Mycobacterium bovis is the pathogenic agent responsible for bovine tuberculosis (bTB), a zoonotic disease affecting mostly cattle, but also transmittable to humans and wildlife. Genetic studies on M. bovis allow to detect possible routes of bTB transmission and the identification of genetic reservoirs that may provide an essential framework for public health action.M. bovis genotypes collected from different regions in Africa with 45 new Mozambican samples. Our analyses, based on phylogeographic and population genetics’ approaches, allowed to identify two clear trends. First, the genetic diversity of M. bovis is geographically clustered across the continent, with the only incidences of long-distance sharing of genotypes, between South Africa and Algeria, likely due to recent European introductions. Second, there is a broad gradient of diversity from Northern to Southern Africa with a diversity focus on the proximity to the Near East, where M. bovis likely emerged with animal domestication in the last 10,000 years.We used a database with 1235 M. bovis in Africa was probably established millennia ago following a concomitant spread with cattle, sheep and goat. Such scenario could translate into long-term locally adapted lineages across Africa.Diversity indices are higher in Eastern Africa, followed successively by Northern, Central, Southern and Western Africa, roughly correlating with the regional archaeological records of introduction of animal domesticates. Given this scenario M. bovis in Africa using the available genetic data, opening the field to further studies using higher resolution genomic data.This work describes a novel scenario for the spread of  Mycobacterium bovis, the responsible for bovine tuberculosis, mostly present in cattle but also transmittable to other animals including humans. This diversity is geographically clustered within the African continent meaning that the genetic diversity was established through independent evolution within different areas. Higher diversity values of M. bovis are found in Eastern and Northern Africa, followed by Central Africa, with Western and Southern Africa displaying the lowest diversity. These levels of diversity correlate well with the introduction of domesticated livestock in the different regions of Africa, following their domestication in the Near East 10,000 ago. We hypothesize that M. bovis emerged in the Near East and it was carried across Africa together with domesticated animals and people that developed herding practices and biological tolerance for digesting milk in adulthood. Such scenario implies a strong evolution and co-evolution of M. bovis across Africa leading to locally adapted strains that could prove a challenge for public health actions.We describe the genetic diversity distribution in Africa of the pathogen  Mycobacterium bovis is the major causal agent of bovine tuberculosis (bTB) in cattle, the world's most neglected zoonotic disease that needs urgent attention, especially in developing countries [ountries , 2. The ountries –5, or evountries .Mycobacterium tuberculosis infection may be bTB. The disease also impacts on international trade of animals and animal products [M. bovis both within the same country as well as cross-borders [In 2017, Olea-Popelka and co-authors raised awareness for the worldwide importance of bTB, particularly in the African continent, where insufficient surveillance, testing and traditional ways of rearing and consuming cattle may be responsible for under-notification of this zoonotic disease . In fact-borders , 8, 9.Little is known about the prevalence and epidemiology of bTB in Africa, and its impact on humans, on livestock and wildlife. In sub-Saharan Africa, few studies describe the transmission status of bTB in regions where multiple hosts are present, but it is believed that sharing of water points plays a key role in the transmission and spread of the disease between animals . Indeed,Different studies have shown that the distribution of bTB in Africa is not uniform, with areas of low and high prevalence , 12. HowIn Mozambique, this scenario is common and bTB is assumed to be an important cause of the economic losses in bovine production evidenced by the rejection of carcasses at the slaughterhouse and limitations on national and international trade. However, little is known about the impact of bTB on humans. Mozambique is one of the countries most affected by the TB, TB / HIV and MDR-TB triad with six other African countries, two of which are countries that share borders with Mozambique .M. bovis have been estimated to at least around 6000 years [The transport of animals might have been a major determinant for the spread of the disease since pre-historic periods. The genetic coalescence of 00 years  and dome00 years . The so-00 years –19.M. bovis allow the possibility to detect possible routes of bTB transmission within and across borders. The main objective of this work is to shed light on the presence of genetic reservoirs of bTB in Africa and to trace historical/prehistoric events in the continent using genetic diversity and phylogeography of M. bovis in Africa.Genetic studies on Institutional permission to conduct the study was obtained from the National Directorate of Veterinary Services in Maputo, Mozambique (Nota 162/ MINAG/DNSV/900/2013) and Ethical Principles in Animal Research adopted by Ethic Committee in the Use of Animals (CEUA) of School of Veterinary Medicine and Animal Science, University of São Paulo (Protocol number CEUAx 6755081216). Sampling and culling were performed as part of the Veterinary Services regular activity for disease control, following the procedures determined by the Mozambican Animal Health Regulation . The slaughter was done in registered abattoirs according to stipulated procedures. All mycobacterial cultures were performed in the National Tuberculosis Reference Laboratory, Ministry of Health Mozambique.Mycobacterium tuberculosis complex (MTBC) strains was based on the use of two sets of multiplex PCR for identifying the genus Mycobacterium [Forty five samples, consisting of tissue/organs containing lesions, were processed in the BSL3 from the National Reference Laboratory of Tuberculosis (LNRT) in Mozambique in duplicate. Cultures grew in Stonebrink (ST) medium with pyruvate and Lowenstein-Jensen (LJ) medium with glycerol. DNA extraction was performed according to the protocol described by Van Soolingen and colleagues . The molacterium  and for acterium .M. bovis samples was performed using three methodologies: spoligotyping, region of difference (RD) analysis and MIRU-VNTR, as described before [Genotyping of the d before .mbovis.org database. RD analysis, a PCR-based method to determine the presence or absence of specific regions of difference (RD) was performed to identify four M. bovis groups named Af1, Af2, Eu1 and Eu2. All samples were genotyped using the MIRU-VNTR 24 loci kit  that includes 24 size-variation markers, although only a few have proven to be polymorphic in M. bovis. The genotypic profiles were reported as a series of 24 numbers corresponding to the number of alleles at each of the 24 loci.Spoligotyping of each sample was determined based on the numerical combination of the presence or absence of the spacers and identified according to M. bovis in Africa. A list of sampled locations and numbers is indicated in M. bovis data that encompasses the 43 spoligotyping markers and 5 VNTR markers that were present in most studies , that previously displayed enough resolution in the African context [M. tuberculosis Complex (MTBC) available at the same genotyping resolution obtained from the reference database of MIRU-VNTR Plus [M africanum, 2 M. canetti, 11 M. caprae, 2 M. pinnipedii, 6 M. microti and 123 M. tuberculosis strains. These genotypes were included in the phylogenetic reconstruction and the dataset of European M. tuberculosis strains were used as an outgroup for the population tree analysis of M. bovis strains.We collected the largest possible database of genotypes for  context , 24. We  context . We alsoNTR Plus  that inchttp://www.fluxus-engineering.com), as we suggested for MIRU-VNTR data in M. tuberculosis [Reconstruction was performed using a matrix of the 48 markers described above and by applying the reduced median algorithm  followedrculosis , 30. Givh, the haplotype diversity Hd (probability that two randomly sampled haplotypes are different), the average number of differences K  and the nucleotide diversity Pi .The diversity of the 48 makers was converted into a binary matrix reflecting genetic distances between each genotype. That binary matrix was used to calculate diversity indices in DNAsp 6 . For eacM. bovis in each population, we used the previously described outgroups (corresponding to genotypes of six other members of the MTBC) to define the root with Network software. The root was used to obtain the ρ statistic for each dataset. The ρ statistic measures the average number of differences from a root  and each of the samples and represents an unbiased model-free diversity measure and time estimator [In order to estimate the coalescence point of stimator .The different statistics were plotted geographically using the Kriging algorithm of Surfer 8 software using the geographic points indicated in Fst distances between pairs of populations were calculated using DNAsp 6 [Fst matrix obtained using MEGA7 [M. tuberculosis genotypes to be used as outgroup in the analysis. DNAsp 6  and a pong MEGA7 . We perfM. bovis across Africa, we performed a detailed phylogeographic and statistical analysis on African diversity of M. bovis. For that we collected 1504 genotypes comprised by 48 markers    to whichM. bovis strains in Africa  and Northern Africa , although some haplotypes with minor frequencies from Central Africa are also present. On an overview analysis of the network, Eastern and Northern Africa seem to display a deeper ancestry of M. bovis compared with other African regions.We reconstructed a phylogeny for the genotypes of n Africa . IncidenM. bovis genotypes radiate into several clades from a single major coalescence point. On that coalescence point it is possible to discern a few longer branches from Ethiopia and Tanzania. There are specific branches from Northern Africa, Mozambique, Southern Africa, Italy and Central Africa, reflecting a strong geographical compartmentalization. Most of the diversity in Central Africa seems present on a single major clade. That clade, defined by the spacer 30, corresponds likely to the common African spoligotype type Af1. Mozambique presents three clades from the main M. bovis radiation point, including one shared with South Africa, but, in general, Mozambique displays, as other locations, a specific local subset of diversity.Most of the In general, the sharing of genotypes across borders is mostly restricted to countries in the vicinity of each other. However, Northern Africa (Algeria) shows common haplotypes with South Africa . Some ofM. bovis in Africa, we obtained four diversity statistics in all populations with more than 20 samples .In order to visualize the distribution of the genetic diversity of  samples  that wer samples . The meaρ, K, Pi), mainly in the case of ρ, followed by Northern Africa (Algeria and Tunisia) that display very high values in terms of K and Pi, comparable to Eastern Africa indices. Countries in Central/Western Africa display lower diversity but in general countries in Southern Africa  display the lowest values. A gradient from North to South is visible in in the maps (M. bovis diversity.It is clear that countries in Eastern Africa (Ethiopia and Tanzania) display the higher values of diversity when measures related with deep ancestry are concerned , followed by Northern Africa , Central Africa , Southern Africa  and Western Africa   .M. bovis is the main organism responsible for bTB, a form of TB present in cattle and other domestic and wildlife species, that can be transmitted to humans through zoonosis. bTB represents a high burden in the African continent. In order to understand how the genetic diversity of this pathogen is distributed across Africa, we performed a genetic analysis on its overall diversity. This allows to establish probable regions that can be genetic reservoirs in the continent and where public health actions should focus aiming at bTB eradication. There are two conclusions that erupt from this analysis:M. bovis introduced by Europeans in South Africa and Algeria.A) One feature is that the transportation of the pathogen across borders is probably more limited than expected–each country displays a subset of diversity that might have been established and maintained for a long period; except for the case of South Africa and Algeria, shared haplotypes seems to occur almost exclusively between neighbour countries. The South African/Algeria sharing of haplotypes can be related with an outside African source. Some of the diversity within these two countries are likely from clades Eu1 and these represent the matches detected, possibly reflecting M. bovis which typically represent more simplistic scenarios for public health than larger genetic reservoirs. However, a real scenario can just be attained by obtaining reliable values of incidence of bTB in different African countries that can be combined with this information. While Mozambique, Tanzania and Zambia show little diversity of circulation genotypes, their estimate of cases that could be related with recent transmission from genetic data is over 70% in the three countries, highlighting probable recent outbreaks.B) Considering the haplotype diversity, the number of circulating strains in different regions differs considerably across Africa. In the present analysis Mozambique, Tanzania and Zambia display the lowest diversity in circulating strains and are minor reservoirs of genetic diversity of M. bovis in Africa raises questions regarding the processes that shaped that diversity. There has been a wide discussion regarding the origin and spread of the Mycobacterium tuberculosis complex that includes M. tuberculosis, the infectious agent responsible for TB, and M. bovis, the causal infectious agent of bTB that mostly infects animals but that is transmitted to humans. One common hypothesis for the origin of the MTBC relates with the emergence of agriculture in the Fertile Crescent in the Neolithic period, over 10,000 years ago [M. tuberculosis from M. bovis the sequencing of both genomes clearly showed that the pathogens are not directly related [M. tuberculosis suggests a coalescence of the organism above 40,000 years ago or above, linking its global spread to a probable co-expansion with the modern humans Out-of-Africa [M. bovis are as recent as 6000 years placing it well within the Holocene and potentially with the expansion of Neolithic or pastoralist societies in Africa.Nevertheless, the distribution of the diversity of ears ago , when ca related  but instf-Africa , 38. On It is possible that bTB arose when animals were kept in larger groups in limited areas, that allowed the disease to be more easily transmitted between individuals. Domestication in Africa occurred through introductions from the Near East, where cattle was domesticated about 10,000 years ago, marking also the initiation of dairy practices. None of the native African animals were domesticated. In that sense we can hypothesize that bTB has a Neolithic origin and it was spread across Africa concomitantly with the introduction of cattle. Domestication of cattle and the maintenance of larger groups of animals is also connected with the use of animals, not only as a source of meat and hides, but also as milk producers, part of the so-called Secondary Product Revolution. That change in societies goes beyond a cultural change since it has deep biological implications, as part of the populations was positively selected through time in order to be able to digest lactose in adulthood. Incidentally, the ability to consume milk facilitates zoonotic transfers as it is clear nowadays that milk consumption is the major mode of transmission of bTB to humans. This suggests a co-evolution of humans, cattle and pathogen that allowed the endurance of the disease. Could the data suggest that these genetic patterns are related with anciently established routes? Two points of evidence support this scenario. The first is the strong geographic clustering of the diversity across Africa that provides a direct evidence that this diversity was established in the past and enough time elapsed for local diversity to be established. The second is that the levels of diversity across Africa, that could illustrate different timeframes for diversity to accumulate in each region, correlate well with the estimated arrival of domesticated cattle and pastoralism into those regions .M. bovis in Eastern Africa.The two major areas with higher diversity in Africa are Eastern Africa and Northern Africa. These were the first analysed areas where domesticated cattle, goats and sheep were introduced from the Near East . FollowiM. bovis genotypes. While some earlier cases of cattle domestication might exist in this area [As mentioned above, North Africa saw an earlier introduction to the area that is nowadays Egypt (8000 years ago), however in Northwestern Africa (location of our samples) pastoralism was introduced about 6000 years ago , 43. Thehis area , 48, clehis area .Populations in Southern Africa, before the massive expansion of Bantu-speaking agriculturists in the last two millennia from Central Africa  that repρ and Pi, the statistics that better reflect time and depth of accumulated diversity. More exactly, both set of statistics Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Clupea pallasii) across 1600 km of coastline, we show that reproductive timing drives population structure in these pelagic fish. Within a specific spawning season, we observed isolation by distance, indicating that gene flow is also geographically limited over our study area. These results emphasize the importance of considering both seasonal and spatial variation in spawning when delineating management units for herring. On several chromosomes, we detected linkage disequilibrium extending over multiple Mb, suggesting the presence of chromosomal rearrangements. Spawning phenology was highly correlated with polymorphisms in several genes, in particular SYNE2, which influences the development of retinal photoreceptors in vertebrates. SYNE2 is probably within a chromosomal rearrangement in Pacific herring and is also associated with spawn timing in Atlantic herring (Clupea harengus). The observed genetic diversity probably underlies resource waves provided by spawning herring. Given the ecological, economic and cultural significance of herring, our results support that conserving intraspecific genetic diversity is important for maintaining current and future ecosystem processes.The timing of reproduction influences key evolutionary and ecological processes in wild populations. Variation in reproductive timing may be an especially important evolutionary driver in the marine environment, where the high mobility of many species and few physical barriers to migration provide limited opportunities for spatial divergence to arise. Using genomic data collected from spawning aggregations of Pacific herring ( What is known stems largely from plants  and inseClupea pallasii, Clupea harengus), estimated to have diverged 2.2 Ma [Reproductive allochrony may be particularly important in explaining the population structure and biocomplexity of Pacific and Atlantic herring . The fisheries are managed spatially, though the geographical extent of spatial units varies by country and administrative area (i.e. state or province). In Washington State, spawning biomass is estimated for individual spawning areas and a limited bait fishery harvests herring outside the spawning season . In BritThe temporal and spatial differentiation in both Atlantic and Pacific herring may be surprising given evidence for some straying between spawning locations  as well . 2a). Spawn timing at these sites ranged from January to June . Restriction site-associated DNA (RAD) sequencing was used to genotype each sample at 6718 polymorphic loci that aligned to the Atlantic herring genome . After filtering for missing data, each individual sample had an average read depth of 75× at these loci.We sampled 1104 Pacific herring from 23 different spawning aggregations across 1600 km on the Pacific Coast of North America a. Spawn p < 0.0001). In a subsequent discriminant analysis of principal components (DAPC) (b), the first discriminant axis explained 38% of the retained variance and separated herring spawning in May and June from all other individuals, while the second discriminant axis summarized 19% of the retained variance and separated samples along a latitudinal and temporal gradient. A pattern of isolation by time [r = 0.51, Mantel p ≤ 0.00099; figure 1c) despite low overall levels of genetic differentiation , indicating limited gene flow between herring that spawn at different times. This isolation by time relationship was even stronger after we removed the highly divergent May–June spawners from the analysis . Additionally, there was also evidence of isolation by distance [r = 0.61, Mantel p ≤ 0.00099; March–April spawners: Mantel r = 0.77, Mantel p ≤ 0.00099; figure 1d).We tested whether subtle differences in spawn timing (in the range of weeks to months) and geographical distance influence the genetic population structure of Pacific herring. A principal components analysis revealed differentiation between populations spawning at different times and in different geographical regions , and the first two principal components were highly significant using the Tracy-Widom statistic (s (DAPC) b, the fidistance  within eA model-based Bayesian analysis of population structure  estimateFST = 0.014), it provided a good foundation for investigating selective differentiation associated with seasonal reproduction and spawn timing diversity. Many temperate fishes initiate reproduction in response to seasonal changes in day length . These SNPs were distributed across 17 different chromosomes, suggesting that spawn timing is a polygenic trait. Differentiation over all samples at these shared outlier loci was an order of magnitude higher than at other loci .As genome-wide differentiation was low across all populations ; in ouroperiod,  that accoperiod, . There wn = 44; 36%) were found within chromosomes 8, 12 and 15 which had high linkage disequilibrium (LD) extending over several Mb . By contrast, LD decayed very quickly on most other chromosomes . Network analysis of LD [figure 3c; electronic supplementary material, figures S7 and S8). This pattern is characteristic of chromosomal rearrangements such as inversions [figure 3d). Within a particular spawning site, tests for Hardy-Weinberg equilibrium (HWE) indicated that individuals were approximately randomly mating with regards to the putative inversions . Hierarchical analysis of molecular variance (AMOVAs) showed that the frequency of putative inversion genotypes (figure 3e) differed between groups with different spawning phenology (January–February versus March–April versus May–June spawners). When all spawning groups were included in the analysis, statistically significant (p < 0.0001) differences in genotype frequency were observed across all hierarchical levels and chromosomes . When only January–February and March–April spawners were included, there was no difference in the frequency of putative inversion genotypes on chromosomes 12 and 15 , suggesting that these spawning groups may share more recent ancestry with one another than with March–April spawners.A subset of these outlier loci  were located on chromosome 15 and mapped to the mRNA encoding SYNE2, a nuclear membrane protein that (among other functions) influences the position of retinal photoreceptors early in vertebrate development [SYNE2 that were strongly correlated with spawning photoperiod (n = 5) were found within the coding sequence of this gene. The SNP with the highest correlation to spawning photoperiod (log10 Bayes factor = 41) was a non-synonymous missense variant (serine to histidine), which is predicted to have a moderate effect on the protein structure. The other outlier SNPs in SYNE2 represented intron variants or splice region variants. Additional genomic regions that were highly associated (log10 Bayes factor > 5) with spawning photoperiod in Pacific herring and may play a role in reproductive pathways include two loci on chromosome 6: SH2D4A, a locus with oestrogen receptor binding activity [SPATA4, a locus associated with spermatogenesis [Outlier SNPs with the strongest correlations to spawning photoperiod  that were highly correlated with reproductive timing variation in both species (a), suggesting that similar genes may underlie highly complex traits such as the timing of reproduction. Furthermore, it is likely that these loci are within chromosomal rearrangements or subject to linked selection in Pacific herring, as LD network analysis showed that SNPs within three of these genes  were found in networks of high LD on chromosome 15, while the SNP within HK3 was found in networks of high LD on chromosome 8.Such mechanistic links provide an opportunity to test for parallel evolution—the evolution of mutations in independent lineages that result in phenotypic similarities . In Atla species a, suggesSYNE2 were among the most highly associated with spawn timing in both species , suggesting a strong effect of that gene on reproductive timing. However, specific SNPs in Pacific herring differed from those in Atlantic herring; the five SNPs within SYNE2 found in Pacific herring were monomorphic in Atlantic herring. This pattern suggests that species-specific polymorphisms arose independently and may indicate parallel evolution of varied reproductive phenologies across sister species. Future studies comparing the two species by conducting targeted sequencing in this genomic region should be able to rigorously test this hypothesis. Interestingly, winter and spring spawning populations of Atlantic cod also exhibit large allele frequency differences [SYNE2, providing additional evidence that this gene or genomic region plays a role in reproductive phenology in a variety of fish taxa.SNPs in ferences  in SYNE2Our results indicate that genetic variation underlying spawn timing structures herring populations in time and space. Given the crucial role of forage fishes in marine ecosystems of converting primary and secondary production to energy for higher trophic levels ,58, it iIdentifying the genetic basis and environmental drivers of variation in reproductive phenology is also important for predicting impacts of climate change and other anthropogenic disturbances on marine food webs. It is currently unknown how photoperiod interacts with other environmental cues  to influence the timing of reproduction in herring. Recent studies of marine phenology indicate that climate change and the warming of surface waters are causing shifts towards earlier larval emergence in some pelagic fishes ,62, a phCurrent fishery management practices  as well . 3(a)n = 1344) were collected by state, federal, or tribal biologists, or permitted subsistence fishers. Subsampled tissue was individually placed in 100% ethanol.Approximately 48 herring were collected from each of 23 distinct spawning sites spanning 1600 km of the Pacific northwest coast of North America . All but one of these sample collections consisted of sexually mature herring caught in pre-spawning aggregations (before 2013) or during active spawning events (after 2013) using nets or hook-and-line fishing gear. Late-stage eggs were collected at one site . Five different locations were sampled twice across different years (range of years separating spawning events = 1–15 years); we hereinafter refer to these samples as ‘temporal replicates'. All herring ((b)et al. [We followed the protocol of Etter et al.  to prepatacks v. 1.46 [owtie2 v. 2.2.6  [IH) and comparing it to a set of clean reference samples [IH values higher than clean reference samples (IH > 0.32), as in Petrou et al. [We used S v. 1.46  to analy v. 1.46  using Boignment)  and only samples . Individu et al. .(c)FST [FIS within each sample collection, and per-locus FSTs were calculated using the R package hierfstat v. 0.04.22 [smartPCA in Eigensoft v. 7.2 [plink v. 1.9 [adegenet v. 2.1.3 [a priori based on sampling location and collection year and the optim.a.score function was used to identify the number of principal components to retain in the analysis.Pairwise FST  between  0.04.22 . We visut v. 7.2 . As inpuk v. 1.9 . We assev. 2.1.3 . Groups FST (FST/(1 − FST) to the number of days separating sampling events. The statistical significance of isolation by time was evaluated using a Mantel test  as implemented in the R package vegan v. 2.5.6 [FST to the geographical distance separating sample collections. The shortest distance (as the crow flies) separating two sampling locations was calculated using the R package geosphere v. 1.5.10 [To test whether the timing of reproduction affected genetic differentiation , we conducted a linear regression of linearized pairwise v. 2.5.6 . We alsov. 2.5.6  by condu. 1.5.10 , and theWe assessed the relative importance of reproductive timing and geographical distance in structuring Pacific herring populations by conducting a hierarchical Bayesian analysis of population structure with the program GESTE v. 2.0  (see the(d)pcadapt 4.3.3 [bayenv2, which accounts for the covariance of alleles owing to hierarchical population structure and sampling error [We identified SNPs that were outliers to overall patterns of population differentiation in our samples using PCA, as implemented in the R package pt 4.3.3 . We evalng error  (see the(e)genetics v.1.3.8 [Emin = 20, φ = 7). Because several chromosomes contained loci in strong LD (r2 > 0.5) over extended distances (greater than 1 Mb), we conducted intrachromosomal PCAs in adegenet to identify whether individuals clustered in the distinct ‘three stripe' pattern characteristic of chromosomal rearrangements such as inversions [k-means clustering to the first two eigenvectors of the PCA using the R function kmeans.We estimated linkage disequilibrium (LD) within each chromosome using the R package  v.1.3.8 . LD netw v.1.3.8  was usedversions  et al. [et al. [et al. [We investigated whether the same genomic regions and SNPs were correlated with reproductive timing in both Atlantic and Pacific herring, which are sister species that diverged approximately 2 Ma and now inhabit different oceans. Atlantic herring SNPs correlated with spawn timing were previously published in Lamichhaney et al.  and Fuen [et al. . Using t [et al. , we asseClick here for additional data file.


Rangifer genealogies spanning from 15,000 years ago to the 18th century, as well as modern samples from the wild Taĭmyr population and from domestic herds managed by Nenetses. The wild and the domestic population are the largest populations of their kind in Northern Eurasia, and some Nenetses hold their domestic reindeer beside their wild cousins. Our analyses of 197 modern and 223 ancient mitochondrial DNA sequences revealed two genetic clusters, which are interpreted as representing the gene pools of contemporary domestic and past wild reindeer. Among a total of 137 different mitochondrial haplotypes identified in both the modern and archaeological samples, only 21 were detected in the modern domestic gene pool, while 11 of these were absent from the wild gene pool. The significant temporal genetic shift that we associate with the pastoral transition suggests that the emergence and spread of reindeer pastoralism in Northwestern Siberia originated with the translocation and subsequent selective breeding of a special type of animal from outside the region. The distinct and persistent domestic characteristics of the haplotype structure since the 18th century suggests little genetic exchange since then. The absence of the typical domestic clade in modern nearby wild populations suggests that the contemporary Nenets domestic breed feature an ancestry from outside its present main distribution, possibly from further South.Just as the domestication of livestock is often cited as a key element in the Neolithic transition to settled, the emergence of large‐scaled reindeer husbandry was a fundamental social transformation for the indigenous peoples of Arctic Eurasia. To better understand the history of reindeer domestication, and the genetic processes associated with the pastoral transition in the Eurasian Arctic, we analyzed archaeological and contemporary reindeer samples from Northwestern Siberia. The material represents  This manuscript demonstrates that the reindeer mitochondrial genome in Northwestern Siberia has gone through massive genetic replacement since medieval times characterized by a significant loss of native mtDNA haplotypes, together with a significant introduction of new haplotypes. The significant temporal genetic shift associated with the pastoral transition suggests that the emergence and spread of reindeer pastoralism in Northwestern Siberia involved the translocation and breeding of a special type of animal with a partly non‐native origin. Just as the domestication of sheep, goat, cattle, and pigs were critical elements in the Neolithic transition in many parts of Western Eurasia  are distributed throughout most of the northern Holarctic region with the domestic form mainly confined to the Eurasian continent. Among the world's total stock of nearly 2,000,000 domestic reindeer, ~2/3 are distributed in Russia and 1/3 in Fennoscandia —have been dated to the same period .The domestic reindeer studied are from nomadic herds managed by indigenous Nenetses Figure . NenetseKrupnik, . BeginniKrupnik, . Today NII and Ib) and Northwestern Russia , the Nenets Autonomous District (NAD), and the eastern portion of the Komi Republic (KR), with ages spanning from ~15,000 years before present (BP) to ~300 BP  is from reindeer close to Malygina Strait at the north end of the I͡Amal Peninsula. Their seasonal migration pattern is mainly within the northern territory of I͡Amal sovkhoz. The Taz‐Nenets samples (sample code 18) were from a herd in the Taz District located directly east of the I͡ANAD on the opposite side of the Ob' Gulf from the I͡Amal Peninsula. These eastern Nenets families move their herds annually from the town of Tazovsk on the Taz River all the way to the western part of the TDND. The Eniseĭ‐Nenets samples (sample code 19) were from the reindeer herding families who travel around the settlements of Tukhard and Nosok located at the western part of TDND. Most of the ~100,000 Nenets reindeer in TDND are situated in this area.DNA from 173 skin, muscle, and blood samples from contemporary domestic and wild reindeer were extracted and analyzed. The four domestic sample sets were from separate and widely spread districts in I͡ANAD and the western part of the Taĭmyr Dolgano‐Nenets District  following the protocol described in Bjørnstad and Røed  following the manufacturer's protocol. A 503‐bp long fragment of mitochondrial CR was amplified and sequenced as given in Kvie, Heggenes, and Røed . All seq2.4arlequin v3.5  of sample sets, that is, codes 1–14 together with 20 versus codes 15–19 on the basis of the among‐group partitioning of molecular variance , nucleotide diversity  and nucleotide pairwise differences  in the recent domestic sample set, compared to the wild sample set  and in Northwestern Russia  and with all other haplotypes radiating from this by one to three mutations  or in the older sample codes 3–13 (n = 37). Subcluster Ib contained three haplotypes in 11 samples, two in modern domestic reindeer and nine in ancient reindeer (sample codes 3–12).The Bayesian phylogenetic tree reconstruction of the 137 haplotypes identified several medium to well‐supported clusters/subclusters Figure . Some ofs Figure . Such a s Figure . This an4Our analysis of the mitochondrial CR in contemporary and archaeological reindeer in the vast Nenets‐occupied region reveals two distinct genetic clusters. The first corresponds to the modern wild reindeer together with the older sample sets ranging from the late Pleistocene to the late medieval periods covering large areas from I͡Amal in the East to the Ural Mountains in the West. The second includes those from widely separated contemporary domestic herds from I͡ANAD and the western part of the TDND together with the relatively recent archaeological site dated to 300 BP. The high proportion of temporal sample overlaps on I͡Amal and the fact that nearly all archaeological sites clustered together with contemporary wild reindeer, and all modern domestic herds assigned to the other cluster, gives strong evidence that these highly differentiated clusters represent the maternal gene pools of wild and modern domestic reindeer respectively. Based on these results, the mitochondrial CR appears to be an appropriate marker to study genetic processes associated to the pastoral transition in Northwestern Siberia.4.1The high levels of genetic variation within a homogeneous genetic structure found within the wild gene pool both temporally and spatially suggest that relatively large populations of wild reindeer occupied the region since late Pleistocene. The significant genetic shift associated with the late‐dated archaeological site Khali͡ato‐1 is striking and points toward a fundamental genetic change on the I͡Amal that seems to have been completed by the start of the 18th century. This change coincides very roughly with the period associated with the onset of the pastoral tradition. Using historical sources, Krupnik  associatIt should be noted that others have interpreted the many artifacts found at sites on the I͡Amal Peninsula as signs of a social complexity and a remarkable inter‐regional standardization of techniques built on large‐scale reindeer transport and large‐scale reindeer husbandry, beginning as early as 2,000 BP  at sites 3, 5, 10 and 13 could indicate that a small number of animals with a genetic constitution similar to those of the domestic reindeer today were present on I͡Amal since as far back as 6,000 BP. If local wild reindeer were tamed , and the small fraction haplotypes belonging to the domestic lineages among the archaeologic sites 1–14, nevertheless suggest that the dominant domestic type of today had its origins in animals imported from other areas.Several mechanisms could have been involved in introducing new maternal genomes to the region. This polar region experiences periodic crashes in the number of reindeer due to poor weather or spring icing events ; data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); resources (supporting); software (supporting); supervision (supporting); writing‐original draft (lead); writing – review and editing (lead). Kjersti Kvie: Data curation (supporting); formal analysis (supporting); resources (supporting); writing‐original draft (supporting); writing – review and editing (supporting). Robert J Losey: Resources (supporting); writing‐original draft (supporting); writing – review and editing (supporting). Pavel A Kositsev: Resources ; validation (supporting); writing‐original draft (supporting); writing – review and editing (supporting). Anne K Hufthammer: Data curation (supporting); resources (supporting); validation (supporting); writing‐original draft (supporting); writing – review and editing (supporting). Mark J Dwyer: Resources (supporting); writing‐original draft (supporting). Vasiliy Goncharov: Resources (supporting); visualization (supporting); writing‐original draft (supporting). Konstantin B Klokov: Resources (supporting); writing‐original draft (supporting). Dimitry V Arzyutov: Resources (supporting); Writing‐Original Draft (Supporting); Writing‐Review & Editing (supporting). Andrei Plekhanov: Resources (supporting); writing‐original draft (supporting). David George Anderson: Funding acquisition (supporting); project administration (supporting); resources (supporting); writing‐original draft (supporting); writing – review and editing .Appendix S1Click here for additional data file.


Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. Dogs are the main peridomiciliary reservoir of L. infantum infected dogs using Leishmania-derived recombinant antigens. We aimed to establish an effective serological assay for the diagnosis of Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health . Further samples from a cohort of 30 dogs were searched. For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use .L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide. For the diagnosis of  Leishmania. In 2017, 94% of new leishmaniasis cases reported by the World Health Organization (WHO) were concentrated in seven countries: Brazil, Ethiopia, India, Kenya, Somalia, South Sudan and Sudan.Leishmania donovani in India, East Africa, Bangladesh and Nepal in an anthroponotic cycle, and L. infantum in China, Central Asia, the Mediterranean, part of Africa and Latin America in a zoonotic cycle.Leishmaniases are a family of infectious diseases caused by obligatory intracellular protozoa of the genus Canis familiaris) are the main L. infantum reservoir in urban and peri-urban areas in several regions of the worldDogs  detecting antibodies against the total L. major-like antigen, for confirming the diagnosis of CanL.,,,,L. major-like-ELISA, since it is prepared from the crude lysate of L. major-like parasites, variability is frequent in performance, with reported sensitivities ranging from 11.59%L. major-like total lysate. It would be desirable to have an ELISA based on a recombinant antigen as it is a more specific protein, and production can be done on an industrial scale. Thus, we propose in this study to evaluate the use of four recombinant proteins  for the serological diagnosis of dogs in ELISA.On the total L. infantum and L. donovani, related to kinesin and highly expressed in amastigotes.Leishmania infection,,Leishmania antibody assay that we know has poor performance.The recombinant protein rK39 is a repeated sequence of 39 amino acids highly preserved in L. infantum rK9, rK26 and rK39 epitopes, presented high sensitivity (99%) and specificity (96%) in the ELISA in samples of dogs from Italy.,The rK28 protein, constructed as a chimeric protein based on the fusion of Leishmania antigens that we include in this project became available, and it would be important to study them in other geographical regions, including Brazil, since regional variation in infection features may present.Presently other promising recombinant L. donovani infected patients with no corresponding homology in humans. Besides, although the identity between KR95 and Trypanosoma cruzi kinesin is 79%, there appears to be no cross-reactivity.The recombinant antigen KR95 is a kinesin-bound protein comprising peptide sequences identified from Leishmania direct agglutination test (DAT) which has good diagnostic performance, both in human and canine VL. In a study with patients from Ethiopia and Brazil, DAT had 100% sensitivity and specificity.Parasitological examinations that require invasive procedures were not performed on canine survey samples. Instead, the sera were subjected to the Leishmania antigens, suggesting the rK39 antigen for the diagnosis of CanL combined with immunochromatographic DPP assay or alone. Although the rK39-ELISA test has been evaluated in European dogs,,In this study, we validated the ELISA with recombinant Samples - Samples from various institutions in different regions of Brazil were used. Samples were provided by Instituto Adolfo Lutz de São Paulo (IAL/SP), Faculdade de Agronomia, Medicina Veterinária e Zootecnia da Universidade Federal de Mato Grosso (UFMT), Instituto da Saúde e Produção Animal da Universidade Federal Rural da Amazônia (UFRA), Escola de Medicina Veterinária e Zootecnia da Universidade Federal da Bahia (UFBA) and Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo (USP). To calculate the sensitivity, specificity and cut-off of the ELISA with recombinant antigens and to determine the cut-off of the DAT, the following samples were obtained from the IAL/SP, where the parasitological examination, DPP and L. major-like-ELISA (characterized samples) were performed:L. infantum and the local presence of the vector for Leishmania  attended at the Zoonosis Control Centers were used. These dogs had a positive parasitological exam in sternum bone marrow and popliteal lymph node aspirates on glass slides that were fixed and stained with a modified Romanowsky staining rapid test kit . In our study, animals with a positive parasitological (gold standard) result were included regardless of the serological result.- Sera from 74 symptomatic dogs from areas with the canine transmission of L. infantum, with the absence of the vector for Leishmania (Jundiaí - SP). These dogs were negative by the tests recommended in the algorithm of the Brazilian Ministry of Health (DPP and ELISA-L. major-like) and DAT.- Sera from 66 healthy dogs from areas without canine transmission of L. infantum and the local presence of the Leishmania vector . These dogs were positive by the tests recommended in the algorithm of the Brazilian Ministry of Health (DPP and ELISA-L. major-like) and DAT.To evaluate whether subclinically VL infected dogs could be detected by recombinant antigen-ELISA, sera from 19 asymptomatic dogs were obtained from areas with the canine transmission of L. infantum infection in municipalities from north, northeast, southeast and mid-west of Brazil, endemic for CanL. Samples were collected from 2,530 dogs from municipalities of the State of Pará , State of Bahia , State of São Paulo  and State of Mato Grosso   .To evaluate if an infection could be detected earlier than the recommended assays currently in use, paired-samples from 30 dogs in the municipality of Bauru (located in the State of São Paulo) were obtained at day 0 (time 1) and again at 180 days (time 2) for evaluation. These dogs used collars impregnated with deltamethrin  to reduce the risk of a new infection during the period of evaluation. The collars were changed according to the manufacturer’s recommendation or replaced in case of losses, in order to avoid infection through contact with the vector.Ehrlichia canis and non-reagents for Borrelia burgdorferi, Anaplasma phagocytophilum, Anaplasma platys and Dirofilaria immitis in SNAP 4DX Plus® test . These animals came from São Paulo, a non-endemic municipality for VL.To evaluate if the recombinant antigens cross-react with other diseases, we selected 10 samples from dogs without clinical signs for CanL but reagents for Sample size calculation - Aiming at a 95% sensitivity for the rK39-ELISA, rK28-, rKR95- and rK18- testsEthical aspects of the research - The project was approved by the Research Ethics Committee on the Use of Animals of the Instituto de Medicina Tropical, Universidade de São Paulo (CEUA - IMT/USP) on 02/06/2015 (Process number 000305A). The necessary care was taken during the collection of samples, preserving the anonymity of the owners and dogs and regarding conservation and correct use of the stored biological material.Serological tests® Leishmaniose Visceral Canina  - The immunochromatographic tests used by the L. infantum Infection Control Program, of the São Paulo Department of Health, and the Ministry of Health from Brazil to screen dogs for infections was used according to the manufacturer’s recommendations.- DPP - TR DPPL. major-like-Elisa - EIE Leishmaniose Visceral Canina  - The ELISA, used by the L. infantum Infection Control Program, of the São Paulo Department of Health, and the Ministry of Health from Brazil to confirm the positive DPP® test, was performed according to the manufacturer’s recommendations. The Ministry of Health recommends that only samples positive by DPP should be tested by L. major-like-ELISA for confirmation. However, in this study, all samples were submitted to this ELISA.- - DAT  - DAT was performed at the Instituto de Medicina Tropical, Universidade de São Paulo, according to the manufacturer’s recommendations. To calculate the agglutination test cut-off, the same groups , as repo2O2 (Tetramethylbenzidine/hydrogen peroxide) chromogen  (50 µL/well) at four different times: 5, 7, 10 and 15 min. The reaction was stopped with 2N sulfuric acid (H2SO4) (25 µL/well). The absorbances were read at 450 nm using an ELISA reader .- ELISA with recombinant antigens- rK39, rK28, rKR95 and rK18 - The recombinant antigens rK39, rK28, KR95 and rK18 were produced at the Infectious Disease Research Institute (IDRI), Seattle, United States. For the ELISA’s standardization, two positive  and one negative  samples were initially used. Leishmania endemic areas - Protocols were distributed to laboratories in Leishmania endemic regions and the technicians trained accordingly. ELISA with recombinant antigens were performed at the laboratory of the IMT/USP, the coordination center, and at laboratories in other locations  and Laboratório de Infectologia Veterinária do Hospital-Escola de Medicina Veterinária da UFBA), where leishmaniasis caused by L. infantum is endemic. The results were compared to ensure the functionality of the tests, regardless of the location, provided they were carried out under appropriate conditions.- Evaluation of recombinant antigen-ELISA in laboratories within Evaluation of samples from a follow-up study with dogs in the endemic area - To evaluate if the studied assays can detect an early infection in animals, samples from 30 dogs from a cohort of 600 dogs from Bauru - SP collected at day 0 (time 1) and 180 days (time 2) of follow-up were evaluated. Samples from this cohort had been previously tested by the DPP + L. major-like-ELISA tests in Bauru - SP. The samples of the 30 dogs selected were submitted to rK39-ELISA, rK28-ELISA, and rKR95-ELISA. These dogs received collars impregnated with deltamethrin  that were changed every 6 months or replaced in case of losses, to avoid infection through contact with the vector during the study.Evaluation of cross-reactivity with E. canis - To evaluate if the recombinant antigens detect antibody related to other diseases, we selected 10 samples from dogs without clinical signs for CanL but reagent for E. canis in SNAP 4DX Plus® test . These samples were non-reagent for B. burgdorferi, A. phagocytophilum, A. platys and D. immitis. The samples of the selected dogs were submitted to DPP, L. major-like-ELISA, DAT, rK39-ELISA, rK28-ELISA and rKR95-ELISA.Statistical analysis - With the results of the absorbances obtained in each assay with specific recombinant antigens, the percentage of absorbance of the positive standard (ABS% Positive) was calculated by dividing the absorbance of each sample by the absorbance of the positive standard serum and multiplying by 100.Kappa index. The stability of the sensitized plates was evaluated by linear regression of each sample. To compare the reactivity indices of samples with the recombinant antigens, the Friedman test (paired samples) was used, followed by Dunn’s multiple comparison test (nonparametric data). The comparison of the sensitivity and specificity of the methods used was made by McNemar’s chi-square test and the comparison between proportions by the Fisher’s exact probability test or the chi-square test. Statistical analysis was performed using the GraphPad Prism 5  and SigmaStat 3.5 analysis system software .Initial sample characterization - The samples were characterized according to the diagnostic algorithm recommended by the Brazilian Ministry of Health (MH). This involves initial evaluation with DPP followed by L. major-like-ELISA of DPP-reactive samples. When the sera of 74 symptomatic dogs with positive parasitological examination were submitted to the MH algorithm, the sensitivity was 93.2% (95% CI: 85.1-97.1%) and the specificity 100.0% (95% CI: 94.6-100.0%) with 66 samples from healthy dogs .L. infantum infection) and negative samples from healthy dogs, generating a sensitivity of 97.3% (95% CI: 90.6-99.7%) and specificity of 100.0% (95% CI: 94.6-100.0%) at a 1/640 cut-off point , while the DAT was positive in 72 (sensitivity 97.3%); one sample did not react in either test . Although the difference between the MH algorithm and DAT sensitivity was not significant, given the greater number of samples positive by DAT, we determined to use DAT as a reference to compare the ELISA performance using recombinant antigens.Performance of recombinant antigen ELISA - In the standardization of each of the ELISA using recombinant antigens, the best conditions were established as: antigen concentration 0.5 µg/mL (rK39) and 1.0 µg/mL ; serum dilution 1/25 (rK18) and 1/100 ; conjugate dilution 1/40,000 , chromogen time 5 min  and 7 min (rK18) (n (rK18) .Based on the ROC curves constructed using the values of the ABS% Positive of the 74 samples of dogs with symptomatic VL and 66 samples of healthy dogs, the optimal cut-off point was 6.19 with rK39-ELISA; 13.42 with rK28-ELISA; 8.92 with rKR95-ELISA; 12.76 with rK18-ELISA. Using these cut-off values, the sensitivity (95% CI) of the ELISA using rK39, rK28, rKR95 and rK18 was, respectively, 97.3% (90.6-99.7%); 95.9% (88.6-99.2%); 95.9% (88.6-99.2%) and 89.1 (79.8-95.2%) and the specificity of 100.0% (94.6-100.0%); 100.0% (94.6-100.0%); 100.0% (94.6-100.0%) and 98.6% (91.8-100.0%) .Considering the 74 parasitologically positive symptomatic VL dogs, no significant difference was observed in the sensitivity of the MH algorithm in relation to the rK39-ELISA ; both the rK28-ELISA and the rKR95-ELISA ; and the rK18-ELISA .Sensitivity of recombinant antigen ELISA with subclinically VL infected dog samples - As a selection criterion, the 19 samples of subclinically infected dogs were positive in the DAT and MH algorithm. In this group of asymptomatic dogs, the sensitivity (95% CI) of the rK39-ELISA, rK28-ELISA, rKR95-ELISA and rK18-ELISA was, respectively, 89.5% (68.6-97.1%); 84.2% (62.4-94.5%); 84.2% (62.4-94.5%) and 57.9% (36.3-76.9%). In this group of asymptomatic animals, we did not compare the sensitivity of the ELISA test with recombinant antigens and the MH algorithm because all animals in the panel had already been considered positive in the tests recommended by the Ministry of Health of Brazil.Comparing the groups of symptomatic (74) and subclinically VL infected (19) dogs, no significant difference was observed in the sensitivity obtained with rK39-ELISA , rK28-ELISA  and rKR95-ELISA . However, lower sensitivity was observed in the subclinically VL infected group with rK18-ELISA .Evaluation of cross-reactivity with E. canis - After verifying the good performance of the tests with the recombinant antigens, we evaluated the possibility of cross reaction with E. canis, a disease transmitted mainly by ticks and very common in the clinical practice of veterinary routine. Of the 10 animals evaluated, 2  dogs were reactive in the rKR95-ELISA, one  in the DPP and non-reactive  in L. major-like-ELISA, rK39-ELISA, rK28-ELISA and DAT.Evaluation of each serological diagnostic strategy for the detection of L. infantum-infected dogs in various localities - The percentages of the positivity of samples from each studied location, using the DAT, MH algorithm (DPP + L. major-like-ELISA), rK39-ELISA, rK28-ELISA and rKR95-ELISA are presented in Evaluation of the prognostic ability of recombinant antigen ELISA - Since ELISA using recombinant antigens showed higher sensitivity than both the DAT and the MH algorithm, we considered the possibility that the ELISA would allow an earlier detection of the infection than the currently recommended assays. To test this, we evaluated samples’ reactivity from a cohort of 600 dogs sampled over an extended period. Considering the MH algorithm results, samples from 30 dogs were selected and distributed in two groups with 15 dogs in each. Group 1 consisted of 15 dogs that at time 1 were considered negative at the initial evaluation for Leishmania infection by the MH algorithm but subsequently became positive six months later (time 2). Group 2 consisted of samples from 15 dogs that seroconverted from DPP negative at time 1 to DPP positive at time 2 but were negative by L. major-like-ELISA at both periods.In Group 1, the rate of positive responses at time 1 were determined to be 66.7% by rK39-ELISA; 80.0% by rK28-ELISA and 60.0% by rKR95-ELISA  . DiffereIn group 2, rK39-ELISA assessed that 47.0% of the dogs were seropositive at time 1. Only one sample was positive by rK28-ELISA and two by rKR95-ELISA  . At timeViability to use rK39-ELISA, rK28-ELISA and rKR95-ELISA in the laboratories of the endemic area - Having demonstrated the utility of these ELISA in our laboratory, we then assessed assay robustness by distributing them to laboratories in areas endemic for CanL after specific training of technicians. The agreement between the UFMT and the IMT was perfect  in the rK39-ELISA, rK28-ELISA and rKR95-ELISA tests and very good  in the rK18-ELISA. The agreement between the IMT and the IAL/SP was perfect  in the rK39-ELISA, rK28-ELISA, rKR95-ELISA, and rK18-ELISA. The agreement between the IMT and the IAL/RP was perfect  in the rK39-ELISA, rK28-ELISA and rKR95-ELISA and very good  in the rK18-ELISA. The agreement between the IMT and the UFBA was good in the rK39-ELISA , rK28-ELISA  and rK18-ELISA .Meanwhile, we ascertained that the plates pre-sensitized with recombinant antigens and stored at 4ºC and -20ºC were stable with no significant decrease in reactivity up to 180 days .L. infantum-infected dogs in control programs. Previous studies show unsatisfactory performance,L. major-like-ELISA), not being used another method for the confirmation of the infection, leaving a margin of doubt about the real infection of these dogs that could thus interfere in the sensitivity results. Importantly, when samples from a group of early-infected still subclinical dogs were evaluated, the accuracy (95% CI) of ELISA with recombinant K39, K28, KR95 and K18 was 97.7% (91.6-99.9%), 96.6% (90.0-99.2%), 97.7% (91.6-99.9%) and 84.1% (74.9-90.4%), respectively. These data suggest the detection of infected dogs before the emergence of overt clinical signs.Considering the fundamental role of dogs in the VL epidemiology, diagnostic algorithms are proposed for routine surveillance to detect L. major-like ELISA by rK39-ELISA in the MH algorithm, then the reactivity reached 17.4%, thus similar, tending to be higher in this comparison. In both evaluations, both in DPP + L. major-like-ELISA and DPP + rK39-ELISA, the concordances with the DAT, the reference test, were good. Since the total antigen production is a limiting factor in producing diagnostic kits for national demand, the use of recombinant antigens for ELISA kits emerges as a real possibility to replace L. major-like-ELISA.As the main purpose of the test is its use in serological inquiry in endemic areas for VL, we conducted a search with 2,530 canine samples collected for serological inquiries in four Brazilian states. Although the parasitological examination is the gold standard for CanL diagnosis, in serological surveys of endemic areas, it becomes impracticable to perform this method due to the demand of execution time, invasive collection material and specialized team. Thus, in our study, we used the DAT, which shows good diagnostic performance in CanL, as a reference test for comparing survey samples. We submitted the samples to the MH algorithm and additionally to the DAT. The reactivities were 28.1% for rK39-ELISA, 27.1% for rKR95-ELISA, and 26.6% for rK28-ELISA, compared with 16.6% with the MH algorithm. These data suggested that the tests with these recombinant antigens detect earlier the dogs with subclinical infections. We evaluated a situation substituting total L. infantum in these locations. The ELISA with recombinant antigens K39, K28 and KR95 showed good and similar performance in the global analysis. Still, analyzing the samples according to geographical regions, we observed variable performances with rK28-ELISA and rKR95-ELISA being worse than rK39-ELISA that showed good performance regardless of the geographic area. Thus, rK39-ELISA appears to be a good assay for serological inquiry in any region. We should consider further that the recombinant antigen K39 is based on the sequence of L. infantum (syn. L. chagasi),E. canis, Toxoplasma gondii and Neospora caninum using the Kalazar Detect™ test, which uses the rK39 antigen. However, in our study, the 10 sera of dogs positive for E. canis did not react in the ELISA with the rK39 and rK28 antigens. Although the number of samples was small, these results may discard the possibility of cross-reactivity at least with E. canis of samples positive for rK39-ELISA and rK28-ELISA.Analyzing the performance of the different tests in samples obtained from different geographical areas, endemic for zoonotic VL, we observed that the presently used assays had non-satisfactory performance, compared with tests based on recombinant antigens , in city 1 (PA), city 2 (PA), city 3 (BA), city 5 (SP), city 7 (SP) and city 9 (MT). The reason may be the intra-specific variability of T. cruzi, no cross-reactivity was seen with human patients’ sera.E. canis.With rKR95-ELISA, although the protein identity of rKR95 was 79% with On the data showing higher positivity of ELISAs with recombinant antigens, around 10% higher than the result obtained with the MH algorithm, we considered indicative of the existence of a considerable number of false-negative results.L. infantum infection submitted to periodic evaluation. The dogs in this part of the study were previously evaluated through physical examination and serology . From the negative diagnosis (DPP + L. major-like-ELISA), the animals received collars impregnated with deltamethrin  that were changed according to the manufacturer’s recommendation or replaced in case of losses to avoid infection through contact with the vector. Thus, the risk of infection by the vector was extremely reduced. Despite being the gold standard in the diagnosis of CanL, the parasitological examination presents variable sensitivity,To address this possibility, we evaluated animals from a cohort of 600 dogs from an endemic area for We selected 15 dogs (Group 1) that were negative using the DPP test at time 1 that became positive using the MH algorithm six months later (time 2). Although the number of samples was reduced, the results were conclusive. Using rK39-ELISA, around 67% of the dogs turned positive among those considered negative by the MH algorithm at time 1. We also evaluated another 15 dogs from the same cohort that were initially negative in DPP in time 1 that turned out to become positive in DPP at time 2 (Group 2). Using rK39-ELISA, around 47% of these samples were positive, bringing concern on the performance of immunochromatographic rapid assays that are in use worldwide, although it was used here in one brand of kit.Leishmania recombinant antigens detect infections earlier than the MH algorithm in use. All animals in group 1 in the second collection were reactive to the tests recommended by the Ministry of Health and we believe that some were already infected since the first collection, since anti-Leishmania antibodies were detected with the antigens used in our study. We may rule out the possibility of false positive results using ELISA with recombinant antigens considering the results of the present study showing above the analysis with positive samples confirmed by parasitological exams, and the high specificity obtained. These data bring concerns on the efficacy of measures to control L. infantum transmission, implying the maintenance of infected dogs in the disease cycle with a consequent risk for the human and canine population.These results suggest that ELISAs with To ensure the ELISAs functionality in other laboratories, we evaluated the viability of applying the tests in laboratories of endemic areas for CanL. For this, we counted on the partnership of collaborating institutions that carried out the tests. The results of the concordance analysis were good to perfect and thus, we conclude that the standardized tests at the coordination center IMT can be safely reproduced by other laboratories provided they are submitted to appropriate test conditions. Further, the antigen-coated plates were stable up to 180 days when maintained at -20ºC and 4ºC.® vaccine is the only one approved for the prevention of Canine Visceral Leishmaniasis, and several debates regarding serological methods for the differentiation of vaccinated and infected dogs have been raised. In our study we did not evaluate groups of vaccinated dogs, but we believe that the antigens validated in the present study do not detect antibodies from animals vaccinated in the ELISA because they have different epitopes. In the study conducted by Campos et al.,® were not reagent to DPP and L. major-like-ELISA in the 30, 180 and 360 days after the vaccination protocol.In Brazil, the Leish-TecLeishmania antigens, suggesting the rK39 antigen for the diagnosis of CanL. This test is efficient, and it seems there is a superiority in sensitivity compared with the tests currently in use in Brazil. Thus, rK39-ELISA used as a single screening and the confirmatory test is a concrete possibility for the diagnosis of dogs infected with L. infantum, both in Brazil and in other locations where the dog is the reservoir. Although the control of CanL in Brazil was our primary interest, considering the increasing importance of VL caused by L. infantum in other parts of the world, the rK39-ELISA here shown with high diagnostic performance can be extended to the diagnosis and management of CanL worldwide.In this study, we validated the ELISA with recombinant


Argyresthia conjugella , is a pre-dispersal seed predator. Larvae feed on rowanberries (Sorbus aucuparia), and when rowanberry seed production is low , the moth switches from laying eggs in rowanberries to apples , resulting in devastating losses in apple crops. Using genetic methods, we investigated if this small moth expresses any local genetic structure, or alternatively if gene flow may be high within the Scandinavian Peninsula . Genetic diversity was found to be high . For three out of ten tetranucleotide STRs, we detected heterozygote deficiency caused by null alleles, but tests showed little impact on the overall results. Genetic differentiation between the 28 sampling locations was very low . Surprisingly, we found that all individuals could be assigned to one of two non-geographic genetic clusters, and that a third, geographic cluster was found to be associated with 30% of the sampling locations, with weak but significant signals of isolation-by-distance. Conclusively, our findings suggest wind-aided dispersal and spatial synchrony of both sexes of the apple fruit moth over large areas and across very different climatic zones. We speculate that the species may recently have had two separate genetic origins caused by a genetic bottleneck after inter-masting, followed by rapid dispersal and homogenization of the gene pool across the landscape. We suggest further investigations of spatial genetic similarities and differences of the apple fruit moth at larger geographical scales, through life-stages, across inter-masting, and during attacks by the parasitoid wasp (Microgaster politus).Knowledge about population genetic structure and dispersal capabilities is important for the development of targeted management strategies for agricultural pest species. The apple fruit moth,  Agricultural insect pest species have considerable negative impacts on crop and fruit yields and consequently, food security. Global climate change may accelerate pest species distribution ranges and population sizes with increasing temperatures and range shifts . Hence, The degree of population genetic differentiation in insect pest species depends on several factors. Among these, distribution and patchiness of the host plants, specialization to host species, active dispersal capabilities, and passive transport by wind dispersal may be important , 7–12. I13C) showed long distance migration across the east coast of North America [Danaus plexippus) could also detect long distance migration in addition to identifying genes and pathways associated with migratory behavior [Detailed knowledge about insect dispersal may help understand population dynamics and develop forecasting systems to alert farmers and foresters to invasions. Outbreak dynamics often occur at large geographical scales and therefore, large-scale assessments are needed to understand population dynamics across the landscape. Both observational and genetic methods have been used to track and detect the migration and dispersal of insects. Direct observational methods like radio telemetry, harmonic radar, vertical looking radar and trapping have been used to estimate the dispersal of butterfly species –21. Capt America . Capture America . In addi America . In cont America , 24–27 a America . DNA seqbehavior .Argyresthia conjugella, Lepidoptera, Yponomeutida, Zeller 1839) is a small species with a body length of 5–6 mm. The distribution range of the apple fruit moth includes Europe, North America, and Asia [Sorbus aucuparia). Females are attracted to unripe rowan berries and lay their eggs on or near the berries. The larvae bore into the fruits, where they live and forage. In late summer, the larvae drop to the ground and pupate. The pupae overwinter in the ground and the adults emerge in May-June the following year [The apple fruit moth  genetic markers [Our main objective in this study was to investigate large-scale genetic structuring of populations of the apple fruit moth as a Lepidoptera model species. Genetic studies of other Lepidoptera species with cyclic outbreaks have shown genetic differences between life stages . However markers .2, see Apple fruit moth larvae were collected from infested rowanberries in natural habitats from a total of 28 locations in Norway (17 locations), Sweden (8 locations), and Finland (3 locations) during August 2016. The study area was located on the Scandinavian Peninsula  according to the manufacturer’s instructions, and the only modification was the elution of DNA in 150μl instead of 50–100μl elution buffer.2 (ABI), 0.5 μM of each primer (Life technologies), 1 U Amplitaq Gold DNA polymerase (ABI), 1x BSA (NEB), and 1 μl template DNA (0.1 to 2.0 ng). PCRs were performed on a 2720 Thermal cycler and the conditions for the PCR were 10 min at 95°C, 26 cycles of 30 s at 94°C, 30 s at 58°C, 1 min 72°C, and final extension for 45 min at 72°C. (ii) multiplex PCRs were developed by testing different primer combinations until a set of four markers amplified successfully with clear chromatograms and without artificial alleles/spikes/primer dimers. The PCR forward primer for each locus was labeled with one of four different fluorescent labels  in such a manner that no two markers with the same fluorescent dye had overlapping allele size ranges. The primer concentrations were optimized to result in equal peak heights (RFU) for all markers within a multiplex panel, and the number of cycles for all PCR reactions was optimized to achieve optimal peak height of alleles . For the final analysis, the STR markers were split into three multiplexes. The PCR reactions were carried out in 10 μl reaction volumes containing: 5 μl 2x multiplex PCR master mix (Qiagen Multiplex kit), 0.05 μg/μl BSA (NEB), and adjusted primer set concentrations. PCR conditions for multiplexes were carried out as: 10 min at 95°C, 24 cycles of 30 s at 94°C, 30 s at 58°C, 1 min 72°C, and final extension for 45 min at 72°C. Separation of fluorescently-labeled PCR products was carried out on an Applied Biosystems 3730xl Genetic Analyzer  and GeneMapper 5.0  was employed to determine allele sizes. Negative and positive controls were included on all 96-wells plates. In addition, we performed a separate re-run with eight different individuals with all multiplex reactions to ensure reproducibility. We excluded individuals with low amount or bad quality of DNA, or individuals with results that showed four or more missing loci, from the study. Concerning potential genotyping errors, we randomly selected three specimens per population and re-amplified those with the 10 STR marker set. Then, we compared the retrieved genotypic profiles with the results of the first amplification round. All genotypic profiles were identical, indicating that STR markers could be reliably amplified and scored. In addition, we were able to rule out any mix-ups of plates that might have led to erroneous statistical results. Based on these results, we concluded that genotyping and scoring errors or other lab-related errors can be excluded as potential causes for obtained genetic patterns in this study. Finally, we also tested the difference in power to detect genetic differentiation and structure with the smaller dataset (n = 64) from the previous study [From our previous work on apple fruit moths , we seleus study , applyinIS) for each STR locus and averages across all STRs per sampling location. Run parameters included the Markov chain method with 10,000 dememorization steps, 5,000 batches, and 10,000 iterations to estimate P-values. Moreover, we calculated Nei’s genetic diversity [Tests for null alleles, scoring errors, and large allele dropouts were conducted with MICRO-CHECKER 2.2.3 . We testiversity  using Poiversity . In addiiversity .We tested for genetic structure using Bayesian assignment analysis with STRUCTURE 2.3.4 , which wSince uneven population sizes can lead to false inferences about the number of genetic clusters in Bayesian assignment tests , the sofST values and significance of pairwise FST values was tested with 9,999 permutations with the same software.We performed a hierarchical Analysis of Molecular Variance (AMOVA) analysis  to estimST [p.adjust function in R to correct for multiple testing of all pairwise genetic differentiation values with the Benjamini-Hochberg correction [In addition, two genetic differentiation estimators were calculated, GST  and JostST  to proviST . We usedrrection .ST- 1)/4 [Gene flow was estimated assuming Nm = (1/FST- 1)/4 . Mantel’ST- 1)/4  was usedST- 1)/4 , 67.ST values using the ENA method; Chapuis and Estoup (2007) [Since null alleles were found to be present at three loci , the anap (2007) ).compareestimator function implemented in RELATED. Subsequently, the best relatedness estimator with the highest correlation coefficient (R) was chosen to compare the observed relatedness values for each sampling site against deviations from random mating expectations, using 1,000 permutations, to determine whether individuals within sampling sites were more closely related than expected.We estimated relatedness within and among sampling sites using the R package RELATED v.1.0 . First, We utilized the software INEST 2.2  to test IS was 0.009 to 0.522  showed consistent and significant signs of null alleles . In contrast, eight of the 28 sampling sites were significantly differentiated from most other populations, namely, locations H, J, K, M, O, T, X, and Z when including ten STR loci in the analysis  showed no genetic differences between males and females . AnalysiEstimated gene flow among the populations was high (Nm = 15.1). Mantel’s test value was very low, but significant , indicating largely a lack of correlation between genetic and geographic distances .Isolation-by-distance tests revealed that there was a weak but significantly increasing genetic differentiation with increasing geographic distance .The Wang (2002)  relatednRegarding bottlenecks, no consistently significant results were found for any of the sampling sites tested, suggesting that recent demographic bottlenecks were absent.In this study, we found that the apple fruit moth showed high genetic diversity and overwhelming genetic admixture within one study year for both sexes, over large areas, and across different climatic zones. We found two major non-geographic genetic clusters in contrast to geographic substructure in 20–30% of the specific sampling locations on the Scandinavian Peninsula. We also detected null alleles among the STRs, which may affect the results. Consequently, we included tests in the study to avoid bias in the population genetic analyses.ST was estimated using the correction method implemented in FREENA [Null alleles in STR markers appear to be common in taxa with high effective population sizes, such as lepidopteran species , 73–76. n FREENA , as welln FREENA , 67, resn FREENA , FREENA n FREENA , 82 in tReducing the number of STRs from 19 to 10 in this study led only to limited loss of genetic differentiation detection power in a test based on a smaller dataset and a smaller geographical area. The more than ten times larger dataset and the high number of alleles in the present study may additionally reduce this loss in power to detect genetic structure. Thus, we conclude that the alternative use of all 19 STRs  may not The substructure that we observed for six  by favoring directed winds and selecting specific flight altitudes [Microgaster politus) population [The lack of genetic differentiation across large areas indicated a high migration rate for the apple fruit moth. A high migration rate has been found to be rather common in Lepidoptera species, e.g. lostella –85, Vanea cardui , Busseolla fusca , Athetislepigone , Ostriniubilalis  and Spodugiperda . Spring-ugiperda , 33, 89.lostella , 90 and ubilalis . The mignd to UK . In nortltitudes . Based opulation , 92, 93.In a study in Norway in 2011 using AFLP data, we found two genetic clusters separated by a mountain plateau , while sTherefore, we are currently unable to fully distinguish between these two hypotheses and cannot exclude the possibility that a mixed effect is present in the apple fruit moth. Hence, future studies with a temporal sampling design are needed to further corroborate these initial findings. Our results further suggest that 2012  was a pHelicoerpa armigersa). Thus, we suggest to further investigate if these spatial genetic similarities and differences of the apple fruit moth persist on larger geographical scales, through life-stages, across the dynamic fluctuations of low rowanberry production (inter-masting), and during attacks by the parasitoid wasp (Microgaster politus).Population structure may also be influenced on a more global scale, as for the relationship between metapopulation dynamics  and seveS1 Table(DOCX)Click here for additional data file.S2 TableST values below the diagonal. Probability, P(rand > = data) based on 9,999 permutations is shown above diagonal. Bold values are significant after Benjamini-Hochberg [The analysis is based on 10 STR loci using the Arlequin software version 2.0 . FST valHochberg  correcti(DOCX)Click here for additional data file.S3 TableST values below the diagonal. Probability, P(rand > = data) based on 9,999 permutations is shown above diagonal. Bold values are significant after Benjamini-Hochberg [FHochberg  correcti(DOCX)Click here for additional data file.S4 TableST values below the diagonal. Probability, P(rand > = data) based on 9,999 permutations is shown above diagonal. Bold values are significant after Benjamini-Hochberg [GHochberg  correcti(DOCX)Click here for additional data file.S5 TableST values below the diagonal. Probability, P(rand > = data) based on 9,999 permutations is shown above diagonal. Bold values are significant after Benjamini-Hochberg [GHochberg  correcti(DOCX)Click here for additional data file.S6 TableEST values below the diagonal. Probability, P(rand > = data) based on 9,999 permutations is shown above diagonal. Bold values are significant after Benjamini-Hochberg [DHochberg  correcti(DOCX)Click here for additional data file.S7 TableEST values below the diagonal. Probability, P(rand > = data) based on 9,999 permutations is shown above diagonal. Bold values are significant after Benjamini-Hochberg [DHochberg  correcti(DOCX)Click here for additional data file.S8 TableStandard; genetic distances were not transformed into linear genetic distances, linear: genetic distances were transformed. In parentheses: results were corrected or uncorrected for null alleles  [CHORD designates the chord distance that was used as this genetic distance metric has been suggested to perform better in a range of circumstances in isolation by distance tests when null alleles are present  [. (2007) . In addil. 2017) . For geo(DOCX)Click here for additional data file.S1 FigA. Results for runs with 10 loci and no LOCPRIOR; B. Results for runs with 10 loci and with LOCPRIOR option.(EPS)Click here for additional data file.S2 FigA. Results for runs with seven loci and no LOCPRIOR; B. Results for runs with 7 loci and with LOCPRIOR option.(EPS)Click here for additional data file.S3 FigA. 10 loci without LOCPRIOR, B. 10 loci with LOCPRIOR, C. Seven loci without LOCRPRIOR, and D. Seven loci with LOCPRIOR.(EPS)Click here for additional data file.S4 FigBayesian assignment was performed using the program STRUCTURE with bar plots for K = 2 to K = 3, without (A) and with (B) LOCPRIOR.(EPS)Click here for additional data file.S5 Fig(EPS)Click here for additional data file.


Plasmodium falciparum have been performed so far. For this reason, this study aims to examine the genetic diversity of P. falciparum by studying samples collected in Grande Comore in 2012 and 2013, using merozoite surface protein 1 (msp1), merozoite surface protein 2 (msp2) and single nucleotide polymorphism (SNP) genetic markers.Despite several control interventions resulting in a considerable decrease in malaria prevalence in the Union of the Comoros, the disease remains a public health problem with high transmission in Grande Comore compared to neighbouring islands. In this country, only a few studies investigating the genetic diversity of msp1 gene as well as allelic families IC3D7 and FC37 of the msp2 gene were determined by using nested PCR. Additionally, 50 out of 151 samples were genotyped to study 24 SNPs by using high resolution melting (HRM).A total of 162 positive rapid diagnostic test (RDT) samples from Grande Comore were used to extract parasite DNA. Allelic families K1, Mad20 and RO33 of the msp1 gene (55%) and the FC27 family of msp2 gene (47.4%). Among 50 samples genotyped for 24 SNPs, 42 (84%) yielded interpretable results. Out of these isolates, 36 (85%) were genetically unique and 6 (15%) grouped into two clusters. The genetic diversity of P. falciparum calculated from msp1 and msp2 genes and SNPs was 0.82 and 0.61, respectively.Two allelic families were predominant, the K1 family of P. falciparum was observed in Grande Comore. This may favour persistence of malaria and might be one of the reasons for the high malaria transmission compared to neighbouring islands. Further surveillance of P. falciparum isolates, mainly through environmental management and vector control, is warranted until complete elimination is attained.In summary, a large genetic diversity of  Plasmodium falciparum remains the most dominant species in Africa and responsible for the most severe clinical forms; it is associated with 95% of malaria-associated deaths [P. falciparum infections, such as the level of acquired immunity of the individual and genetic characteristics of the parasite [Despite several interventions to prevent, control, and eliminate malaria, 219 million cases and 435,000 deaths occurred worldwide in 2018, mainly in children under 5 years and pregnant women in Africa [P. falciparum remains an obstacle to achieve malaria elimination. Previous vaccine trials have shown limited efficacy of interventions in time and different geographic regions due to genetic polymorphisms [P. falciparum [P. falciparum is characterized by substitutions of single nucleotides occurring at specific positions in the genome, referred to as single nucleotide polymorphisms (SNPs). Approximately 112,000 SNPs have been described in the genome of P. falciparum and are considered useful molecular markers for barcoding to identify and characterize genetic diversity of P. falciparum [The genetic diversity of orphisms , 6. The lciparum –9. The glciparum –12.Plasmodium species. First, because high genetic diversity has been associated with high levels of malaria transmission [P. falciparum isolates can provide important information on how to improve strategies to reach malaria elimination.In the Comoros archipelago, which is composed of four islands in the Indian Ocean between Madagascar and the African coast, malaria remains a major public health problem with high transmission and mortality rate among children under five years old . Since 2smission , a measusmission . Second,P. falciparum strains in the Union of Comoros where the mean expected heterozygosity (He) was 0.63 in Grande Comore and the multiplicity of infection (MOI) was 1.39 from microsatellite data [msp genes correlated with a decrease in malaria transmission in Grande Comore.A 2010 study revealed a significant diversity of ite data . Since tite data . The resP. falciparum populations in other sites in Grande Comore using msp1, msp2 and SNPs to determine the genetic diversity and MOI.As a follow-up of that study, the aim of the present study was to characterize P. falciparum were collected from health centres. RDTs that were negative or invalid were excluded from this study. All RDTs were stored at room temperature until they were used for DNA extraction.A total of 162 rapid diagnostic tests (RDTs) were collected in 2012 and 2013 in three sites of Grande Comore . Malaria transmission varies in these three sites: hypoendemic in Moroni, mesoendemic in Mitsamiouli, and hyperendemic in Mbeni [. [Parasite DNA stored on the nitrocellulose membrane of RDTs was extracted according to the protocol developed by Cnops et al. . DNA extMsp1 and msp2 genes were amplified by nested PCR as described in earlier studies [® DNA polymerase-reaction buffer , 0.5 μM of each primer, and 11 μl reagent grade water. The cycling conditions for the primary PCR were as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 58 °C for 2 min, 72 °C for 2 min, with a final extension cycle of 72 °C for 3 min. The cycling conditions for the nested PCR were as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 61 °C for 2 min, 72 °C for 2 min, with a final extension cycle of 72 °C for 3 min. Positive controls and negative control were run in each PCR. PCR products were analysed by gel electrophoresis on 2% agarose gels and visualized under ultraviolet trans-illumination (Biorad Gel Doc™ XR + System with Image Lab). A 100 bp DNA ladder (Quick-Load) was used to determine the size of PCR products. studies , 20. Allmsp1 gene were between 150–300 bp, 130–230 bp, and 150–200 bp for K1, MAD, and RO33, respectively. For the msp2 gene, the expected sizes were between 290–450 bp for FC27 and between 450–700 bp for IC3D7.The expected sizes of PCR products for the P. falciparum were genotyped by using high-resolution melting (HRM), as previously described [Twenty-four SNPs of escribed . PCR ampmsp1 and msp2 genes of the 3 populations  was determined by the number of alleles per locus and by Nei [Genetic diversity of d by Nei  unbiasedd by Nei .\documeni allele.where n is the sample size and Genetic diversity index based on SNPs, equivalent to the expected heterozygosity index (He), was estimated by ty index , implemety index . Phylogety index .msp1 and msp2 genes in three regions of the Grande Comore and He. The MOI is the mean number of fragments detected for each msp gene and each allelic family. A sample with one clone for the msp genes was considered monoclonal, while a sample with at least 2 clones was considered polyclonal. Mann–Whitney U test was used to compare mean MOI. The significance threshold was fixed at α = 0.05.Fisher’s exact test was used to compare the allelic frequencies of msp1 gene and 9 alleles for msp2 gene.Of 162 samples, 151 were successfully genotyped: 25 from Moroni, 49 from Mitsamiouli, and 77 from Mbeni. Among them, 21 individual alleles were found, including 12 alleles for msp1, 7 K1 type alleles (150–300 bp), 3 MAD20 type alleles (190–230 bp), and 2 RO33 type alleles (150 and 200 bp) were observed  and 6 IC3D7 type alleles (450–700 bp) were obtained. For FC27, the most frequent type allele was 400 bp fragment, and the most frequent type allele for IC3D7 was 500 bp fragment  than in Moroni  or Mbeni . RO33 msp1 allelic family was poorly represented (2.4–4.0%) in all three sites.In all three sites, the K1 family was predominant (55%) for ne Table . There wmsp2 gene, IC3D7 allelic family was predominant in Moroni (65%). Its frequency was lower in both Mitsamiouli  and Mbeni . The FC27 family was found to be predominant in Mbeni (60.5%), in comparison to Moroni  and Mitsamiouli .For  samples showed polyclonal infection with at least two clones, resulting in mean MOI of 1.40 Table . In Mitsmsp genes, the representativeness of our sub-sample was confirmed.In this study, 50 of 151 (33%) samples were genotyped for 24 SNP markers. Among these, 42 samples  and 21 SNPs yielded interpretable results  different genotypes were obtained from 42 samples. Six of 42 (14.3%) isolates grouped into two clusters, defined as a group of parasites with identical SNP barcode: one cluster consisting of 2/42 (4.8%) parasite populations from Mitsamiouli  and another cluster consisting of 4/42 (9.5%) samples from Mbeni (defined by the barcode TATTCCGTTGCCACTCGATTG). Moreover, among 36 genetically unique isolates, several had a strong linkage since they only differed from each other by a few nucleotides , SNPs (clusters and unique barcodes) were classified as monoclonal or polyclonal. Analysis showed that all clusters were polyclonal. Among the parasites with unique barcodes, 7 of 36 (19.4%) were monoclonal, and 29 of 36 (80.6%) were polyclonal.To investigate whether there is an association between barcode and Data on allelic frequency showed that several nucleotides predominated at the study sites. At P2 position, adenine (A) number predominated at all sites. At P5 position, cytosine (C) number predominated. At positions P21 and P24, adenine (A) and guanine (G) numbers were seen in the majority of isolates collected in Mitsamiouli and Moroni, respectively. The lowest minority allelic frequency (MAF) was found in position 20 in 5 of 42 (11.9%) isolates calculated from guanine number  (Table . The highest genetic diversity was found at Mitsamiouli (He = 1) and the lowest at Mbeni (He = 0.41).Genetic diversity (He) was estimated from s) Table . There ws) Table . For SNPmsp and SNP were compared in both sites using Fisher's exact test. The He index based on msp gene was higher than the index based on SNP in Mbeni (p < 0.01), but an opposite but insignificant trend was observed in Mitsamouili (p = 0.08). Comparison in the whole populations did not show any difference (p = 0.9)  Table .Table 4CP. falciparum has so far received limited attention and needs to be further investigated. In Grande Comore, genotyping of msp1 and msp2 genes revealed a high allelic polymorphism (He = 0.83), comparable to areas of high malaria transmission in Africa [msp1 gene, the K1 family was dominant (55%). For 9 alleles in the msp2 gene, the FC27 family was most represented (47.4%). These results are similar to those obtained in Madagascar [The Union of Comoros has entered the phase of malaria elimination after the implementation of several strategies, such as the massive use of insecticide-impregnated bed nets (ITNs), artemisinin-based combination therapy (ACT) for confirmed malaria cases, intermittent preventive treatment in pregnant women (IPTp), and mass drug administration (MDA) with artesunate-mefloquine and primaquine. However, the impact of this intervention on genetic diversity in n Africa –29. For dagascar  and in Gdagascar . Interesdagascar .msp1 and msp2 allelic families using agarose gel electrophoresis used in the present study has limitations due to difficulties in defining specific alleles with accuracy for each isolate. To circumvent this drawback, 24 SNPs were used to determine the genetic diversity of P. falciparum in Grande Comore for the first time. These 24 SNPs are able to identify different parasites with a high minor allelic frequency [P. falciparum on the island (He = 0.61). The results of SNP genotyping confirm those of msp1 and msp2 markers .Fragment analysis of requency . After Smsp and barcode showed that all clusters were polyclonal. Similarly, among isolates with unique barcodes, the majority (78.9%) were polyclonal as well although data obtained in the present study suggested that the barcode of 21 SNPs cannot predict mixed infections. MOI based on msp remains the most powerful tool for determining the number of distinct P. falciparum populations in an infection. The average MOI found in this study for all sites was 1.36 for msp1 and 1.05 for msp2. These results are similar to those obtained in Grande Comore in isolates collected in 2013–2016, suggesting a decreasing malaria transmission intensity on this island [.  [P. falciparum. This may be due to the choice of msp markers used, which are biased due to immune pressure.The comparison of results from the studies concerning s island . Moreovemalaria) . HoweverP. falciparum is more frequent in areas of high transmission [Moreover, the considerable level of genetic diversity was found in this study despite trends in decreased malaria transmission in Comoros. Indeed, it has been established that a high genetic diversity of smission . This obsmission . Indeed,smission . TherefoP. falciparum showed high genetic diversity in Grande Comore. This might be one of the reasons for the high malaria transmission in this island, in comparison to neighbouring islands, despite the implementation of several malaria elimination strategies. Further studies on genetic diversity are essential to determine the impact of past interventions. Additionally, they may contribute to the adoption of future strategies for the achievement of malaria elimination in the Union of the Comoros.Clinical isolates of Additional file 1: Table S1. Molecular barcodes for sequenced P. falciparum isolates. Reference P. falciparum clones 3D7/unknown origin , Dd2/Indochina, HB3/Honduras, and Tm90/Thailand were controls used to differentiate parasite genotypes. MAF, minority allelic frequency. N denotes the number of mixed alleles. X denotes missing nucleotide data. The frequency of minority alleles was determined by counting the number of minority allele in each position.Additional file 2: Figure S1. Phylogenetic tree of SNPs. Samples C13 and C34 are from Mitsamiouli and had the same nucleotide sequence. Samples C54, C48, C44, and C41 had the same sequence. They were all collected in Mbeni. Several isolates were strongly linked. For example, C22 and C24 had a single nucleotide difference. Phylogenetic tree based on SNPs data was constructed using MEGA X UPGMA algorithm.