the maximum risk changes over the years.
Residents of this region in eastern Switzerland appear to be exposed to bites by ticks infected with B. microti. The serologic result is unlikely to reflect low specificity of the assay, as previous evaluation of this IFA has demonstrated high specificity (19,20). As part of this evaluation, we tested 50 sera from residents of Iceland, where ticks capable of transmitting B. microti are absent; none of the sera reacted with B. microti antigen (19). We recently repeated testing of these sera and obtained similar results. This finding suggests a satisfactory positive predictive value Positive predictive value (PPV)
The probability that a person with a positive test result has, or will get, the disease.
Mentioned in: Genetic Testing
positive predictive value of the serologic test even in settings of low prevalence.
In the northeastern United States, B. microti seroprevalence has varied in endemic regions, from 3.7% in Red Cross blood donors on Cape Cod, Massachusetts (21), to 2.5% and 9.5% in Connecticut residents who were seronegative seronegative /se·ro·neg·a·tive/ (-neg´ah-tiv) showing negative results on serological examination; showing a lack of antibody.
adj. and seroreactive, respectively, to Borrelia burgdorferi (22). Our findings of high local prevalence of B. microti infection in I. ricinus ticks may seem counterintuitive coun·ter·in·tu·i·tive
Contrary to what intuition or common sense would indicate: "Scientists made clear what may at first seem counterintuitive, that the capacity to be pleasant toward a fellow creature is ... given the lower seroprevalence in residents of our study area. The findings may, however, reflect a high degree of spatial clustering of transmission with a low average risk (18). Alternatively, the low seroprevalence may be the result of low test sensitivity resulting from antigenic differences between North American B. microti strains, which were used for the serologic testing, and the European strains to which our study population had been exposed.
At least locally, the potential for zoonotic transmission of B. microti by I. ricinus is considerable, which explains serologic evidence in human beings of exposure to this agent in parts of Europe. The lack of recognized human pathology associated with European strains of B. microti, despite exposure to infectious tick bites, may be a consequence of a lower virulence of European strains than those of North American. Disease episodes due to B. microti, on the other hand, may be overlooked because of the relative nonspecificity of signs and symptoms and the presumption that this agent rarely infects I. ricinus.
Table 1. Babesia microti infection in nymphal Ixodes ricinus ticks as determined by polymerase chain reaction, eastern Switzerland Sampling point or line Pool 1 Pool 2 Pool 3 Pool 4 B1 11 11 12 -- B1-2 10 10 10 -- B2 4 3 -- -- B2-3 3 3 3 -- B3 3 -- -- -- B3-4 8 (a) 8 -- -- B4 1 -- -- -- B4-C2 5 5 4 5 C1 1 -- -- -- C1-2 9 9 9 9 C2 7 7 5 -- C2-3 5 5 5 5 C3 10 -- -- -- C3-4 4 -- -- -- C4 4 -- -- -- C4-5 7 6 -- -- C5 8 7 6 -- C5-D1 8 7 7 8 D1 5 5 6 -- D1-2 8 7 -- -- D2 6 6 -- -- D2-3 5 5 5 6 D3 4 -- -- -- D3-4 6 6 5 -- D4 11 11 10 -- D4-5 3 -- -- -- D5 0 -- -- -- Total Sampling No. of No. of point or line Pool 5 pools ticks B1 -- 3 34 B1-2 -- 3 30 B2 -- 2 7 B2-3 -- 3 9 B3 -- 1 3 B3-4 -- 2 16 B4 -- 1 1 B4-C2 -- 4 19 C1 -- 1 1 C1-2 -- 4 36 C2 -- 3 19 C2-3 5 5 25 C3 -- 1 10 C3-4 -- 1 4 C4 -- 1 4 C4-5 -- 2 13 C5 -- 3 21 C5-D1 -- 4 30 D1 -- 3 16 D1-2 -- 2 15 D2 -- 2 12 D2-3 6 5 27 D3 -- 1 4 D3-4 -- 3 17 D4 -- 3 32 D4-5 -- 1 3 D5 -- 0 0 Total 64 408 (a) Pools in bold are positive; dashes represent the absence of further pools. Table 2. Global and local prevalence estimates of Babesia microti infection in nymphal Ixodes ricinus ticks as determined by polymerase chain reaction, eastern Switzerland Point estimate (%) 95% confidence interval MLE (a), overall 3.6 0.2 to 9.06 MLE, section B 1.04 0.10 to 9.01 MLE, section C 0.77 0.08 to 8.47 MLE, section D 10.99 6.17 to 17.71 MLE, sections B,C 0.82 0.61 to 2.65 MLE, sections C,D 5.25 2.96 to 10.43 (a) MLE (maximum likelihood estimate) of the point estimates are shown with 95% bootstrap confidence limits. Table 3. Reactivity of sera tested against Babesia microti as determined by indirect immunofluorescent assay Titer No. of sera (%) <1:64 391 (98.7) 1:64 1 (0.3) 1:128 1 (0.3) 1:256 1 (0.3) 1:512 2 (0.5) Total 396 (100.0)
We thank Alexandra Weld for technical assistance.
This study was partly supported by grants from the National Institutes of Health (AI 42402, 37993, 39002). The serologic survey was partly supported by a grant from Immuno AG Switzerland.
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1. chronic, asymptomatic infection with protozoa of the genus Babesia.
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Zoonosis, also called zoonotic disease refers to diseases that can be passed from animals, whether wild or domesticated, to humans. in Europe. Folia fo·li·a
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1. A piece composed for the development of a specific point of technique.
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Dr. Foppa is assistant professor of epidemiology at the Norman J. Arnold School of Public Health, University of South Carolina
• • , Columbia. His main interests include quantitative methods in infectious disease epidemiology and dynamics of vector-borne zoonoses.
Address for correspondence: Ivo M. Foppa, Department of Epidemiology and Biostatistics, Norman J. Arnold School of Public Health, 800 Sumter Street, Columbia, SC 29208, USA; fax: 803-777-2524; e-mail: email@example.com
Ivo M. Foppa, * Peter J. Krause, ([dagger]) Andrew Spielman, * Heidi Goethert, * Lise Gern, ([double dagger]) Brigit Brand, ([section]) and Sam R. Telford III *
* Harvard School of Public Health, Boston, Massachusetts, USA; ([dagger]) Connecticut Children's Medical Center and University of Connecticut School of Medicine, Hartford, Connecticut, USA; ([double dagger]) Universite de Neuchatel, Institut de Zoologie, Neuchatel, Switzerland; and ([section]) Kantonsspital, Blutspendezentrum, Chur, Switzerland