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Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n = 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-119 and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P = 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n = 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.
While large populations of the world are at risk of malaria (30, 62), the brunt of mortality caused by Plasmodium falciparum continues to be borne by children in sub-Saharan Africa. It is estimated that in this region alone, nearly 1 million children under the age of 5 years died as a direct consequence of malaria in the year 2000 (59). An effective vaccine is urgently needed but has proved challenging to obtain. In endemic areas, older children and adults develop naturally acquired immunity to severe and life-threatening malaria but remain susceptible to infection (37). Classical experiments in which passively transferred antibodies from immune adults were successfully used to treat children with severe P. falciparum malaria (14, 40) provide the strongest evidence that antibodies are important mediators of naturally acquired immunity. Clinical symptoms of malaria result from the asexual blood stage of the infection, in which potential antibody targets include merozoite antigens involved in invasion (18) and parasite-derived surface antigens on infected erythrocytes (8).
Studies on protective immunity to malaria involve monitoring subjects in endemic communities for variable durations of time to measure the incidence of infection or clinical disease. Associations between the presence of a specific immune response to a target antigen and an outcome determine whether an immune response to the specific antigen appears to be “protective.” These immuno-epidemiological studies have often provided conflicting data, with responses to the same antigen appearing to be protective in some studies but not in others (2, 5, 10, 15, 21, 24, 31, 52, 57). Most antibody-based analyses of protection are tethered on seropositivity (usually defined as the mean plus 3 standard deviations of non-malaria-exposed sera) and do not take into account the continuous, quantitative nature of antibody concentrations. Furthermore, the majority of studies have concentrated on associations between responses to a single or a limited number of antigens and protection from clinical malaria, despite the fact that individuals living in endemic areas are simultaneously and repeatedly challenged with numerous malaria antigens. Few studies have examined the interactions between specific antibody responses against multiple malaria antigens (32, 41) and whether these might be synergistic, antagonistic, or neither with regards to protection.
To test whether either the number of important target antigens to which antibodies are made or the levels of such antibodies in serum are associated with protection from malaria, we analyzed naturally acquired antibodies to five leading P. falciparum merozoite-stage vaccine candidate antigens (apical membrane antigen 1 [AMA1], merozoite surface proteins 1, 2, and 3 [MSP-1, MSP-2, and MSP-3], and erythrocyte binding antigen [EBA-175]), as well as P. falciparum schizont extract, in a cohort of Kenyan children who were monitored longitudinally for mild (uncomplicated) clinical malaria (Chonyi cohort). We also examined combinations of, and interactions between, antigen-specific antibodies to determine the combination(s) that predicted the strongest protection from clinical malaria. These antigens were selected for study because of the cumulative evidence that the presence of antibodies to these antigens may be associated with protection (10, 15, 42, 50, 53-56, 65, 66), backed by evidence that polymorphisms in their sequences are maintained by natural selection (16), and their biological plausibility (3, 13, 20, 25, 36, 46, 60). The analytical approaches were developed using data from the Chonyi cohort, and the methods subsequently were validated in an independent case-control study whose end point was malaria severe enough to require admission to hospital.
Cohort study.This study was conducted in Kilifi, a rural district along the Kenyan coast. Details of the study area and population have been published elsewhere (44), along with a description of a cohort comprising both adults and children from Chonyi village in Kilifi. This area typically experiences two seasonal peaks in malaria transmission (June to August and November to December) and has an average annual entomological inoculation rate of approximately 20 to 100 infective bites/person/year (38). Following a cross-sectional bleed at the start of a malaria transmission season in October 2000, the cohort was followed for clinical episodes of malaria in the ensuing 6 months. Asymptomatic parasitemias were not subjected to drug clearance at the beginning of the study or during follow-up. Active detection of mild clinical malaria (outcome) was achieved through weekly visits to participants' homes, where a morbidity questionnaire (investigating symptoms occurring in the preceding week) was administered and the presence or absence of fever was ascertained (axillary temperature of >37.5°C). Participants found to be unwell were referred for free treatment to a dedicated outpatient clinic at the local district hospital, where they also had open access as required at any time during the study (passive case detection). Clinical episodes of malaria were treated with sulfadoxine-pyrimethamine, as per the Kenyan national treatment guidelines at the time the study was conducted. Age-specific criteria for defining clinical episodes of malaria were developed previously for this area as follows: children under 1 year, fever plus any parasitemia; children older than 1 year, fever plus a parasitemia of greater than 2,500/μl (44). Participants were only included in the study if they were present for all but three of the weekly visits during the 6 months of follow-up. For analytical purposes, only the first clinical episode was counted, although all children continued to be monitored until the close of the study. Within the cohort, children aged 10 years and less (n = 280) accounted for nearly 90% of all the clinical episodes. The current analysis focused on children who were slide positive (they were all asymptomatic) at the time the cross-sectional blood sample was collected (n = 119; details are provided below in the description of the statistical analysis). Data on antibody responses to AMA1, MSP-2, and MSP-3 were previously published (50, 53, 54), while data on the remaining antigens (EBA-175, MSP-119, and MSP-1 block 2) are reported here (see Fig. S1 in the supplemental material).
Case-control study.The findings in the Chonyi cohort were validated in a separate, spatially and temporally distinct group of children investigated in an analysis of parasite antigens on the infected red blood cell surface that was previously conducted in Kilifi (7). Children were recruited from an area immediately surrounding the administrative town of Kilifi, with an entomological inoculation rate of approximately 1.5 to 8 bites/person/year (39). In May 1995, a cross-sectional survey was carried out during which finger prick blood samples were collected from 4,783 children (aged 1 to 5 years), from which sera were separated (stored at −70°C) and thick blood smears were prepared and examined for parasites. Over the following 8 months, children who were part of this survey and presented to hospital with malaria that was severe enough to warrant pediatric ward admission were identified (passive case detection). A total of 89 children from the survey were admitted during that period and were frequency matched, allowing for age and location of children (n = 298) who took part in the cross-sectional survey but did not present to hospital with malaria. Sera from the cross-sectional survey for cases and controls (n = 387) were analyzed for immunoglobulin G (IgG) antibodies to AMA1, MSP-2, MSP-3, EBA-175, MSP-1 block 2, MSP-119, and P. falciparum schizont extract. Ethical approval was granted by the Kenya National Research Ethics Committee.
Recombinant antigens.The recombinant antigens were expressed in Escherichia coli as glutathione S-transferase-fusion proteins (MSP-2_Dd2 and MSP-2_CH150/9) (66), MSP-1 block 2 (RO33, Palo Alto, 3D7, MAD20, and Wellcome) (12), and MSP-119 (9), His-tagged (AMA1_3D7) (23) and EBA-175_F2_CAMP (51), or as maltose-binding protein-fusion proteins (MSP-3_K1 and MSP-3_3D7 (56). Recombinant AMA1_FVO (35) was expressed in Pichia pastoris, while EBA-175_F2_3D7 (19) is a baculovirus-expressed product. Details on these antigens are provided in Table S1 of the supplemental material.
Antibody assays.Enzyme-linked immunosorbent assays (ELISAs) against each recombinant antigen and against parasite schizont extract were performed according to a standard protocol as previously described (50, 53, 54). Individual wells of Dynex Immunolon 4HBX ELISA plates (Dynex Technologies Inc.) were coated with 50 ng of antigen per 100 μl of carbonate coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.3). Wells were coated with P. falciparum schizont extract (the A4 strain for the Chonyi cohort and Wellcome strain for the hospital cohort) in phosphate-buffered saline (PBS) according to the method of Ndungu et al. (45). Plates were incubated overnight at 4°C before washing four times in PBS-Tween (PBS-0.05% Tween 20) and blocking for 5 h at room temperature with 1% skimmed milk in PBS-Tween (blocking buffer). Following this, wells were washed again and incubated overnight at 4°C with 100 μl of test serum (1/1,000 dilution in blocking buffer). Plates were then washed four times and incubated for 3 h at room temperature with 100 μl of horseradish peroxidase-conjugated rabbit anti-human IgG (Dako Ltd.) at a 1/5,000 dilution in blocking buffer before final washing and detection with H2O2 and o-phenylenediamine (Sigma). The reaction was stopped with 25 μl of 2 M H2SO4 per well, and absorbance was read at 492 nm. The same positive controls (hyperimmune sera) were run in duplicate on each day of the experiment, on each plate, to allow for standardization of day-to-day and plate-to-plate variations. Single-dilution serum ELISA optical density (OD) values were used as proxies for antibody titers, as they correlate closely with full endpoint antibody titrations when used at appropriate dilutions (22, 67).
Statistical analysis.All data analyses were performed with STATA version 9.2 (StataCorp, College Station, TX). Models were first developed using data from the Chonyi cohort and subsequently validated in the case-control study with some modifications (below). The primary analysis was on the subgroup of 119 children from the Chonyi cohort (n = 280) who were asymptomatically parasitized at the time of serum collection in October 2000, because in previous analyses, P. falciparum parasitemia at the time of serum collection modified the effects of antibodies to both variant red blood cell surface (6) and merozoite (50, 53, 54) antigens on the risk of disease. The confounding effects of exposure on antibody responses were controlled for by adjusting both for age as well as antibody reactivity to parasite schizont extract in multifactorial analyses.
The probability of a clinical episode for each antigen (and each allelic form) for given antibody levels was estimated by logistic regression, fitting ELISA OD values for the antigen as a linear covariate and adjusting for age (in 2-year categories). The logits from these models were converted into probabilities (43) to give estimates of risk (see Fig. 1, below). These analyses established that for most antigens and antibodies, higher antibody levels were associated with a lower risk of disease and that allelic versions of the same antigen (or the same allelic family for MSP-1 block 2) generally gave similar patterns of protection. The probability plots were used to define a threshold (cutoff) for high versus low/undetectable antibodies as the OD level above which the risk of disease was lower than the population's average risk of 33.6% (i.e., the risk of disease assuming no role for any antibodies) (see Fig. 1, below; see also Table S2 in the supplemental material). The suitability of the logistic model was confirmed by examining the residuals when the OD data were fitted in quintiles (data not shown). The individual effects of high levels of each antibody on the risk of disease were then reanalyzed, fitting antibody level as a factor rather than as a linear covariate (Table 1) for ease of interpretation and to facilitate analyses of the breadth and the interactions between antibodies. To avoid the lack of convergence commonly encountered in conventional binomial regression analyses, data were fitted to a modified Poisson regression model with robust error variance, which tends to provide conservative results (69).
The predicted probability of an episode of malaria in children decreases with increasing antibody titer for most antigens (n = 119). Each panel represents the allelic antigens tested at each locus, as well as parasite schizont extract. The red horizontal line represents the risk of an episode without taking antibody responses to any antigen into account. The final panel combines antibodies to one allelic form of each antigen (and one antigen from each of the three main allelic families of MSP-1 block 2). The lines, from top to bottom, represent MSP-1_B2_Wellcome, MSP-3_K1, MSP-2_Dd2, schizont extract, AMA1_3D7, EBA-175_F2_3D7, MSP-1_B2_3D7, MSP-1_B2_RO33, and MSP-119.
Antibodies to different allelic forms of most antigens (AMA1, MSP-2, MSP-3, and the F2 subdomain of EBA-175) and to the main allelic types of MSP-1 block 2 (K1 and MAD20 types) were highly correlated (see Table S3 in the supplemental material). Consequently, high levels of antibodies to only one allelic form of each antigen were considered for the analysis of antibodies to multiple antigens. Antibodies to MSP-1 block 2 (MSP-1_B2) were highly correlated only within the main allelic families, and so for this antigen, antibodies to one antigen from each of the three main allelic families (MAD20-like, K1-like, and RO33-like) were included to give an overall MSP-1 block 2 response (any of MSP-1_B2_Wellcome, MSP-1_B2_3D7, or MSP-1_B2_RO33). The criteria used to select antibodies to a single allelic form of each antigen have been outlined (see text in the supplemental material). The antigens retained for further analyses (breadth and combined responses) were AMA1_3D7, MSP-2_Dd2, MSP-3_K1, EBA-175_3D7, MSP-119, and MSP-1 block 2 (overall response).
Breadth was analyzed in an age- and schizont extract-adjusted modified Poisson regression model that compared the risk of disease among children who had high levels of antibodies (fitted as a fixed level factor) to between one and six antigens to those who had low/undetectable antibodies to all six antigens. The combination of antibodies that was associated with the lowest risk of clinical malaria was determined by analyzing all pair-wise combinations, investigating interactions between antigens by fitting a model with two main effects and an interaction term. Interaction as presented here refers to statistical interaction where the estimate of risk obtained for antibodies to two antigens is significantly lower than expected (i.e., lower than the product of the individual risk ratios). It does not exclude biological interaction. To make certain that we were not simply measuring correlated antibodies arising from shared exposure, we separately included antibodies to all antigens in a single regression model, together with age and reactivity to schizont extract, dropping each out sequentially in decreasing order of their P values. Antigens that remained significant in this model at the P < 0.10 level were MSP-2, MSP-3, and AMA1.
Data on children from the hospital cohort were analyzed essentially as described above with minor modifications. Models were fitted to data from the entire hospital cohort (not only the subgroup that were parasitemic at the time of serum collection) because it appeared that frequency matching of cases and controls for location (and therefore exposure) successfully eliminated the interaction between the antibody's protective effect and parasite infection status.
Magnitude of antibody response and protection.The probability of developing an episode of clinical malaria for a given value of measured antibody level (OD) was estimated for each antigen. We found that the levels of serum antibodies to some, but not all, vaccine candidate antigens were inversely related to the probability of developing malaria (Fig. 1). Increasing OD levels to MSP-2, MSP-3, AMA1, and the MAD20-like antigens of MSP-1 block 2 (denoted Wellcome and MAD20) were associated with reduced probability of malaria morbidity, while those to MSP-119, EBA-175, and the K1- and RO33-like antigens of MSP-1 block 2 had little effect. Within these loci (and within the main allelic families for MSP-1 block 2), the patterns were similar for the different allelic forms. Increased antibody titers to whole parasite schizont extract were also associated with a reduced probability of clinical malaria.
Breadth and protective efficacy of antibody response.The probability plots (Fig. 1) were used to define a threshold (cutoff) for high versus low/undetectable antibodies for each antigen. This threshold varied both by antigen and by population (the Chonyi cohort and the case-control study), ranging from relatively low OD values for the MSP-1 block 2 antigens to high values for MSP-2 (see Table S2 in the supplemental material). Children who had high levels of antibodies to one, two, three, four, five, or six unrelated (nonallelic) antigens were compared with those who did not have high levels to any antigen to test the hypothesis that the breadth of specificities for unrelated antigens in the antibody response is important for protection. The risk of malaria was inversely associated with increasing breadth of antibody specificities in both study groups (Fig. 2). None of the children in the Chonyi cohort who had high-titer antibody responses to five or more antigens (17/119; 15%) experienced a clinical episode (P = 0.0006 by Fisher's exact two-tailed test). Similarly, in the case-control study, none of the children who had high-titer responses to five or more antigens (23/298; 7.7%) was admitted to hospital with severe malaria (P = 0.004, Fisher's exact two-tailed test).
Protective efficacy increased with increasing breadth of response in children from the Chonyi (parasitemic children, n = 119) (a) and hospital cohorts (all children, n = 387) (b). Each bar represents the comparison between individuals making high-titer responses to n number of antigens with those who make no responses to any antigen. Proportions above each bar are the percentage of individuals making high-titer responses to n antigens. The effect of high-titer responses to P. falciparum schizont extract is also shown.
Breadth of antibody specificity increases with age and concurrent parasitemia.The breadth of high-titer antigen-specific responses increased with age in both groups of children (Fig. 3). Parasite positivity at the time of serum collection significantly increased the breadth of the response. In the Chonyi cohort, nearly three times as many children who were parasitemic at the time of serum collection had high antibody titers to three or more antigens, compared to those who were aparasitemic (47% [56/119] versus 17.3% [28/161]; Pearson's chi-square, 28.67; P < 0.001). This difference was more marked in the case-control study, with over five times as many children who were parasitemic at serum sampling having high-titer responses to three or more antigens compared to those who were not parasitemic (57% [102/176] versus 10.4% [30/287]; Pearson's chi-square, 120.77; P < 0.001).
The breadth of antibody specificity increased with age in both the Chonyi cohort (n = 119) (a) and the case-control study (n = 387) (b).
Combinations of antibodies and protection.Interactions between antibodies were investigated to determine which combination(s) was associated with the lowest risk of clinical episodes in the Chonyi cohort. High levels of antibodies to combinations that included MSP-2, MSP-3, and AMA1 were associated with a lower risk of disease compared to their individual effects (Table 2). While the combined effects of antibodies were always greater than each of the individual effects, there was no statistical evidence of synergism or antagonism, i.e., more or less protection, respectively, than expected from the combination of the two antigens acting additively. The strongest protection was associated with high levels of antibodies to both MSP-2 and MSP-3. Thirty-three children (of 119) had high antibody levels to both MSP-2 and MSP-3, and none of them experienced any episodes of disease (P < 0.001 by Fisher's exact two-tailed test; still highly significant after a Bonferroni correction , allowing for multiple comparisons, P = 0.003). This finding was validated in the case-control study, where admission to hospital with malaria was the end point. Children who had high levels of antibodies to both MSP-2 and MSP-3 were significantly less likely to be admitted to hospital with malaria (odds ratio, 0.26; 95% confidence interval, 0.08 to 0.81; P = 0.020) (see Table S5 in the supplemental material).
We found that in two independent studies conducted in both high-transmission (Chonyi cohort) and low-transmission (case-control study) settings at different times, both the breadth of specificity for distinct merozoite antigens and the magnitude of antibody responses to these antigens provide robust predictors of the immune status of children. High titers of antibodies to combinations of three merozoite antigens in particular (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection from clinical episodes of malaria compared to other putative “protective” merozoite antigens (MSP-1 or EBA-175).
Out of the panel of malaria vaccine candidate antigens studied here, high levels of antibodies to combinations that included AMA1, MSP-2, and MSP-3 were the most strongly associated with protection. This is consistent with other studies in which naturally acquired antibodies to each of the three antigens individually have been associated with protection from clinical malaria in this and other populations (1, 41, 42, 53, 54, 61, 63, 65, 66). Recently, long-term clinical protection was associated with IgG3 isotype antibodies to MSP-3 in Senegalese children (58). In contrast, antibodies to MSP-1 block 2, which have been associated with protection in two cohorts in West Africa (10, 15, 55), were not similarly protective in the two cohorts we studied from Kilifi, Kenya. Antibodies to MSP-119 have been associated with protection from clinical malaria in some studies, but not in others (5, 10, 15, 21, 24, 31, 52, 57). This may be explained in part by the finding that the fine specificity of anti-MSP-119 antibodies appears to be more important with regards to protection (17, 48). A separate study found that individuals with high-titer anti-MSP-119-specific invasion-inhibitory antibodies were protected from infection (33) and underscored the importance of developing robust functional assays for malaria. Antibodies to the F2 subdomain of EBA-175 were not associated with protection from clinical disease in our studies, as has been found in other parts of Africa where this (49) and other subdomains of EBA-175 (32, 47, 49) have been studied. To date, only one study has reported significantly higher antibody levels to EBA-175 peptide 4 (residues 1062 to 1103, within region V) in children protected from clinical attacks of malaria compared to susceptible children (68).
The importance of allele-specific immunity was highlighted in the Combination B malaria vaccine trial in Papua New Guinea. Children who received this vaccine (containing a combination of P. falciparum ring-infected erythrocyte surface antigen, MSP-1, and the 3D7 allele of MSP-2) were less likely to be infected with parasites bearing the homologous allele of MSP-2 (28), suggesting (as was later confirmed) that the vaccine had induced primarily allele-specific MSP-2 antibodies (26). In the context of naturally acquired infections, while some data suggest that parasites bearing specific genotypes induce allele-specific antibodies (11, 34, 53), to our knowledge no studies have examined the protective effects of preexisting allele-specific antibodies on subsequent disease caused by parasites bearing homologous alleles. We found that for most antigens tested, responses to allelic forms of each antigen had similar effects on the probability of mild or more severe malaria, suggesting the possibility that there may be significant cross-allele protection to clinical episodes.
In a study conducted in The Gambia, Gray et al. (29) found that while antibodies to a similar panel of individual antigens were only weakly correlated with protection, those to the combinations of AMA1 and MSP-2 were significantly associated with protection from clinical malaria. There are two important differences between this Gambian study and the results reported here from Kenya. First, k-means clustering and phylogenetic networks were used to investigate associations between antibody reactivity profiles and clinical status in the Gambian cohort. These methods independently identified the group of children who were asymptomatic (asymptomatic parasitemia, splenomegaly, or both) at the end of the study and who had not apparently experienced clinical disease. That end point differs from that of the studies reported here, in which outcome was simply defined as mild (Chonyi cohort) or severe (case-control study) malaria during the period of observation. Second, the magnitude of responses was not taken into account, mainly because this generates increased individual differences, impairing cluster analysis. One other longitudinal study, carried out among children in Burkina Faso, examined antibodies to a different set of blood-stage malaria antigens (glutamate-rich protein, P. falciparum exported protein 1, and MSP-3) and, like our studies, they found that the simultaneous presence of antibodies to more than one antigen was associated with a lower frequency of malaria episodes (41). However, in a separate study on protection from malaria infection as opposed to clinical episodes in Kenyan adults, John et al. (32) found that high antibody titers to multiple blood-stage antigens were not protective (though there was evidence of protection for responses to preerythrocytic antigens). Our data suggest that the combination of blood-stage antigens analyzed in these Kenyan adults (AMA1, EBA-175, and MSP-119) may not have been optimal. While these studies are difficult to compare directly due to differences in study design, study populations and end points, antigens tested, and analytical methodologies, the picture that nevertheless emerges clearly is that antibodies to key combinations of multiple parasite targets are more strongly associated with protection from clinical malaria than are antibodies to individual antigens.
With the completion of the P. falciparum genome, numerous new (and old) antigens of the parasite have been identified and are being characterized. High-throughput assays employing suspension array technology (27) or microarrays (29, 64) now allow for simultaneous analysis of antibodies to multiple antigens using minimal amounts of sera. This technology has not been matched with equivalently efficient tools for identifying protective immune responses. Robust concurrent analyses of numerous responses in relatively small studies, where children have been monitored longitudinally over a limited time period for disease episodes, remain challenging. The pair-wise analyses of combinations of high-titer antibody responses as presented here have obvious limitations when numerous antibodies are to be analyzed. Other analytical techniques, such as clustering and the use of phylogenetic networks (29), while attractive for screening of potential vaccine candidates, similarly become more complex when increasing numbers of responses are analyzed and may well obscure genuinely “protective” responses. New strategies to identify protective responses in humans are urgently needed.
Studies of associations between immune responses and clinical malaria need to take into account the possibility that any given response is merely a marker of cumulative exposure (which is itself necessary to induce immunity) or of a response to an as-yet-unidentified antigen(s) that elicits strongly protective immunity. In our study, the fact that antibodies to specific antigens were more strongly predictive of protection than those to whole-schizont extract (containing all the specific antigens and many other blood-stage antigens) (Fig. 1) suggests that specific responses do not merely reflect exposure. The finding that protective efficacy increased with increasing breadth of antibody specificity indicates that the effect of any one apparently protective response does not simply result from correlation with responses to other antigens (Fig. 2) and argues for the interpretation that these are truly protective responses. Ultimately, the critical test of any such hypotheses will be to achieve equivalent protection through vaccination. Our demonstration of strong protection against malaria associated with high antibody levels to AMA1, MSP-2, and MSP-3 lends support to the development of vaccines based on combinations of these key malaria antigens.
We thank Chetan Chitnis for provision of the recombinant antigen representing the F2 subdomain of EBA-175. We also thank Moses Mosobo for his invaluable assistance in the laboratory and Britta Urban for helpful comments on the manuscript.
This work was supported by a research training fellowship for F.O. from the Wellcome Trust, grant no. 073591.
Returned for modification 9 February 2008.
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matches: V. V. V. V. V. V.


Every year there is a freak out over some decision from the U.S. Supreme Court. Sometimes, it's mostly a tempest in a teapot and that seems to be the case with this year's big paroxysm, Birchfield v. North Dakota, JUN16, USSC No. 14-1468.
Birchfield is a consolidation of three different drunk driving cases. It's poorly organized and requires at least a couple readings before you can suss out its organization and ruling. Everyone seems to be jumping to the false conclusion that it changes things an incredible amount, but if you actually read it rather than just listening to third person hype it doesn't (at least not in Virginia).
The cases now before us involve laws that go beyond that and make it a crime for a motorist to refuse to be tested after being lawfully arrested for driving while impaired. The question presented is whether such laws violate the Fourth Amendment’s prohibition against unreasonable searches.
Put more succinctly, can the government impose criminal sanctions for a person refusing testing to see if that person is intoxicated?
The answer? Yes for breath tests. No for blood draw tests.
The case rambles around, but it is organized into three possible rationales. First, do exigent circumstances justify the testing? Second, does search incident to arrest justify testing? Third, does implied consent justify testing? These three are confused in that the case is addressing both breath test and blood draws across the three questions.
To begin with, the court reiterates its holding in Missouri v. McNeely, APR13, USSC No. 11-1425, that while there are circumstances, to be determined on a case-by-case analysis, that justify an exigent blood draw the fact that the body metabolizes and removes the intoxicant is not enough by itself to make an exigent circumstance.
A breath test is allowed as a valid search incident to an arrest because there is no expectation of privacy in air in the lungs and it can only provide evidence of drunkeness.
A blood test is not a valid search incident to arrest because the breath test is available, it is a significant bodily intrusion, and it can be used to develop evidence of more than drunkeness.
Our prior opinions have referred approvingly to the general concept of implied-consent laws that impose civil penalties and evidentiary consequences on motorists who refuse to comply. Petitioners do not question the constitutionality of those laws, and nothing we say here should be read to cast doubt on them.
So, under implied consent, non-criminal penalties for refusing a blood test are still valid. However, the court makes short shrift of the argument that criminal penalties should still be allowed.
Motorists cannot be deemed to have consented to submit to a blood test on pain of committing a criminal offense.
First, let's set out what the rule of this case is: A government cannot criminalize the refusal to submit to a blood test in order to prove drunk driving.
§ 18.2-268.3(D) - A first violation of this section is a civil offense and subsequent violations are criminal offenses. For a first offense the court shall suspend the defendant's privilege to drive for a period of one year. This suspension period is in addition to the suspension period provided under § 46.2-391.2.
If a person is found to have violated this section and within 10 years prior to the date of the refusal he was found guilty of any of the following: a violation of this section, a violation of § 18.2-266, or a violation of any offense listed in subsection E of §18.2-270, arising out of separate occurrences or incidents, he is guilty of a Class 2 misdemeanor and the court shall suspend the defendant's privilege to drive for a period of three years. This suspension period is in addition to the suspension period provided under § 46.2-391.2.
If a person is found guilty of a violation of this section and within 10 years prior to the date of the refusal he was found guilty of any two of the following: a violation of this section, a violation of § 18.2-266, or a violation of any offense listed in subsection E of § 18.2-270 arising out of separate occurrences or incidents, he is guilty of a Class 1 misdemeanor and the court shall suspend the defendant's privilege to drive for a period of three years. This suspension period is in addition to the suspension period provided under §46.2-391.2.
The green part is clearly allowed. The parts in yellow are allowed for breath tests, but not for blood tests. It's as simple as that. Here's the only thing which needs to change in the way in which officers handle DUI's in Virginia: When the officer reads implied consent for a blood draw he cannot mention any possibility of a criminal prosecution. If he does and the suspect relents and allows the blood draw it will be suppressed by the courts.
Otherwise, everything is just the same as it was. Carry on. Nothing to see here. At least until the Supreme Court goes back in session this Fall and people find some other tempest in a teapot to make them start screaming about falling skies again.
Sadly, one of the statutes just put in place by our legislature is one putting a $250 fine on fake use of a dog under claim that it is a service dog. You'd think this wouldn't be much of a problem, but I know that I have spoken with more than one inn keeper about people claiming that all sorts of dogs are service animals in order to keep Spot with them in their hotel room. So, I thought I'd try to put down what the state of Virginia law is in this matter.
Any person who knowingly and willfully fits a dog with a harness, collar, vest, or sign, or uses an identification card commonly used by a person with a disability, in order to represent that the dog is a service dog or hearing dog to fraudulently gain public access for such dog pursuant to provisions in § 51.5-44 is guilty of a Class 4 misdemeanor [up to $250 fine].
E. Every totally or partially blind person shall have the right to be accompanied by a dog in harness trained as a guide dog, every deaf or hearing-impaired person shall have the right to be accompanied by a dog trained as a hearing dog on a blaze orange leash, and every mobility-impaired or otherwise disabled person shall have the right to be accompanied by a dog, trained as a service dog, in a harness, backpack, or vest identifying the dog as a trained service dog, in any of the places listed in subsection B without being required to pay an extra charge for the dog, provided that he shall be liable for any damage done to the premises or facilities by such dog.
Notice that there are only three types of service dogs allowed and each has to be wearing some sort of identifying device. This is the only real identifier that an inn keeper could use to differentiate Spot the pet from Spot the service dog. To be fair, one would expect that in the vast majority of cases it will be clear if someone has vision, auditory, or mobility problems. However, the "otherwise disabled" leaves a lot of wiggle room. And if anybody wanted to cheat in that area it is ridiculously easy to buy the gear which proclaims a dog is a service animal.
The only real limit here is that "[t]he provision of emotional support, well-being, comfort, or companionship shall not constitute work or tasks for the purposes of this definition." Va. Code § 51.5-40.1.
In the end, I have to conclude that hotels and motels should be within their rights under Virginia law to refuse service to people without the obvious handicap who don't have the dog properly outfitted. Beyond that, if anyone is caught faking they could end up coming to court to get fined. If the innkeeper is suspicious he should call local law enforcement to investigate.


text:
Crabapples (Malus spp.) are popular ornamental trees in the commercial and residential landscape. Over a 33-year period at the Secrest Arboretum, Wooster, OH, 287 accessions of ornamental crabapple were evaluated for their resistance to apple scab caused by the fungus Venturia inaequalis. Of these 287 accessions, 31 had no symptoms of scab for longer than a 10-year period and were identified as resistant to the disease. Of these 31 resistant accessions, 14 eventually displayed symptoms, presumably as a result of infection by one or more newly present races of the pathogen in the trial plot. Notable resistance breakdowns in accessions previously classified as resistant include the development of scab on M. × ‘Prairifire’, M. × ‘Bob White’, M. × ‘Red Jewel’, and M. floribunda. Corresponding to these changes of resistance is the putative development of new V. inaequalis races in North America: Race 5, possessing virulence to the Vm gene in ‘Prairifire’; Race 3 that infects M. × ‘Geneva’ but not M. baccata ‘Dolgo’; and the first identification and report of scab on a M. floribunda population that was reported as resistant even before the first 25 years of the evaluation. The detection of scab on this species suggests the presence of Race 7 in North America for the first time. Five named accessions remained free from scab for the entire 33-year trial: M. sargentii ‘Sargent’, M. baccata ‘Jackii’, M. × ‘Beverly’, M. × ‘Silver Moon’, and M. × ‘White Angel’ and may serve as sources of durable resistance in crabapple and commercial apple breeding in the Midwest.
The genus Malus includes both commercial apple and crabapple with the primary difference between them being fruit size; crabapple fruit is less than 2 inches in diameter, whereas commercial apples have fruit greater than 2 inches. Although apple (Malus ×domesticus) arose primarily from M. sieversii, many commonly used crabapple species such as M. prunifolia (Willd) Borkh., M. baccata (L.) Borkh., M. mandshurica (Maxim) Kom., and M. sieboldii (Regel) Rehder may have hybridized with M. sieversii (Luby, 2003). Many of these potential progenitors to the commercial apple are used in the breeding of both crabapple and commercial apple and serve as a source of durable resistance to scab and other major diseases of Malus (Fiala, 1994; Shay et al., 1962).
Crabapples are among the most widely cultivated ornamental trees in the northeastern and midwestern regions of the United States and in southern Canada. Crabapple trees vary in size and shape and can provide four seasons of interest: spectacular displays of single, double, and semi-double flowers in shades of pure white to clear red in the spring; foliage that exists in a variety of shapes and colors for the summer and fall; fall fruit of varying size and color and persistence; and strong architectural forms in the winter. This variation in size, tree structure, bloom, and fruit is a testimony not only to the popularity of crabapples, but also to the diversity of genetic backgrounds that has been selected and bred to create superior selections of crabapples. Proof of this popularity is readily observable; over 700 varieties of crabapple have been named within the nursery industry (Dirr, 1990; Fiala, 1994). Many of these selections and much of the breeding effort have been directed to evaluate resistance to apple scab caused by the fungal pathogen Venturia inaequalis (Cke). In scab-susceptible crabapples, symptoms of infection include defoliation by early summer coupled with loss of winter hardiness and even death resulting from repeated defoliation and attack by opportunistic insects or pathogens. The identification of scab-resistant cultivars is important for both commercial producers and retailers of crabapples (Romer et al., 2003) and for commercial apple breeding programs that seek to develop scab-resistant apples (Janick, 2002; Shay et al., 1962).
Host plant resistance is considered one of the most efficient and effective methods to control plant diseases with much of the breeding effort within the genus Malus directed to evaluate resistance to apple scab. The V. inaequalis–Malus interaction is one of the earliest examples demonstrating gene-for-gene interactions (Boone, 1971; Hough, 1944; Williams and Shay, 1957). The gene-for-gene theory states that for every major gene conferring resistance (R) in the host, there exists a corresponding avirulence (AVR) gene in the pathogen (Flor, 1956; Hammond-Kosack and Jones, 1997). Disease resistance results only when the corresponding product of a dominant resistance gene (R) recognizes a dominant Avr gene product from the pathogen. Disease results when the loss or alteration of the pathogen avirulence gene (now denoted as avr) fails to trigger recognition by the corresponding product of the host resistance (R) gene (Hammond-Kosack and Jones, 1997). This model suggests a strong selection pressure exists against avirulence (AVR) within a pathogen population with any loss of avirulence (avr) resulting in new virulent races that are identified only when cultivars previously scored as resistant succumb to disease (MacHardy, 1996). This phenomenon is referred to as “resistance breakdown” (McDonald and Linde, 2002). However, it is better understood and more correctly stated as “avirulence breakdown,” as the pathogen population has shifted, whereas the host genotype remains fixed (McDonald and Linde, 2002).
In both agricultural and horticultural crops, fungal pathogens are under considerable selection pressure to infect resistant host cultivars. In a mixed population of crabapple, differences in pathogenicity and virulence would be expected to develop over time, and these differences would manifest as loss of resistance within the population of crabapple. On resistance “breakdown,” the successful infection by one ascospore creates a founder effect for that successful individual pathogen; in V. inaequalis, this successful infection quickly amplifies itself through asexual reproduction resulting in thousands of conidia reinfecting the once-resistant host plant (Guerin and Le Cam, 2004). Over time, these differences in the ability to infect one cultivar but not another are identified as physiological races (Bagga and Boone, 1968a; MacHardy, 1996). The term “physiological race” is used to describe a subpopulation of a pathogen possessing a specific pattern of virulence and avirulence on specific cultivars of apple (Malus ×domestica), termed differentials (MacHardy, 1996).
In crabapple, classical major genes conferring resistance to scab have been identified and include Vf (from Malus floribunda 821), Vm (from M. micromalus 245-38 and M. ×atrosanguinea 804), Vb (from M. baccata ‘Hansen's baccata #2’), Vbj from M. baccata ‘Jackii’, Vr from M. pumila R12740-7A, and Va from Antonovka PI172623 (or “true” Va resistance as per Dayton and Williams, 1968; Hough et al., 1970; Lespinasse, 1989; as explained by Gessler et al., 2006). Resistance breakdown resulted in the identification of races of scab capable of infecting different cultivars and species of apple (Table 1) (Dayton and Williams, 1968; Schmidt, 1938). Historically, Race 1 is described as a well-sporulating isolate on popular domestic apple cultivars (M. ‘Gala’, M. ‘Fuji’, and M. ‘Red Delicious’) but elicits a hypersensitive response without sporulation on M. baccata ‘Dolgo’, R12740-7A, and M. × ‘Geneva’ (Shay and Williams, 1956). Race 2 can sporulate on M. baccata ‘Dolgo’, M. × ‘Geneva’, and certain offspring of M. ‘R12740-7A’. Race 3 is characterized as being able to only sporulate on common commercial apple cultivars and M. × ‘Geneva’, but not M. baccata ‘Dolgo’, and Race 4 differs from Race 1 by sporulating on some (but not all) progeny of M.‘R12740-7A’. Race 5 has the ability to sporulate on carriers of the Vm resistance gene, whereas Race 6 can infect some (but not all) progeny of M. floribunda 821. Race 7 is capable of infecting M. floribunda 821, the source of Vf resistance that has been introgressed into scab-resistant commercial apples worldwide. Additional races have been described as well (Bus et al., 2005). Many of these genes are also present in commercial apples, and their use is of increasing importance as growers adopt organic approaches to disease control (Crosby et al., 1992; Janick, 2002). However, a greater genetic diversity exists within the crabapple population, because tree appearance drives selection rather than fruit size and flavor.
To develop long-term, durable disease management (McDonald and Linde, 2002), plant breeding must be integrated with plant pathology to understand the evolution of pathogen populations and resistance breakdown. The purpose of this study is to use over 30 years of scab evaluation data from 287 accessions of crabapple planted at the Secrest Arboretum at the Ohio Agricultural Research and Development Center in Wooster, OH, for the purpose of identifying durable scab resistance and to examine the evolution of races of V. inaequalis concomitant with the loss of avirulence/development of resistance breakdown. This report examines and documents the breakdown of scab resistance in popular crabapple cultivars in one location in North America resulting from the development of new races within the scab population over time.
The crabapple evaluation was initiated in 1959 by L.C. Chadwick at the Secrest Arboretum at the Ohio Agricultural Research and Development Center (OARDC) in Wooster, OH. Confirmation of tree identity is solely based on taxonomic and cultivar release descriptions.
The crabapple research plot is located at Secrest Arboretum on the OARDC campus. Early ratings (before 1983) involved between two and five replicates per accession. In 1983, the National Crabapple Evaluation Project (NCEP) plot (Crablandia I) used three replicates per accession in a completely randomized design. The second NCEP trial in 2001 (Crablandia II) uses five replicates of each accession in a completely randomized design. The soil type is silt loam.
Apple scab is an endemic problem in the Midwest, and no additional inoculations were required to drive the disease. Many trees included in the trial after 1982 were part of the NCEP. From 1972 to 2005, 287 crabapple species, hybrids, and cultivars were evaluated at different times between June and August. Apple scab susceptibility ratings and observations were conducted in 1972, 1978, 1982 to 1991, and 1993 to 2005 during the months of June through August by the following individuals: P.C. Kozel, Thomas Dugan, Elton Smith, Sharon Treaster, James Chatfield, Erik Draper, Kenneth Cochran, Peter Bristol, Charles Tubesing, and David Allen. Their findings over this 33-year period were compiled for this report. These observers provided descriptive climate information for each period evaluated. Additional data to confirm observers’ assessments of the conditions at the Wooster Experiment Station were obtained from the National Climatic Data Center at http://www7.ncdc.noaa.gov/CDO/normalsproduct for years 1972 to 2001. Data from 2001 to 2005 were obtained by the OARDC and are available online at http://www.oardc.ohio-state.edu/newweather/.
To standardize 33 years of data collecting with different scales used by different observers, noted observations were defined with the following numerical values to the described symptoms: 0 = highly resistant to immune, no scab; 1 = resistant but with a trace of scab; 2 = susceptible to minor scab infection but without defoliation; and 3 = highly susceptible to scab and extensive defoliation. Scores were averaged among at least three trees per season, and scab assessments were performed between one and five times per season.
A low incidence of tree loss occurred as a result of trunk cankers and fire blight. Some cultivars were deaccessioned resulting from lack of ornamental interest, only to be “reaccessioned” on recognition of superior scab resistance. Data collected from all 287 accessions with respect to scab are presented in Table 1. Year-to-year variation in scab severity occurred regularly with more severe scab outbreaks reported in 1983, 1984, 1989, 1990, 1991, 1993, 2000, and 2003.
Apple scab is difficult to assess in the field (Croxall et al., 1952). As a result, there are several different published scales to evaluate scab susceptibility (Croxall et al., 1952; den Boer and Green, 1995; Sandskar and Liljeroth, 2005; Shay and Hough, 1952). With different scales used by at least 11 people during the 33 years of evaluations, we began by converting the observations to a basic scale that recognized only four classes of infection (Table 1). This resulted in greater consistency of scab resistance scores in cultivars evaluated over the span of many years (data not shown). Despite the broader categories in scoring resistance, qualitative assessments remained intact; trees described as highly resistant to immune to scab retained a score of zero and trees that could be infected received scores of 1, 2, or 3 depending on scab severity.
Accessions with changes in resistance status.
Over the 33-year period of evaluation, numerous cultivars exhibited increased susceptibility, including M. × ‘Adams’, M. × ‘Bob White’, M. × ‘Coralburst’, M. × ‘Liset’, M. ‘Mary Potter’, M. × ‘Molten Lava’, a selection of M. sieboldii, M. × ‘Ormiston Roy’, M. × ‘Purple Prince’, M. × ‘Selkirk’, M. × ‘Sentinel’, M. × ‘Sugar Tyme’, M. sieboldii (formerly designated as M. zumi) ‘Calocarpa’, M. × ‘Red Jewel’, M. halliana ‘Parkmanii’ and M. floribunda. Most other named selections either were initially identified as scab-susceptible or were deaccessioned as a result of fire blight, animal damage, or other major disease problems (Table 2).
A summary of cultivars identified in this study with 10+ years resistance to scab.
Periods conducive to scab outbreaks coincided with putative changes in the scab population corresponding with a loss of avirulence/resistance.
Susceptibility of previously resistant accessions occurred in the high rainfall years of 1989 (M. × ‘Adams’), 1990 (M. × ‘Molten Lava’), 1993 (M. × ‘Liset’, M. × ‘Professor Sprenger’, M. × ‘Selkirk’, M. × ‘Sugar Tyme’, M. halliana ‘Parkmanii’), 2000 (M. × ‘Prairifire’), and 2003 (M. × ‘Lancelot’, M. × ‘Purple Prince’, M. × ‘Sentinel’). Breakdown of other cultivars in 1994 (M. × ‘Mary Potter’) and 1996 (M. floribunda) occurred during average to low rainfall years. These data are highlighted in bold in Table 1.
Species, hybrids, and cultivars with durable resistance.
Crabapple trees defined here as having durable resistance possessed resistance at the end of the study or for greater than 10 years. Throughout the entire study, M. baccata ‘Jackii’ [resistance gene Vbj (Gygax et al., 2004)], possessed durable resistance, although occasional trace infections were reported in 1986 and 1987 (Table 2). M. × ‘Silver Moon’ exhibited no scab for the entire 33 years. M. baccata ‘Dolgo’ possessed very stable resistance, although infection was noted in 2003; M. sargentii ‘Sargent’ possessed solid resistance/immunity for the entire trial, and named M. sargentii cultivars like M. × ‘Firebird’, M. × ‘Rose Low’, and M. × ‘Tina’ possessed resistance for the entire time they were evaluated. M. ×robusta and M. ×robusta ‘Percifolia’, the peachleaf crabapple, showed no incidence of scab for almost 20 years of the trial until deaccessioned as a result of fire blight. M. × ‘Rosseau’ showed no incidence of scab for 20 years before being deaccessioned as a result of fire blight. M. × ‘Strawberry Parfait’ possessed solid resistance for almost 25 years before scab infection. M. tschonoskii, a rare crabapple grown mostly in arboreta, was scab-resistant for over 20 years until other foliar diseases and fire blight necessitated its removal. M. yunnanensis ‘Veitchii’, an unusual crabapple with excellent scab resistance, but very susceptible to fire blight, was removed in 1992. See Table 2 for a list of durable, scab-resistant accessions.
Races of scab present based on susceptible cultivars.
In evaluating Table 1, it must be noted that the inclusion of the universal susceptible cultivar M. × ‘Royal Gala’ or M. × ‘Red Delicious’ for identification of Race 1 (Bus, 2006) from commercial apples did not occur. However, M. × ‘Hopa’, a universally known scab-susceptible cultivar wherever crabapples are grown in North America, serves as an adequate substitute that demonstrates reliable susceptibility throughout the entire 33-year study.
Race 2, capable of infecting M. baccata ‘Dolgo’ and M. × ‘Geneva’ (Shay and Williams, 1956), was not observed over the course of this study. The existence of stable resistance in M. baccata ‘Dolgo’ suggests the absence of Race 2 at Secrest Arboretum. However, the presence of Race 3 is suggested by the susceptibility of M. × ‘Geneva’ that developed in 1986, whereas M. baccata ‘Dolgo’ essentially remained uninfected. Race 4, capable of infecting some progeny of M. ‘12740-7A’, cannot be considered in this study as a result of an absence of differentials for this race.
Unlike Europe, where Races 5, 6, and 7 were found, only Races 1 through 4 have been reported in North America (Dayton and Williams, 1968). The recurring presence of scab on M. × ‘Prairifire’ suggests the development of Race 5 in North America. M. × ‘Prairifire’, a Vm-resistant crabapple (Mattison and Nybom, 2005; S. Korban, personal communication), first developed scab in 2000. There are no differentials present at the Secrest Arboretum to identify the presence of Race 6. Race 6 is not able to infect the M. floribunda 821.
The presence of scab on a M. floribunda selection that was reliably scab-resistant for at least 40 years suggests, but does not conclusively demonstrate, the development of Race 7 in North America. An overview of Venturia race breakdown and susceptible hosts at Secrest can be seen in Table 3.
The Malus species, hybrids, and named cultivars in this study represent a tremendous diversity of genetic background, aesthetic characteristics, and scab resistance and included Malus species from Asia (M. floribunda, M. halliana, M. spectabilis, M. toringoides, M. transitoria, M. tschonoskii, M. yunnanensis), North America (M. coronaria, M. ioensis), and Europe (M. florentine, M. pumila). It is important to note that the delineation of species within the genus Malus has been problematic (Luby, 2003; Robinson et al., 2001). Accessions were reported as recorded with additional notes provided by the authors if changes of taxonomy have occurred as is the case for M.sieboldii/M. zumi varieties (Fiala, 1994; Luby, 2003). Unfortunately, as noted, “scores were averaged between at least three trees per season”; no effort was made on the part of any of the observers to note the novel occurrence of scab on any previously “resistant varieties” until 2005 (Chatfield et al., 2005).
The results from this study indicate that various levels of both qualitative (major) and quantitative (minor) resistance to V. inaequalis exist within the genus Malus. Aderhold (1899) previously observed that V. inaequalis could be divided into distinct physiological races distinguishable by their different ability to induce sporulating lesions or only flecks on various cultivars. These differences of V. inaequalis pathogenicity patterns were identified as regional in nature as early as 1938 (Schmidt, 1938). This regionality of races was again observed with M. sieversii seed collected in Central Asia and grown and screened in the United States. When M. sieversii seed (collected in Central Asia) were raised in the United States, these seedlings had high levels of apple scab resistance that varied with the geographic region where the seedlings were screened; from 27% of the population exhibiting resistance in New York to 65% of the population screened in Minnesota. The parents of these seedlings (wild apples from Central Asia, specifically those from the Karatau mountains) were without scab; the Zailisky site was 35% scab free, and the Tarbagatai site did not have observable scab (Forsline et al., 2003). These sites are considered xeric, and the absence of free water for germination would reduce the incidence of scab. This suggests the differences in scab evaluations in the United States [e.g., Jacobs and Spravka (1996) compared with the data here] may be the result of: 1) changes in environment between arid Central Asia and the wet and humid eastern half of the United States; and 2) regional differences in races of scab present at those locales.
The study of V. inaequalis–Malus genetics began before scab resistance breeding programs (Keitt and Langford, 1941; Keitt and Palmiter, 1938) or even an understanding of gene-for-gene systems (Flor, 1956). Boone and Keitt (1957) identified seven R–Avr interactions involving moderately resistant cultivars and identified several multigenic gene-for-gene relationships. In these studies on V. inaequalis, the avirulence-resistant gene interaction was designated “p-x,” with p describing the pathogenicity gene and x indicating the allele number. In keeping with previous literature describing avirulence (Boone and Keitt, 1957; Williams and Shay, 1957), only the p-8 avirulence locus is present in the population at Wooster, OH (Table 3). The loss of avirulence genes in this population includes p-10 through p-19 (Bagga and Boone, 1968a, 1968b; Williams and Shay, 1957). This information is included in Table 3 for the sake of completion. Based on the increased understanding of the V. inaequalis–Malus gene-for-gene interactions, it is apparent that a new system of designating the nomenclature of these interactions is needed because the current one is both confusing and inadequate (Bus, 2006).
In evaluating the composition of scab races at the arboretum, M. × ‘Hopa’ serves as our universally susceptible cultivar and demonstrated reliable susceptibility throughout the entire 33-year study. This cultivar is a Race 1 indicator despite previously being identified as possessing p-14 and p-15 avirulence factors (Bagga and Boone, 1968a). It should be stressed that even the Race 1 differential used in apple breeding, M. ‘Royal Gala’ possesses some resistance factors (Parisi et al., 2004). In reality, Race 1 exists as a highly complex, structured population with an undetermined number and frequency of avirulence alleles that prevent this race from infecting previously identified differentials (Bus et al., 2005; Gessler et al., 2006).
Race 2, capable of infecting M. baccata ‘Dolgo’, M. × ‘TSR34T132’, and M. × ‘Geneva’ (Gessler et al., 2006), was not observed over the course of this study. This race has been reported in Sweden on some segregants of the Russian cultivar R12740-7A (Sandskar and Liljeroth, 2005), but no work has been performed to confirm that M. baccata ‘Dolgo’ and these accessions carry the same resistance gene. Race 2 was reported previously in South Dakota (Shay and Williams, 1956) and has also been observed in Minnesota (J. Beckerman, personal observation). The existence of stable resistance in M. baccata ‘Dolgo’ suggests the absence of Race 2 at Secrest Arboretum. However, the presence of Race 3 is suggested by the susceptibility of M. × ‘Geneva’ that developed in 1986, whereas M. baccata ‘Dolgo’ essentially remained uninfected.
There is no evidence that Race 4, capable of infecting some progeny of M. ‘12740-7A’, is present in the arboretum as a result of an absence of differentials for this race. Tremendous confusion exists in the literature regarding Race 4 (Bus, 2006). This confusion arises from Shay et al. (1962) who stated that the original Russian apple accession contains “at least 3 gene pairs, only one of which is resistant to all known races” without referencing any specific races of V. inaequalis. However, it is safe to assume that Races 1 through 3, the only ones identified to that point, are understood as “all known races.” Based on this report (Shay et al., 1962), none of the accessions in this study could knowingly serve as reported differentials for Race 4, although other susceptible selections of M. pumila were included in this location (e.g., M. pumila ‘Niedzwetzkyana’, M. pumila ‘Elise Rathke’, and M. pumila ‘Paradise Foley’) and were susceptible to scab throughout most of the evaluation.
The Vm resistance gene, derived from either M. micromalus 245-38 or M. ×atrosanguinea 804 (Hough, 1944), is one of several resistance genes found in the small-fruited, Asiatic species of crabapples (Williams and Brown, 1968). A tightly linked DNA marker for Vm, SCAR B12, was previously used to identify Vm in M. × ‘Prairifire’ (Mattison and Nybom, 2005; S. Korban, personal communication). Cheng et al. (1998) and Patocchi et al. (2005) have identified markers tightly linked to Vm resistance in M. × hartwigii, M. halliana, M. hupehensis, M. fusca, and in M. × ‘Prairifire’ (S. Korban, personal communication). However, not every accession of these species was found to possess the markers for Vm (Cheng et al., 1998; Patocchi et al., 2005). M. ×atrosanguinea developed scab as early as 1978, and M. ×micromalus ‘Midget’ was deaccessioned in 1993. The breakdown in resistance in M. × ‘Prairifire’ was first reported in 1997 (P. Pecknold, personal communication) in Indiana but was not detected at the Secrest Arboretum until 2000. Unfortunately, other carriers of the Vm gene, M. ×hartwigii, M. halliana, M. hupehensis, and M. fusca were deaccessioned as a result of fire blight in the early 1990s. M. × ‘Mary Potter’, a hybrid of M. sargentii ‘Rosea’ × M. ×atrosanguinea, developed scab in 1994. Work is currently underway to examine these cultivars for the existence of the linked Vm marker and to look at M. × ‘Liset’, a multibred with M. ×atrosanguinea in the background, to determine if Vm exists in this cultivar as well (Patocchi et al., 2005) and if the breakdown of resistance was attributable solely to the development of Race 5 at Secrest. Curiously, Race 5 has only been reported in Europe (Dayton and Williams, 1968); the development of scab on M. × ‘Prairifire’ suggests a race with virulence similar to Race 5 has developed North America. A cultivar recorded as M. ×atrosanguinea was evaluated as scab-susceptible for the entire 10 years it was included in the trial; it is unknown if this selection possesses the Vm gene or if it is M. ×atrosanguinea 804. M. atrosanguinea 804 was included in the release of cultivars by The Arnold Arboretum in the early 1900s (Crandall, 1926).
Race 6 isolates, capable of infecting some Vf scab-resistant commercial apples, including M. × ‘Prima’, M. ‘Judeline’, and M. × ‘Florina (querina)’ (Parisi and Lespinasse, 1996; Parisi et al., 1993), could not be scored as a result of the absence of these cultivars in the trial. Other M. floribunda selections or hybrids include M. × ‘Exzellenz Theil’, an M. floribunda × M. prunifolia ‘Pendula’ hybrid (Fiala, 1994) that was very susceptible to scab when evaluated, M. ×arnoldiana, and M. ×Scheideckeri. There is no evidence to suggest that any of these cultivars possesses Vf resistance.
M. floribunda, the Japanese flowering crabapple, is considered one of the finest crabapples in cultivation and was introduced into Europe by von Siebold in 1853 from Nagasaki, Japan. The place where this tree grows wild still remains unknown (Fiala, 1994). The selections at Secrest match the taxonomic description of Crandall (1926). Selection M. floribunda 821 was identified as resistant in 1943 by Hough (1944) and Hough et al. (1953) and has been used extensively as a source of resistance in commercial cultivars (Crosby et al., 1992; Janick, 2002). Concomitantly, many of these selections were regularly distributed to arboreta throughout the United States in the 1940s to 1960s for crabapple evaluations (Fiala, 1994); unfortunately, many were misidentified or misnamed (Jefferson, 1970). Records and morphology indicate that this tree is, in fact, M. floribunda, although whether it is a scion of M. floribunda 821, the parent tree that provided both the Vf and Vfh resistance genes that were introgressed into most scab-resistant commercial apples (Janick, 2002), remains undetermined. The identification of Race 7 in the United Kingdom parallels its potential discovery here; a backyard crabapple was identified as a M. floribunda with scab. Inoculation of M. floribunda 821 with scab from the backyard crabapple demonstrated susceptibility and confirmed the development of Race 7 (Roberts and Crute, 1994). The work to conclusively establish the host range and specificity of this M. floribunda-infecting isolate is currently underway.
Until recently (Parisi et al., 1993), Vf was considered to be the most durable source of resistance with Vf-derived cultivars resistant to scab for over 50 years. Not all selections of M. floribunda possess this allele and that selfed M. floribunda 821 had a segregation of resistance that indicates that Vf is a dominant resistance gene (Hough et al., 1953). However, the fact that resistance in this isolate was effective for at least 40 years at Secrest (Chadwick, 1965; this publication) under intense selection pressure supports the contention that it is a selection of M. floribunda with the Vf gene, if not in fact the original M. floribunda 821 (J. Janick, personal communication). The resistance observed at Secrest before 1997 was complete immunity (Score 0), not the light infection (Score 1) described by Parisi et al. (1993) with respect to the identification of Race 6 on commercial apples with Vf resistance. This is consistent with the descriptions by Hough et al. (1953) and Janick (2002). Furthermore, the breakdown of this resistance was absolute with defoliating scab occurring by 2003 and continuing in subsequent years. Current work is underway to confirm the presence of the Vf resistance gene, HcrVf2, in this isolate, the resistance gene that was identified as sufficient to confer scab resistance in M. ‘Gala’, a universally susceptible cultivar for Race 1 differentiation (Belfanti et al., 2004).
Despite the apparent breakdown in Vm and Vf resistance, this study confirms anecdotal reports from nursery managers and landscapers regarding the resistance of M. sargentii selections (‘Sargent’; ‘Firebird’®, = ‘Select A’ PP 12,621, and ‘Tina’), all of which demonstrate considerable resistance to scab. Buttner et al. (2000) identified M. sargentii as a potential source of scab resistance for commercial apple in Europe, although it should be noted that, in addition to small fruit, it is fairly susceptible to fire blight (Richter et al., 2005; Shay et al., 1962). Nursery managers also report the durable resistance of M. baccata ‘Jackii’, an observation confirmed in this study. However, scab has been reported on M. baccata ‘Jackii’ in Sweden (Sandskar and Liljeroth, 2005), so resistance breakdown of this selection is possible in North America in the future.
The breakdown of scab resistance in several hybrid Malus taxa is interesting. M. × ‘Bob White’, introduced in 1876 (Jefferson, 1970), is a suspected M. sieboldii var. zumi hybrid and would therefore be expected to possess comparable polygenic resistance of other M. sieboldii var. zumi selections (e.g., M. var. zumi ‘Wooster’ and var. zumi ‘Calocarpa’). M. × ‘Strawberry Parfait’, a cross between M. hupehensis and M. ×atrosanguinea, also was resistant in 1994, developing scab the same time as M. × ‘Mary Potter’, the offspring of a M. ×atrosanguinea × M. sargentii ‘Rosea’ cross (Fiala, 1994), strongly suggesting the development of a new race of scab concomitant with Vm resistance breakdown. The long-term scab resistance and subsequent development of scab on this selection suggests a minimal oligogenic resistance or that re-evaluation is warranted of the dogma that polygenic resistance is more durable than monogenic resistance. Other crabapples with durable, and probably multigenic, resistance include: M. × ‘Beverly’, a large, upright crabapple with pink flower buds, fragrant, white, five-petaled flowers, bright green leaves and small, red, persistent, glossy fruits strongly suggestive of M. baccata parentage; M. × ‘Silver Moon’, another probable M. baccata hybrid with white flower buds that was described as scab-susceptible by Nichols but did not develop scab at Falconskeape (Fiala, 1994), or throughout our study or in the study by Jacobs and Spravka (1996); and M. × ‘White Angel’, a chance seedling of putative M. sieboldii parentage (Fiala, 1994), also reported by Jacobs and Spravka (1996) as resistant.
In conclusion, data presented in this article demonstrate the durability of resistance of M. baccata ‘Jackkii’, M. sargentii selections, including ‘Sargent’, which was introduced to The Arnold Arboretum in 1892 from Japan, M. × ‘Beverly’, M. × ‘Silver Moon’, and M. × ‘White Angel, and the identification of other potentially stable resistance genes in plants with susceptibility to fire blight or other diseases (Table 2). Over a 33-year period, the evolution of races of V. inaequalis can be observed and the corresponding breakdown of resistance of at least one and possibly two major genes, Vm and Vf/Vfh. The absence of Races 2 and 4 within this population is noteworthy. Work is underway to confirm the presence of HcrVf2 in the susceptible M. floribunda trees at Secrest and conclusively demonstrate the existence of Race 7 and the breakdown of Vf/Vfh resistance in North America. With further integration of the plant pathology and plant breeding efforts, ultimately, the durability of host resistance may be predicted by careful evaluation of pathogen population structure and can result in more effective and durable disease control.
ChatfieldJ.A.DraperE.A.HermsD.A.CochranK.A.2005Apple scab on crabapple at Secrest Arboretum: 2005. The Ohio Agricultural Research and Development Center11712111 Mar. 2009<http://ohioline.osu.edu/sc197/pdf/sc197.pdf>.
Crandall C.S. 1926. Apple breeding at the University of Illinois. Bulletin No. 275.
1 To whom reprint requests should be addressed; e-mail janna@purdue.edu.

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