dynamics of the pyrethroid knockdown resistance allele in ... - CiteSeerX

Am. J. Trop. Med. Hyg., 70(6), 2004, pp. 591–596 Copyright © 2004 by The American Society of Tropical Medicine and Hygiene

DYNAMICS OF THE PYRETHROID KNOCKDOWN RESISTANCE ALLELE IN WESTERN KENYAN POPULATIONS OF ANOPHELES GAMBIAE IN RESPONSE TO INSECTICIDE-TREATED BED NET TRIALS ARAM D. STUMP, FRANCIS K. ATIELI, JOHN M. VULULE, AND NORA J. BESANSKY Center for Tropical Disease Research and Training, Department of Biologic Sciences, University of Notre Dame, Notre Dame, Indiana; Center for Vector Biology and Control Research, Kenya Medical Research Institute, Kisumu, Kenya

Abstract. Permethrin and DDT resistance in Anopheles gambiae s.s. associated with a leucine-serine knockdown resistance (kdr) mutation in the voltage-gated sodium channel gene was discovered recently in western Kenya where a large scale permethrin-impregnated bed net (ITN) program has been implemented. Collections of An. gambiae s.l. were made from intervention and control villages prior to and after onset of the program. The kdr genotypes were determined using allele-specific polymerase chain reaction diagnostic tests. In An. gambiae s.s., the frequency of the kdr mutation prior to ITN introduction was ∼3−4% in western Kenya and zero in samples from the coast. After ITN introduction, the kdr mutation increased in ITN and neighboring villages from ∼4% to ∼8%, but remained unchanged in villages at least 20 km distant and was not detected in coastal Kenya. The identical leucine-serine mutation was found in a single An. arabiensis individual among 658 tested. The leucine-phenylalanine kdr mutation common in west African An. gambiae populations was not detected in An. gambiae s.l. from Kenya. Implications for the population structure and control of An. gambiae are discussed. INTRODUCTION

In western Kenya, intense perennial malaria transmission is maintained by a relay among all three major African vector species. In the Asembo area near Kisumu, a large-scale ITN project was initiated in 1997, which was preceded by several smaller trials in villages northwest of Asembo dating back to 1990.14 As early as 1991, after one year of small-scale ITN use, there was a reduction in permethrin susceptibility in An. gambiae from ITN villages but not from villages without ITNs.15 Subsequent studies showed that reduced susceptibility was due both to increased detoxification caused by elevated cytochrome P450 monooxygenase activity, and to target site insensitivity caused by the L1014S kdr allele.6,13 A preliminary survey of collections made in 1999 from four villages northwest of Asembo suggested that the L1014S kdr allele was relatively rare (∼3.6%), and that its appearance predated ITN use,13 but its frequency in and around Asembo in response to the major ITN project had not been investigated. The Asembo ITN project covered an area of 200 km2 and a population of ∼55,000 persons.14 After distribution of ITNs to half the population in early 1997, ITNs were distributed to the remainder in early 1999, so that all sleeping spaces were covered by ITNs. Insecticide re-treatment has been maintained since 1999 through organized mass-campaigns, ensuring continuous high-coverage (Hawley WA, unpublished data). To determine if large-scale and long-term ITN use in the Asembo area had impacted the frequency of the L1014S kdr allele, we genotyped both An. gambiae and An. arabiensis from collections made before and after the implementation of ITNs from ITN and non-ITN villages in western Kenya. In addition, we genotyped An. gambiae collected in coastal Kenya at similar times. Finally, we screened An. gambiae s.l. samples for the presence of the west African L1014F kdr allele, which may be spreading eastward.

Malaria results in 500 million clinical cases and at least one million deaths annually, 90% of which occur in Africa.1 Insecticide-treated bed nets (ITNs) have shown promise in reducing malaria morbidity and mortality.2 The bed net is a physical barrier against vector mosquitoes such as Anopheles gambiae s.l. and An. funestus, which generally bite at night. The insecticide, a synthetic pyrethroid such as permethrin, acts not only by killing mosquitoes but also as an irritant that repels them from the net, improving the barrier when the net drapes directly upon the inhabitant or becomes torn.3 When ITNs are implemented at high coverage in communities and are re-treated with insecticide at least biannually, they also confer area-wide affects on the mosquito population, protecting those living in houses lacking nets.4,5 Because the insecticide is the key component of this strategy, there is concern that resistance in mosquito populations could reduce the effectiveness of ITNs. Although the impact of pyrethroid resistance on ITN efficacy in malaria control is not yet clear,6,7 resistance is certainly present in several different anopheline vector species, including the major Afrotropical malaria vectors An. gambiae, An. funestus, and An. arabiensis.6,8–10 In An. funestus, pyrethroid resistance was detected in South Africa.8 The mechanism appears to be increased rates of detoxification through elevated levels of mixed function oxidases.11 Metabolic resistance to permethrin has also been found in An. gambiae from Kenya.6 An additional resistance mechanism is found in An. gambiae that is conferred by mutations in the voltage-gated sodium channel, which represents the target site for DDT and pyrethroids. Permethrin resistance associated with target site insensitivity, known as knockdown resistance (kdr), has arisen independently at least twice in this species. Widespread permethrin resistance in west Africa is due to a leucine-phenylalanine substitution at position 1014 of the sodium channel gene (L1014F kdr allele), in the S6 hydrophobic segment of domain II.12 A different mutation at the same amino acid position, causing a leucine-serine substitution (L1014S kdr allele), was associated with permethrin resistance in An. gambiae from Kenya.13

MATERIALS AND METHODS Mosquito sampling. The distribution of villages from which mosquitoes were collected in western and coastal Kenya is shown in Figure 1. In May 1987 before ITN trials began in western Kenya, indoor resting adult female Anopheles gambiae s.l. were sampled by manual aspiration from most of

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FIGURE 1. Distribution of Anopheles gambiae collection sites in western Kenya near Kisumu town. Black circles represent approximate locations of the centers of villages, which consist of widely dispersed family compounds. Not shown are Muhroni, which is located ∼65 km east of Ahero in western Kenya, and Vanga, which is located < 0.5 km from Jego on the Kenyan coast.

these villages (Asembo, Kisian, Wathorego, and the escarpment above it, Ahero, Nyakach, Muhoroni, Jego, and Vanga) and allowed to oviposit. Offspring were reared and frozen at −80°C as adults. One adult from each family was chosen at random for genetic analysis. Following the limited ITN trials that began in 1991 and the extensive trial in Asembo and Gem initiated in 1997,14 additional collections were made in western Kenya. In 2001, indoor resting collections by manual aspiration were made in Kisian (April), Rota and Miwani (August), and Ahero (March). Because of the very significant reduction in indoor resting densities in the ITN area, sampling from Ahero and Seme in January−March and November 2001 and May 2002 was by spray sheet collection using 0.025% pyrethrum extract synergized with the mixed function oxidase inhibitor piperonyl butoxide (0.1%) as previously described.7 Specimens were stored desiccated on silica gel at room temperature. During 1996, Tovi Lehmann (Centers for Disease Control and Prevention, Atlanta GA) collected indoor resting mosquitoes in Jego by manual aspiration and kindly provided DNA extracted from individual specimens. Molecular analysis. Specimens morphologically identified as belonging to the An. gambiae complex16 were assigned to species using a standard ribosomal DNA polymerase chain reaction (PCR) assay.17 The DNA was isolated from individual specimens using DNeasy tissue kits (Qiagen, Inc., Va-

lencia CA) and resuspended in 50 ␮L of elution buffer. Before use, DNA was diluted 1:7 in TE buffer (10 mM Tris, 1 mM EDTA). The kdr genotypes were determined using two allele-specific PCR assays as described by the investigators: one diagnostic for the L1014S kdr allele found in east Africa,13 and the other for the L1014F kdr allele found in west Africa.12 For the exceptional An. arabiensis individual carrying the L1014S kdr allele as judged by results of the allelespecific assay, a portion of the sodium channel gene containing this mutation was PCR amplified and directly sequenced using the primers Agd1 and Agd2.12 Following purification of the PCR product (QIAquick PCR Purification kit, Qiagen, Inc.), sequencing was performed using the ABI PRISM Dye Terminator Cycle Sequencing kit (Applied Biosytems, Foster City, CA) and the ABI 3700 sequencer (Applied Biosystems). For 19 An. gambiae kdr carriers collected from the same house in Seme (see Results), the nucleotide sequence of 539 basepairs of the mitochondrial ND5 gene (positions 70227560 in the An. gambiae reference sequence18) was PCR amplified using primers 19CL and DMP3A, as described elsewhere.19 Direct sequencing of purified PCR products was performed as described earlier in this report using primers 6848 and DMP3A. The DNA sequence alignment was performed using Lasergene software (DNASTAR, Inc., Madison, WI). RESULTS The L1014S kdr allele was detected over a 15-year period in An. gambiae samples from the Kisumu area of western Kenya, but was not found in coastal Kenya. The frequency of the L1014S kdr allele in the western Kenyan An. gambiae samples is shown in Table 1. This allele was never found in the homozygous state in any of 1102 genotyped specimens. In agreement with an initial study,13 this kdr allele was present at low frequency (∼3–4%) in samples from six villages in western Kenya in 1987, before any ITN use in the area. While still relatively low, the kdr frequency had doubled to ∼8% by 2001 in the Asembo ITN area, a significant increase over 1987 levels (P ⳱ 0.04, by one-tailed Fisher’s exact test of a 2 × 2 contingency table). One year later in Asembo, the kdr allele was present at the same elevated frequency (∼8%). In all other samples from non-ITN villages in western Kenya except one, the kdr frequency remained stable at ∼3−4% between 1987 and 2002. The exception was the village of Seme, adjacent to Asembo. Both villages consist of family compounds dispersed widely but homogeneously;14 as measured from approximately central locations, they are ∼5 km apart. Despite

TABLE 1 Knockdown resistance frequencies in Anopheles gambiae s.s. populations in Kenya* Locale Year

Asembo

Seme

Kisian

Western Kenya

1987 2001

0.038 (n ⳱ 160) 0.082 (n ⳱ 366)

0.028 (n ⳱ 36) 0.028 (n ⳱ 316)

0.025† (n ⳱ 162) 0.027§ (n ⳱ 410)

2002

0.078 (n ⳱ 64)

– 0.029‡ (n ⳱ 312) 0.475¶ (n ⳱ 40) 0.074 (n ⳱ 204)

* Sample number (n) is the number of alleles. † Pooled results from Kisian, Wathorego, escarpment above Wathorego, and Nyakach and Muhoroni villages. ‡ Results from at least 63 houses excluding house X. § Pooled results from Kisian, Rota, and Miwani. ¶ Results from a single house (X).

–

DYNAMICS OF THE AN. GAMBIAE KDR ALLELE IN KENYA

the absence of ITNs in Seme, the kdr frequency reached the same level as in Asembo (∼8%) by 2002. The kdr insects were sampled in Seme not only from houses within ∼500 meters of the Asembo border, but also from houses as distant as 1.6, 2.7, and 4.8 km from the border. In all but one collection from western Kenya in 2001−2002, individuals carrying the L1014S kdr allele were distributed in an apparently random pattern among houses within a village. The exception was the spray catch collection made in 2001 from at least 64 houses in Seme. Of the 28 heterozygous kdr insects detected in the total sample, 19 originated from a single house (Table 1). The other nine were distributed among at least six houses, a pattern typical of other collections. To control for the increased influence of the subsample represented by that house (hereafter X), the 2001 Seme collection was partitioned into two groups: one containing all An. gambiae collected in house X, and the other containing all An. gambiae collected elsewhere in Seme (Table 1). The unusual clustering of kdr carriers found in house X could be most simply explained if they were more closely related to one another than expected by chance. We hypothesized that they represented the F1 offspring of one or few mothers. If true, these individuals should share one or only a small number of mitochondrial DNA (mtDNA) sequence haplotypes. If these individuals were no more related to one another than to the population at large, the expected number of haplotypes should be nearly as high as the sample size and the incidence of shared haplotypes should be quite low, according to the results of previous mtDNA studies of An. gambiae at the house level within Asembo.19 To examine this question, the sequence of a polymorphic segment of the mtDNA ND5 gene was determined successfully for 18 of 19 kdr carriers captured in house X. Only two distinct haplotypes were detected, with five polymorphic positions overall (Table 2). The two haplotypes were found in 7 (39%) and 11 (61%) of the kdr carriers. These results indicate a degree of genetic relatedness that is much higher than the norm for An. gambiae in comparable samples, and suggest that the kdr carriers in house X represent two groups of siblings descended from two mothers. If true, the adjusted kdr frequency in the 2001 Seme collection would be 3.5% (11 of 314). Anopheles gambiae and An. arabiensis were found in sympatry in most samples from western Kenya. The only exception was Ahero, a village situated amid large-scale rice cultivation, where only An. arabiensis has been collected for many years20 (Atieli FK, unpublished data). Anopheles arabiensis captured in western Kenya during 2001 were screened for the presence of the L1014S kdr allele. This kdr allele was absent from all An. arabiensis sampled from Ahero (n ⳱ 346 alleles). It was also absent in Seme (n ⳱ 266 alleles), where its frequency in synchronous populations of An. gambiae was 3−4%

TABLE 2 Mitochondrial DNA haplotype composition for Anopheles gambiae knockdown resistance carriers sampled from house X in Seme, Kenya during 2001* Haplotype no.

Frequency

7027

7099

7105

7108

7255

1 2

11/18 7/18

T C

C T

T C

G A

G A

* Only the polymorphic positions are shown, numbered according to the reference sequence.18

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in 2001. Interestingly, of 572 An. arabiensis alleles sampled from Asembo, one L1014S kdr allele was identified using the allele-specific PCR assay.13 The sodium channel gene sequence of this individual at amino acid position 1014 confirmed that it carried the same nucleotide substitution as the L1014S kdr allele found in An. gambiae. Subsamples of Kenyan An. gambiae s.l. were screened for the presence of the L1014F kdr allele often found at a relatively high frequency (> 80%) across geographically widespread regions of west Africa.21–24 To date, this allele has not been detected farther east than the Central African Republic22, although its distribution may be expanding eastward. The L1014F kdr allele was not found among 50 An. gambiae randomly chosen from each of the 2001 collections in Asembo and Kisian. Similarly, the L1014F kdr allele was absent from the 50 An. arabiensis examined in each of two collections from 2001 in Asembo and Ahero. DISCUSSION The L1014S kdr allele was found in An. gambiae samples collected in 1987 from villages within 50 km of Kisumu in western Kenya. These samples predated the implementation of even small-scale ITN trials in the region. However, prior instances of indoor residual insecticide spraying directed against malaria vectors in the area involved not only the organophosphate fenitrothion and the cyclodiene dieldrin, but also DDT, which was applied from 1945 to 1949 to ∼4,000 huts across a 110-km2 area centered on Kericho.20,25 In addition, DDT has a long history of usage in Kenya against pests of cotton, maize, and cattle, as well as in tsetse fly control.26 Although banned for use in agriculture in 1986, its persistence in the environment and continuing availability to farmers raises the possibility that if selection pressure were responsible for maintenance of the L1014S kdr allele prior to 1987, it may have been exerted by insecticides used for agriculture rather than those used for public health. In An. gambiae populations, the frequency of the L1014S kdr allele doubled in the ITN test village and its nearest neighbor from 1987 to 2001, but not outside of this area. This suggests that ITN use has further selected for the kdr mutation. However, although its frequency increased, the mutation is still relatively rare even where selection is presumably occurring, and no kdr homozygotes were found, in marked contrast to the west African L1014F kdr allele.21–24 Several factors that are not mutually exclusive might explain the low frequency of the kdr allele in Kenya. The first factor is methodologic. In Asembo and its neighbor Seme in 2001−2002, An. gambiae densities were so low that sampling by manual aspiration was not practical, and a formulation of permethrin synergized by piperonyl butoxide (to overcome metabolic resistance) was used for spray catches. In all other villages, sampling was manual. Because of target site resistance conferred by the kdr allele, we cannot rule out the possibility that some kdr insects (heterozygotes and especially homozygotes) either escaped being knocked down, or recovered and flew off before capture. Therefore, our estimates of the frequency of the L1014S kdr allele in Asembo and Seme are conservative. There are also biologic factors that could explain the low frequency of the kdr allele. It is possible that a constant influx

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of susceptible alleles from the areas surrounding Asembo ensures that nearly all individuals carrying the kdr allele will be heterozygous. If true, this raises the question of why similar population dynamics have not prevented the west African kdr allele from sharply increasing in frequency within locales and spreading geographically among them within the S molecular form of An. gambiae. The joint operation of additional factors in Kenyan populations may help address this question: an alternative resistance mechanism independent of target site insensitivity, lower levels of permethrin resistance conferred by L1014S than L1014F, and higher fitness costs associated directly or indirectly with the L1014S mutation. If metabolic resistance conferred by cytochrome P450s is the more important resistance mechanism in these Kenyan populations,6,13 the kdr genotype would be a poor indication of phenotypic (behavioral or physiologic) resistance to permethrin. It is also important to consider that while L1014S may confer high levels of resistance to DDT, cross-resistance to permethrin could be much lower. Moreover, there may be significant reductions in fitness associated with this kdr allele, which might explain why no kdr homozygotes were found. Among insect species where kdr mutations have been associated with resistance, the leucine to serine substitution is much less prevalent than the leucine to phenylalanine, possibly indicating a greater physiologic cost to the altered sodium channel. Another explanation may be that the kdr allele is tightly linked to a recessive lethal gene. Preliminary evidence supporting this prediction comes from single pair crosses involving a permethrin resistant strain (RSP) colonized from the Asembo area.15 (Ranson H, unpublished data). Previous population genetic studies of An. gambiae using both mtDNA and microsatellite markers have suggested relatively shallow structure extending from the microgeographic to the macrogeographic scale.19,27,28 Confounding the interpretation of these results is evidence for relatively recent population expansion,29 which poses difficulties for inferring rates and patterns of gene flow by most indirect genetic methods.30 Monitoring the spread of insecticide resistance genes is not only an essential component of operational research on bed net efficacy,31 but also can provide important insight into contemporary gene flow. Despite very high frequencies across west Africa extending into the Central African Republic, the L1014F allele has not yet reached populations in Uganda22 and we did not find it in western Kenya. Moreover, we did not find the L1014S kdr allele in coastal Kenya. The Great Rift Valley approximately bisects Kenya into east and west, and it represents a significant barrier to gene flow in An. gambiae.32,33 The presence of this kdr allele on one side of the Rift and its absence on the other is further evidence for the genetic division in An. gambiae populations. Interestingly, the L1014S allele is not limited to western Kenya; it has been found in both Burundi and Uganda, at frequencies even higher than those reported here in the latter country (van Bortel W, unpublished data). At a more local level, it is worth noting that the L1014S kdr allele increased in frequency in Seme in parallel with Asembo by 2002, despite the absence of ITNs in Seme. Indeed, An. gambiae densities were as low in this non-ITN village as they were in Asembo. This suggests that the geographic area occupied by a deme is at least 5 km, and that migration from surrounding untreated areas was insufficient to reverse the effect of the ITNs. The 20 An. gambiae sampled in 2001 from a single house in

Seme were noteworthy because they had a kdr frequency much higher than was found in the general population. One possible explanation is that they were closely related. A 1997 survey of the An. gambiae mtDNA ND5 gene carried out in Asembo found that specimens collected from the same house have very high haplotype diversity and based on these and corresponding microsatellite data, the investigators concluded that mosquitoes collected within a house were not more closely related to one another than those collected from the same or nearby villages.19 In the Seme sample from house X, we found only two haplotypes among 18 kdr carriers (Table 2), indicating an unusually high level of relatedness. The explanation for this phenomenon is not apparent, but it may be a consequence of the severely reduced population density, and presumably lower effective population size in the bed net area. The presence of the L1014S kdr allele in a single An. arabiensis individual in our samples could represent an independent mutation event in this species, or it might have been transferred from An. gambiae into An. arabiensis through introgressive hybridization. F1 hybrids between the two species have been previously described from east Africa,34–36 and in the course of this study we found hybrids at a similar frequency (3 per 1,535). The reproductive fate of these hybrids was not known, but recent evidence suggests that they serve as bridges for some nuclear as well as mtDNA gene flow across species boundaries,27,37,38 allowing for the passage of alleles such as kdr from one species to the other. Alternatively, an independent mutation event could have created the same kdr codon sequence in An. arabiensis. In theory, these alternative scenarios may be distinguished by determining sequence from intron 1 of the sodium channel gene, where there are fixed differences between species;22 unfortunately, this experiment was thwarted by repeated and unexplained PCR amplification failures. The extreme rarity of the L1014S kdr allele in An. arabiensis populations could be due to very recent introduction by mutation or introgression, or it could be due to fitness costs directly or indirectly associated with the kdr locus. It is also possible that behavioral differences between the species in the area such as increased exophily and zoophily in An. arabiensis35,39 reduce the selection pressure caused by ITNs. The relatively low frequency of the L1014S kdr allele in western Kenya despite significant ITN use is encouraging, but does not fully reflect the potential for permethrin resistance already present in An. gambiae. The cytochrome P450-based resistance found in the RSP strain is also present in An. gambiae populations from the bed net area, as indicated by bioassays and enzyme assays.6 Those assays, together with the population genetics of kdr reported here, suggest that P450based resistance is more important than kdr-based resistance in this region. The P450s are a large and diverse family of enzymes in insect genomes, including An. gambiae.40 Because P450-based resistance is usually due to changes in the expression of one or more P450 genes rather than to changes in the coding sequence, the responsible mutations are more difficult to identify relative to mutations conferring target-site insensitivity. Recently, a resistance-associated mutation leading to the overexpression of P450s was identified in Drosophila melanogaster,41 and progress characterizing An. gambiae P450s40,42,43 will soon lead to identification of the relevant mutations in this species. Until then, the lack of knowledge

DYNAMICS OF THE AN. GAMBIAE KDR ALLELE IN KENYA

about the specific mutation(s) causing P450-based resistance to permethrin makes it impossible to study its population genetics as we have done here for the kdr allele. Both target site insensitivity and metabolic resistance to permethrin are present in An. gambiae populations from western Kenya, and this study has shown that these mechanisms have become more prevalent apparently in response to ITN use.6 Nevertheless, their impact on the effectiveness of the large scale ITN-based malaria control strategy implemented in Asembo has been minimal or non-existent to date.6,7,44 Vector densities and sporozoite rates were significantly reduced not only in ITN villages but also in nearby non-ITN villages.7 The reasons behind these counterintuitive results remain unclear, but may involve higher physiologic costs, reduced blood feeding and parity rates, and lower life expectancies for resistant insects. A recent study of carriers of the west African L1014F kdr allele suggested that large proportions of kdr homozygous females actually were killed by prolonged contact with pyrethroids due to diminished sensitivity to the excito-repellent effect of the insecticide.45 The use of genetic and enzyme assays to monitor the presence, frequency, and geographic distribution of insecticide resistance genes remains an important component of operational research on bed net efficacy. Future research should complement such surveys by linking genotype information with behavioral studies so that the significance of resistant genotypes to ITN-based malaria control programs can be predicted. Received August 31, 2003. Accepted for publication October 10, 2003. Acknowledgments: Tovi Lehmann generously provided An. gambiae DNA samples from Jego in coastal Kenya. Frank Collins and Neil Lobo kindly provided access to the sequencing facility and assisted with sequencing. We thank Bill Hawley, Wim van Bortel and Hilary Ranson for helpful comments on the manuscript. For permission to cite unpublished data, we thank W. van Bortel and H. Ranson. We are grateful to John Gimnig for providing a map of our study sites and the Global Information System coordinates for houses. We acknowledge the entomology staff at the Center for Vector Biology and Control Research of the Kenya Medical Research Institute for assistance with mosquito collections. This paper has been published with the permission of the director of the Kenya Medical Research Institute. Financial support: This work was supported by grants from the National Institutes of Health (AI-44003) to Nora J. Besansky, from the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (TDR) (980101) to John M. Vuvule, and by an Arthur Schmidt PhD Fellowship to Aram D. Stump. Authors’ addresses: Aram D. Stump and Nora J. Besansky, Center for Tropical Disease Research and Training, Department of Biologic Sciences, PO Box 369, Notre Dame, IN 46556. Francis K. Atieli and John M. Vulule, Center for Vector Biology Control Research, Kenya Medical Research Institute, PO Box 1578, Kisumu, Kenya. Reprint requests: Nora J. Besansky, Center for Tropical Disease Research and Training, Department of Biologic Sciences, PO Box 369, Notre Dame, IN 46556, Telephone: 574-631-9321, Fax: 574-631-3996, E-mail: [email protected].

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