P. armeniaca var. ansu were analyzed based on SSR markers. The aim was to provide scientific basis for conservation, efficient utilization, molecular marker assisted breeding and improved variety selection of P. armeniaca var. ansu germplasm resources. The results showed that the level of genetic diversity within the population was high. Among the 30 SSR markers, the mean number of observed alleles was 11.433, the mean number of effective alleles was 4.433, the mean of Shannon information index was 1.670, and the mean of polymorphic information content was 0.670. Among the eight provenances, Tuanjie Township, Xinyuan County, Xinjiang had the highest genetic diversity. The observed alleles, effective alleles, Shannon information index and Nei’s gene diversity index among provenances were higher than those within provenances. Based on Bayesian mathematical modeling and UPGMA cluster analysis, 86 P. armeniaca var. ansu accessions were divided into three subpopulations and four groups, which reflected individual differences in provenances. Subpopulations classified by Bayesian mathematical modeling and groups classified by UPGMA cluster analysis were significantly correlated with geographical provenance (Sig<0.01) and the provenances significantly impacted classification of groups. The provenances played an important role in classification of groups. The genetic distance between Tuanjie Township of Xinyuan County and Alemale Township of Xinyuan County was the smallest, while the genetic relationship between them was the closest and the degree of genetic differentiation was small.The genetic diversity and genetic structure of  Prunus armeniaca L. refers to both the wild progenitor and the cultivated species, which belongs to the family Rosaceae , bu= 0.774)  and Prun= 0.792) . The lev = 0.63) , but a l = 0.72) , China ( = 0.72) , and Pak = 0.72) . Plant g = 0.72) . Among t = 0.72) .P. armeniaca var. ansu population was divided into 3 subpopulations with a Bayesian model , which is similar to earlier results seen in Prunus sibirica [P. armeniaca [P. armeniaca var. ansu geographic groups showed a high level of genetic differentiation (Fst = 0.255> 0.015) [P. armeniaca var. ansu (0<Fit<1) [P. armeniaca var. ansu was likely caused by mating among close relatives rather than self-pollination. Considering the low gene flow in the P. armeniaca var. ansu population (Nm = 0.731<1) [Genetic structure results from the joint action of mutation, selection, migration, and drift , and causibirica , P. armermeniaca  and mostrmeniaca . Neverth> 0.015) , which s0<Fit<1) , and the0<Fit<1) . Therefo0.731<1) , migratiP. armeniaca var. ansu. With the intensification of inbreeding depression, forest productivity and population survivability will decline, which may lead to the extinction of this species, so it is necessary to preserve P. armeniaca var. ansu genetic resources. Considering the decentralized and wide distribution of P. armeniaca var. ansu, according to the existing genetic diversity and genetic structure of its populations, we propose a conservation strategy that combining in situ protection and ex situ protection. Measures that can be taken for in situ protection include establishing nature reserves, forest reserves, prohibiting grazing, controlling the utilization degree of wild resources, and encouraging vigorous promotion of P. armeniaca var. ansu resources in suitable areas. However, it is difficult to implement in situ protection for all species, so ex-situ protection should be given more attention. In addition, increasing the tending management of stands could also contribute to the protection of P. armeniaca var. ansu resources [Prunus species, which requires seeds and scions from each population for ex-situ protection. We will also collect germplasm resources in greater breadth and depth in the future. Based on the results of genetic diversity of P. armeniaca var. ansu from different provenances, the resources of Tuanjie Township, Xinyuan County, Xinjiang should be protected first.In the forest ecosystem, the extinction of a tree species can produce a chain reaction, which can lead to the extinction of some local appendage species . Our resesources . The maiesources . We haveP. armeniaca var. ansu population had a high level of genetic diversity, with those from Tuanjie Township and Xinyuan County being the most diverse. The level of genetic diversity among provenances was higher than diversity within provenances, and there was genetic differentiation within and among provenances. The genetic variation of P. armeniaca var. ansu mainly occurred within provenances, with a small degree present between them. The genetic relationship between Tuanjie Township, Xinyuan County and Alemale Township, Xinyuan County was the closest, and the degree of genetic differentiation was the smallest. Provenances played an important role in the classification of groups, while geographical distance was closely related to genetic difference. These results highlight the importance of accounting for provenances in future breeding efforts. Taken together, the results of our study provide a new scientific basis for conservation, efficient utilization and breeding of P. armeniaca var. ansu germplasm.The S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen which mainly causes diarrhea, dehydration and death in nursing piglets, threatening the global swine industry. Moreover, it can infect multiple animal species and humans. Hence, reliable diagnostic assays are needed to better control this zoonotic pathogen. Here, a blocking ELISA was developed using a recombinant nucleocapsid (N) protein as the coating antigen paired with an N-specific monoclonal antibody (mAb) as the detection antibody. The percent inhibition (PI) of the ELISA was determined using 384 swine serum samples, with an indirect immunofluorescence assay (IFA) as the reference method. Through receiver operating characteristic analysis in conjunction with Youden’s index, the optimal PI cut-off value was determined to be 51.65%, which corresponded to a diagnostic sensitivity of 98.79% and a diagnostic specificity of 100%. Of the 330 serum samples tested positive via IFA, 326 and 4 were tested positive and negative via the ELISA, respectively, while the 54 serum samples tested negative via IFA were all negative via the ELISA. The overall coincidence rate between the two assays was 98.96% (380/384). The ELISA exhibited good repeatability and did not cross-react with antisera against other swine pathogens. Overall, this is the first report on developing a blocking ELISA for PDCoV serodiagnosis.  Porcine deltacoronavirus (PDCoV) is a new type of porcine enteropathogen which mainly infects piglets and causes severe diarrhea, vomiting, dehydration, emaciation and eventual death . Pigs ofIn 2012, Woo et al. identified the complete genome sequence of PDCoV for the first time from pig rectal swabs which were collected during 2009–2010 . In 2014Deltacoronavirus within the Coronaviridae family  × 100%.A checkerboard titration was used to determine the optimal antigen-coating concentration and working concentration of the mAb 1A3 using the two reference swine sera S23 and S0. Specifically, 96-well ELISA plates  were coated with 100 μL/well of 2-fold serially diluted PDCoV N protein in the range of 0.15625–5 µg/mL for 12 h at 4 °C. After being washed three times with PBST, the plates were blocked with 2% BSA diluted in PBST for 2 h at 37 °C. After another three washes with PBST, 100 μL/well of undiluted positive and negative reference swine sera was separately added to the odd and even columns of the plates, which were then incubated for 1 h at 37 °C. After washing, 100 μL/well of the 2-fold serial dilutions of PDCoV N-specific mAb 1A3 (supernatant of the hybridoma cells) ranging from 1:10 to 1:320 was added to each well. The plates were then incubated for 1 h at 37 °C. Upon washing, 100 μL/well of HRP-conjugated Goat anti-Mouse IgG at 1:4000 dilution was added to each well, and the plates were incubated for 1 h at 37 °C. After a final wash, 100 μL/well of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to the plates for a 10 min chromogenic reaction at room temperature in the dark. After stopping the reaction by adding 50 μL/well of 2 M Hc{Sensitivity(c) + Specificity (c) − 1} , which is one of the most accurate methods for the calculation of the optimal cut-off point for diagnostic methods [The cut-off value of the developed N-based blocking ELISA was determined using 384 swine serum samples collected from individual piglets with known PDCoV antibody status, which was confirmed using the reference method—IFA. The detection results of the blocking ELISA were compared with those obtained from the IFA. Receiver operating characteristic (ROC) analysis was conducted to analyze the blocking ELISA results obtained with the positive- and negative-testing serum samples in order to determine an optimized cut-off value that maximizes both the diagnostic specificity and diagnostic sensitivity of the assay. The PI value of each serum was analyzed by the ROC curve using GraphPad Prism software  to present the area under the curve (AUC) at a 95% CI. Furthermore, the IFA test result of each serum sample and its corresponding PI value were also analyzed by the ROC curve using IBM SPSS 26.0 software  to present the corresponding diagnostic sensitivity and specificity values constituting the ROC curve. The resulting sensitivity and specificity values were then used to calculate the Youden index, defined as max methods ,30. The Two-fold serially diluted PDCoV antibody-positive reference sera in the range of 1:8–1:1024 were used to evaluate the analytical sensitivity of the developed blocking ELISA. One hundred microliters of each dilution were tested in octuplicate via the ELISA under the optimal conditions. The maximum dilution of the reference antiserum that still produced a PI value greater than the positive cut-off point was defined as the analytic sensitivity of the blocking ELISA. For comparison, IFA was performed in parallel on the same dilutions of the antiserum. Furthermore, an in-house indirect ELISA developed to detect PDCoV antibodies, using the recombinant PDCoV N protein as the coating antigen and paired with a cut-off value of 0.290 , was alsFirstly, the specificity of the mAb 1A3 used in the blocking ELISA was analyzed via IFA to see whether it cross-reacted with other important pathogens causing diarrhea in pigs, including PEDV, TGEV, PRoV, and PEAV. Subsequently, the analytic specificity of the developed blocking ELISA was evaluated by testing its cross-reactivity with the antisera against PRoV, PEDV, TGEV, and PEAV. Three replicates of each antiserum were run on the same occasion under the optimal conditions. Moreover, for an analytical specificity comparison, the foregoing antisera were also tested using the in-house indirect ELISA .To evaluate the intra-assay repeatability and inter-assay reproducibility of the developed blocking ELISA, three PDCoV antisera representing strongly, moderately and weakly positive samples were tested using the ELISA on one plate in one run or on three distinct plates in three independent runs. Each serum was tested in triplicate. The coefficient of variation (CV) was used to quantify the degree of variation of the blocking ELISA, which was calculated by dividing the standard deviation (SD) by the mean PI value of each antiserum.Software of GraphPad Prism and SPSS 26.0 were used to perform ROC analysis and Youden’s J statistic.E. coli Rosetta (DE3) competent cells, which were then induced with 1 mM IPTG for 6 h at 37 °C. The expressed PDCoV N protein was analyzed via SDS-PAGE and Western blot analyses. As shown in The recombinant plasmid pET-28a-PDCoV-N was transformed into 450nm ratio and was thus selected as the optimal antigen-coating concentration and detection antibody dilution of the developed blocking ELISA  produced the maximum P/N ODng ELISA A. Furthep < 0.0001) with a 95% CI of 0.9982 to 1.000, indicating that the blocking ELISA had a significant coincidence rate with IFA. With the help of SPSS software, the diagnostic sensitivity and specificity values constituting the ROC curve were presented (data not shown), and it was shown that only when the diagnostic sensitivity and specificity were 98.79% and 100.00%, respectively (After optimizing the major reaction conditions of the blocking ELISA, a total of 384 swine serum samples were used to evaluate the performance of the assay. Before testing with the blocking ELISA, all swine serum samples were analyzed via IFA to confirm the PDCoV seronegative and seropositive status. Upon detection via IFA, 330 and 54 serum samples were confirmed to be positive and negative for PDCoV antibodies, respectively. Subsequently, all the samples were tested using the developed blocking ELISA, and the PI value of each sample was calculated. A ROC curve statistical analysis was performed to determine the PI cut-off value and to evaluate the diagnostic sensitivity and specificity of the assay. As shown in ectively A, was thectively B. Based ectively . Compareectively B, which Before assessing the analytic specificity of the developed blocking ELISA, we first analyzed the specificity of the mAb 1A3 which would be used as the detection antibody in the blocking ELISA. As shown in Two-fold serial dilutions of the positive reference swine serum S23 in the range of 1:8–1:2048 were simultaneously detected using the developed blocking ELISA and the IFA. As shown in In the repeatability analysis, three strongly positive, medium positive and weakly positive PDCoV antisera were tested using the developed blocking ELISA on one plate in one run. In the reproducibility analysis, the three antisera were tested using the blocking ELISA on three different plates in three independent runs. As shown in As a newly discovered coronavirus , PDCoV hAs one of the most commonly used serological assays, the ELISA is capable of identifying a previous exposure to a pathogen, evaluating the kinetics of antibody response to the pathogen and facilitating the progression of an epidemiological investigation of the infection. Although a series of N-, S1-, and M-based indirect ELISAs have been established and tried to be applied to diagnose PDCoV infection in pigs ,24,25,26Furthermore, it is necessary to point out that although the N, S1 and M proteins of PDCoV can all be used as the diagnostic target, and indirect ELISAs based on these proteins have indeed been established ,24,25,28In order to obtain the optimal PI cut-off value for our developed blocking ELISA, a panel of 384 swine serum samples collected from piglets experimentally infected with PDCoV were simultaneously detected via the ELISA and IFA, which was used as the reference method to confirm the PDCoV antibody status of all serum samples. The resulting PI values of the 384 sera were then used to calculate the optimum cut-off point via ROC curve analysis in conjunction with Youden’s J statistic . The YouCompared with the IFA, although the blocking ELISA we developed exhibited a slightly lower diagnostic sensitivity, its main advantages include fast detection speed, easy performance, cost effectiveness, high-throughput detection, and easy commercialization. Undoubtedly, our developed blocking ELISA is a robust tool for the detection of PDCoV antibodies.By preparing a recombinant PDCoV N protein and its specific monoclonal antibody, a blocking ELISA was developed to detect PDCoV antibodies. The diagnostic sensitivity and specificity of the ELISA were determined using 384 swine serum samples with known PDCoV antibody statuses, which were confirmed using the reference method—IFA. The blocking ELISA exhibited excellent repeatability in the detection of PDCoV antibodies and did not cross-react with antisera against other pathogens causing diarrhea in pigs. To our knowledge, this is the first report on the development of a blocking ELISA for PDCoV serodiagnosis.


Castanea sativa Mill.) cultivar diversity is essential for managing and conserving the genetic resources of this fruit tree species in Southern Italy. To this goal, the present work investigated the feasibility of mapping, through spatial representation, the distribution of genetic diversity of traditional chestnut varieties in the area of the Roccamonfina Regional Park in the Campania Region. After Principal Coordinates Analysis (PCoA) of molecular-genetic data, chestnuts formed varietal groups in a leopard spot on PCoA plots with a relatively high degree of genetic diversity. Successively, a Geographic Information System (GIS) tool utilized these molecular-genetic data to create a genetic divergence surface by geospatial interpolation on the geographic map of the Regional Park corresponding to each chestnut variety. The regions containing more biodiversity richness resulted in differentially colored from those containing cultivars less genetically distant from each other; thus, the area in study was consistently colored according to the allelic richness as evaluated by molecular-genetic markers. The combined use of tools for molecular and spatial analysis allowed for drafting genetic landscapes with the aim of extracting useful information for the safeguarding of the chestnut biodiversity at risk.Knowledge of the spatial distribution of European chestnut ( Castanea sativa Mill.) is a tall tree species, belonging to the Fagaceae family, of considerable agroforestry importance worldwide [The European or sweet chestnut  has been threatening the survival of traditional chestnut varieties, especially those not very represented on the regional territory [Since 2005, the accidental entry into the Campania Region  of the invasive alien species Hymenoptera: Cynipidae erritory ,12. At territory ,14,15. ILandscape genetics would allow a correct evaluation of the geographic patterns of genetic diversity using reliable molecular tools together with spatial statistics. The routine identification of chestnut cultivars is mainly based on morphological traits, which are unreliable indicators of the specific chestnut genotype as they can be influenced by environmental conditions. Recent research has investigated the distribution of genetic diversity of the chestnut on a large scale at the European level ,17. HoweFor all these reasons, in the current study, a survey of molecular-genetic tools and Geographic Information System (GIS) applications available for mapping the distribution of chestnut biodiversity on the territory of Roccamonfina Regional Park was carried out. We verified the possibility of combining previously acquired molecular data on the genetic diversity of cultivated chestnuts  with spaIt is notoriously recognized that Campania Region is an area with a strong tradition of chestnut cultivation, where chestnuts are present in very diverse environmental and edaphoclimatic conditions that contribute to the formation of specialized and selected chestnut orchards ,9,22. InDNA-based tools were previously tested on the main chestnut varietal genotypes cultivated and appreciated for the quality of their fruits in the protected area of Roccamonfina in Campania Region , i.e., ‘The genetic diversity values were pairwise calculated for the 17 chestnut samples and then elaborated by PCoA for RAPD and KASP markers, respectively. Both PCoA evidenced the genetic distribution of the analyzed genotypes on the first two axes, explaining the 79% of the variability detected by RAPD markers A and theBoth panels coherently evidenced that some pairs of cultivars are more similar, such as ‘Napoletana’/‘Paccuta’ or ‘Mercogliana’/‘San Pietro’, while other cultivar pairs are more distant, such as ‘Marzatica’ and ‘Tempestiva’. However, there are some differences peculiar for each molecular marker. In particular, of all these cultivars in loci could give the same amount of information obtained using about 12 highly polymorphic markers as RAPD [The two molecular methods allowed us to have a raw estimation of the genetic distances existing among the analyzed cultivars in order to test the GIS application. In particular, as RAPD markers were obtained via PCR amplification on random regions of chestnut genomic DNA by using several arbitrary primers, they include much information on the existing genetic variability, resulting in specific patterns of amplification . KASP anThe GIS analysis allowed visualizing the distributions of the genetic distance RAPD A and KASNevertheless, the potential and the limits of this method of analysis for mapping the chestnut agro-biodiversity of a territory are particularly evident in the two maps of landscape genetics shown in loci should be useful for large-scale genetic analysis at the regional level in this economically important fruit species. Consequently, these innovative genetic-spatial tools will allow us to better evaluate the landscape through models of genetic diversity targeted to the conservation, protection, and management of chestnut varieties traditionally cultivated in the Campania Region that are currently at high risk of genetic erosion.Similarly, by combining microsatellite markers and geo-statistical methods, other research identified Italy as a hotspot of natural genetic diversity in large-scale European studies on wild chestnut forests ,17. SincC. sativa  descriptors by Nunziata et al. (2020) [In this study, we utilized chestnut sampling carried out in our previous work by Nunziata et al. (2020)  from the. sativa  located DNA profiling of the chestnut cultivars by RAPD analysis was previously performed by Nunziata et al. (2020) . We usedIn our previous work, 37 SNPs were selected for developing KASP assays designed for molecular discrimination of chestnut cultivars, and analyses were previously conducted by Nunziata et al. (2020) . We usedGIS is a powerful tool for spatial analysis, data visualization, and mapping . In thisIn the present pilot study, we used a multidisciplinary approach combining molecular-genetic and geolocation data to characterize the chestnut trees of Campania. The resilience and resistance capacity of chestnut forests to environmental factors depends on the specific genotypes present on the territory. Traditional cultivars are a major source of biodiversity; therefore, open and controlled access to chestnut germplasm should be one of the pillars of sustainable agricultural development. Considering that the native chestnut germplasm is of paramount importance for public and private sectors, the possibility that stakeholders have available maps of the genetic distribution of local cultivars will provide them with new potent tools for the management and conservation of these genetic resources. To this end, extensive molecular characterization and geo-spatial cataloging of the chestnut resources in traditionally vocated areas is desirable, in order to activate targeted protection and decision-making processes for effective and efficient use of the chestnut heritage.


Melozone leucotis, and the urban dweller house wren Troglodytes aedon. We used seven microsatellite loci and sampled five locations with differing levels of urbanization in Costa Rica. We found considerably higher genetic structure in white-eared ground-sparrows than in house wrens. Circuit theory analyses proved a higher isolation from urban resistance for the white-eared ground-sparrow than for house wrens. These results support that urbanization is a significant barrier for gene flow in urban avoiders, in contrast to urban dweller species that showed little to no impact. Differences could be attributed to a higher plasticity in habitat and nesting site preferences in the house wren, and significant dispersal limitation for the white-eared ground-sparrow. These results emphasize the need for conservation strategies towards white-eared ground-sparrows and other urban avoider species whose habitat and connectivity have been reduced by the recent urban expansion.Gene flow in birds can be affected by urbanization depending on natural history traits and adaptability to habitat change. Contrasting results can be expected when comparing species with opposite resilience to urbanization. In this study, we assessed genetic diversity and structure for two bird species, the urban avoider white-eared ground-sparrow,  The adaptive response to such drastic changes varies widely among species. Susceptible species experience isolation and population decline, and may become locally extinct7. For some species (“urban avoiders”), buildings, streets, highways, and other human settlements restrict their movement, reducing the connection within the metapopulation10. These barriers are expected to reduce gene flow and genetic diversity, increasing inbreeding, allele fixation and genetic structure of those species trapped in isolated vegetation fragments11. However, contrasting results are expected for those species for which population size and connectivity increase in urban areas (“urban exploiters”)12.The rapid expansion of urbanized areas has converted large extensions of natural habitats into a concrete jungle that surrounds some small natural or seminatural fragments of vegetation13. In birds, a species tolerance and adaptability to urban environments determines the impact of negative effects from habitat fragmentation and isolation12. In wrentits (Chamaea fasciata), a species with low dispersion capability, major roads restricted gene flow between fragments in urban landscape, producing a strong genetic structure across populations14. Similarly, in a species with high dispersion capability, as the song sparrow (Melospiza melodia), urban land cover has also shaped its genetic structure15. In this case, higher genetic structure occurred in sites isolated and surrounded by dense and old urban development, compared to more recently developed urban sites or even between forest relics15. However, in low-density urbanized sites, song sparrows showed no evidence of population genetic structure, suggesting that this species is capable of maintaining sufficient gene-flow between sites in non-saturated urban landscapes16. In great tits (Parus major), differences in patch size, population size and fragmentation may have different effects over genetic structure levels18. The distance between patches within an urban matrix has reduced connectivity between patches, and increased the genetic structure of the Singaporean yellow-breasted babbler (Mixornis gularis), a tropical edge-tolerant species19. These examples suggest that the effects of urbanization on structuring population genetics vary within and between species.The effect of urbanization on gene flow and other population genetic parameters will depend on each species’ mobility and life history traits20. This method focuses on random walking and multiple-choice path algorithms to uncover current flow in a resistance matrix developed from geographic information systems (GIS)20. Circuitscape models the probability that individuals disperse or move between populations depending on the permeability of the landscape. With this algorithm landscape resistance hypotheses can be proposed, assigning different resistance values to each cell, or portion of the landscape such as forest cover, altitude or topological characteristics; based on each species natural history and habitat use. Values of cumulative resistance among populations may be correlated with genetic structure15.It is possible to use different approaches to evaluate the effects of urbanization on gene flow and genetic structure. Methods based on circuit-theory are well fitted to evaluate the effect of the complex connectivity structure within urban habitats on the genetic structure of mobile individuals such as birdsMelozone leucotis) inhabit thickets, coffee plantations and secondary forest edges23. Historically, coffee plantations expanded in the early twentieth century, and presumably provided greater connectivity for white-eared ground-sparrow; but in recent years, this habitat type has been gradually transformed into urban settlements and the remaining habitat of the white-eared ground-sparrows has been converted into fragments that vary in isolation26. On the contrary, the second species included in this study, the house wren (Troglodytes aedon), is commonly found in open and semi-open areas, forest edges and it has presumably benefited from urban development28. Their territories are smaller in more urbanized areas, and this has been associated with an increase in nesting and food resources in these areas27. To date, there is a paucity of studies that evaluate the impact of recent habitat fragmentation and habitat loss on genetic diversity and structure of tropical species that differ in their response to the changes imposed by urbanization.In this study, our main objective was to evaluate the effect of urbanization on genetic diversity and gene flow patterns of two neotropical bird species with different levels of resilience to urbanization, across a heterogeneous landscape of urbanization and agriculture in Costa Rica. The white-eared ground-sparrow (Asµ18 and ThPl-01) across all populations, so we removed them from further analyses. For the white-eared ground-sparrow, only Mme7 deviated from Hardy Weinberg Equilibrium (HWE) as expected for a sex-linked marker. To include it in further analysis, the missing allele was coded as missing for females and juveniles15. Locus heterozygosity (Ho) ranged between 0.347 and 0.903 and He ranged 0.368–0.891 for white-eared ground-sparrows  than for house wrens , though confidence intervals of diversity estimates overlapped between species. Inbreeding was not observed in any species, but we found a heterozygote excess in the white-eared ground-sparrow in the UCR subpopulation  for the white-eared ground-sparrow, and θWC = 0.0610 (95% CI = 0.0422 and 0.0736) for the house wren. Pairwise FST differed significantly between all but one comparison (UCR and HDA) for white-eared ground-sparrows  likelihood corresponds to K = 1  and less tolerant great tit, had similar genetic diversity levels in an urban area, even though genetic structure was significantly higher for the great tit than for the blue tit30. Other studies have reported similar genetic diversity estimates between urban and rural populations of the same bird species 32. Therefore, our results may be explained by large effective population sizes in both species. The recent urbanization of our study area (< 60 years)33, has not allowed sufficient time to pass to reduce population sizes and consequently genetic diversity through drift. This is emphasized by the fact that that habitat fragmentation has occurred gradually33, which probably allowed remnant forest patches to act as stepping-stones for gene flow in the last three decades34.Genetic diversity estimates were similar for both species because confident intervals overlapped. These results contradict our initial predictions, since we expected a higher genetic diversity for house wren which is an urban tolerant species that shows great plasticity in habitat and nesting site preferences35. Multiple studies have shown how urban areas increase fragmentation and isolation of avian populations with its corresponding effects on genetic diversity30. However, the magnitude of the effects of urbanization varies among species. For example, differences in sensitivity to urbanization resulted in higher genetic structure for great tits than for blue tits in Poland, in which fragmentation (urban vs. forest) and isolation by urban areas, limited gene flow for great tits but did not for blue tits30. The authors attribute these differences to the greater migration and dispersal ability of blue tits in urban populations30. Tan et al.19 also found that the effective population size of the forest-edge tolerant Singaporean yellow-breasted babbler, correlates with the habitat contraction caused by urbanization. For the babbler, genetic differences in urban subpopulations were attributed to small effective population sizes, limited dispersal, and a lack of connectivity between patches19. The differences we observed on genetic structure between white-eared ground-sparrow and house wren may also be explained by contrasting tolerance to urbanization between these two species27. White-eared ground-sparrows and house wrens differ in how they can benefit from urban habitats. White-eared ground-sparrows feeds in the humid leaf litter and requires thickets to build nests, while house wrens feeds on arthropods on a wide array of substrates and can build nests in buildings and human made cavities29. A large proportion of the original habitat occupied by white-eared ground-sparrows in Costa Rica is now located within the Greater Metropolitan Area (GAM), characterized by a recent, rapid, and unplanned urban development36. For the earlier half of the twentieth century, a large portion of the GAM was covered by coffee plantations and second growth vegetation, but recent urban development has reduced coffee plantations and natural vegetation cover to small, isolated fragments26. Hence, the preferred habitat of the white-eared ground-sparrow has been constantly reduced; threatening the viability of this species’ populations38.Clustering algorithms suggest that urbanization imposes a barrier to gene flow in white-eared ground-sparrow, genetically structuring its populations. In contrast, sampled populations in our study of the house wren in Costa Rica may be considered a large panmictic population. Habitat loss and fragmentation often limit connectivity and gene flow and, consequently, increase differences in allele frequencies between populations29. Accordingly, we were not able to find evidence of isolation by distance or by habitat resistance. However, we found intermediate levels of genetic structure likely attributed to its low natal dispersal, because young individuals do not need to move long distances to find suitable habitat39. However, this hypothesis does not explain why we did not find significant IBD. This issue will be discussed below in further detail.House wrens inhabits a wider array of habitats and can move between populations across large, urbanized areas27. JBL is separated from HDA and UCR by a large, urbanized area of approximately 2280 ha40, and by a mountainous densely forested terrain which likely limited migration and gene flow. These results are supported by the high correlation between urban resistance and genetic differentiation for the white-eared ground-sparrow. MTV is geographically the most distant site, but clustered closer to HDA and UCR, due to large patches of coffee plantations located among these sites37. Contrary to white-eared ground-sparrow, our results suggest that house wrens may be a single panmictic metapopulation. This is supported by the lack of IBD, and the weak correlations between genetic differentiation with urban resistance and climatic resistance. These results support our initial expectations that this species benefits from the recent urban development which increases the availability of nesting sites and habitat use, thus increasing connectivity among populations29. In contrast we expect gene flow to decrease for white-eared ground-sparrow in future generations. This is expected for city populations of an urban avoider that may reach the lowest densities in urban areas27.From the four populations of white-eared ground-sparrows, two ; University of Costa Rica Campus, San José Province ; and Lankester Botanical Garden, Cartago Province . Both Lankester Botanical Garden and University of Costa Rica Campus are urban areas with small patches of secondary vegetation immersed in an urban matrix. HDA had a lower urban coverage, surrounded by both urban zones and coffee plantations. The other sampling site was Monteverde, in the Puntarenas province , characterized by low density population, coffee plantations, and large forest patches that connect with more extensive mature forests.We selected four sites for white-eared ground-sparrow Fig. , three l, we collected individuals in five localities , and is characterized by extensive oil palm plantations.For house wrenies Fig. ; three o50. Anesthesia was not used, and all birds were released unharmed. Procedures were conducted in accordance with the current laws in Costa Rica. Research permits and animal handling protocols were approved by the Research Committee of Biology School and by the Animal Care Committee (ACC) of Universidad de Costa Rica under the project approval code B9-469 and C1-085 of Vicerrectoría de Investigación. All procedures were conducted following the regulations of the ACC.We captured individuals of white-eared ground-sparrow using mist nets upon song playback in 2012, 2013, and 2019–2021, capturing between 12 to 19 individuals per site. House wren individuals were captured using mist nets upon song playback in 2019–2021, capturing between 11 and 16 individuals per site. We color banded all collected individuals, sexed all adults, and categorized juveniles as indeterminate. We collected 10–30 µL of blood from the brachial vein for each individual and stored it in 95% ethanol or Lysis buffer for molecular analyses, we amplified eight primers: Mme2, Mme7, Mme8, Asµ15, Asµ18, Escµ6, Gf01 and Gf0554. For house wren, we amplified eight primers: ThPl-01, ThPl-14, ThPl-17, Ta-C6-7, Ta-B4-2, Ta-A5-2, Ta-A5-15 and Ta-C3(B)-256. Forward primer of each pair was 5’ fluorescently labelled. PCR thermal profiles and primer mixes are described in Supplementary Table Gf01. We used Veriti™ thermal cycler  to perform the PCR reaction. Capillary electrophoresis was done in a 3500 Genetic Analyzer (Applied Biosystems) at Escuela de Biología, UCR, using Hi-Di™ Formamide and GeneScan™ 500 LIZ™ dye Size Standard (Applied Biosystems). We scored genotypes using GeneMarker 1.91 .We extracted DNA from blood and muscle tissue using the DNeasy blood and tissue Kit . For white-eared ground-sparrow57, using the RStudio 1.3.180 GUI58. We calculated expected heterozygosity (He), observed heterozygosity (Ho), inbreeding coefficients (FIS) and allelic richness (Ar) per population (to compensate for differences in sampling size per population) using the “hierfstat” package59. We calculated Weir and Cockerham FST (θWC)60, expected heterozygosity values and 95% confidence interval of both estimates using 9999 bootstrap with boot.vc function of the “hierfstat” package. We also calculated pairwise θWC between populations with pairwise.WCfst function and it’s 95%CI using boot.ppfst by 9999 bootstrap among loci using the “hierfstat” package.We conducted population genetics analyses in R 4.0.261. To determine the most suitable number of PC axis to include in the analysis, we performed cross-validation using the xvalDapc function, with 30 repetitions. We then narrowed the search window to the nearest six PC surrounding the previous selected PC while increasing replicates to 999. The Bayesian clustering methods in Structure 2.3.4 allowed us to assess the possible genetic clustering of individuals into a K number of clusters62. The admixture model and correlated allele frequencies were used, with 300,000 markov chains and a burn-in of 30 000. Phase information was added to include Mme7 marker in the analysis, since previous evidence has shown this is a Z-linked marker53. We tested 1 to 6 clusters with 20 repetitions for each cluster. We determined the most likely number of K clusters using Structure Harvester 0.6.9463 and the Evanno64 and Ln(K) methods. Then, we conducted another structure analysis after determining the most likely K, increasing generations and the burn-in to 1 000 000 and 100 000, respectively.We visualized individuals' similarity using a discriminant analysis of principal components (DAPC) as implemented in the “adegenet” packageTo test for isolation by distance (IBD) we estimated the correlation between pairwise Euclidean genetic distances and geographic linear distances among individuals using a Mantel test after 9 999 permutations. The same coordinates of the sampling sites were assigned to all individuals within each population.65. To create the resistance raster, we used a 30 m resolution land coverage of 2005 shapefile layer of continental Costa Rica created by the Earth Observatory Systems Laboratory of the University of Alberta, based on Landscape 7 satellite image classification and interpretation into 16 categories  using Circuitscape 4.0.568. We obtained the occurrence of each species from the eBird database (www.ebird.org). We then removed duplicated data, erroneous observations, and randomly thinned spatial data to 1 km for white-eared ground-sparrow and 5 km for house wren. We used all 19 bioclimatic data available at www.worldclim.org at a 1 km2 resolution. We delimited the area of calibration to 25 km radius around each occurrence point. We sampled 10 000 background points and used the spatially block partition method. We set regularization multiplier parameters from 0.5 to 4, with 0.5 increments and used 5 feature class combinations . We evaluated the models using the area under the curve (AUC) and average 10th percentile omission rate. To convert it into a resistance matrix input for Circuitscape, we performed a cloglog map prediction and set it as conductance in our Circuitscape model. We developed pairwise modeling between all sampling locations of white-eared ground-sparrow and house wren with a log10 transformation for the cumulative current map. We performed a Mantel test with 9999 permutations between individual genetic Euclidean distance and resistance values.To test for isolation by environment (IBE), we developed a maxent species distribution model using the “Wallace” R librarySupplementary Information.


Bos taurus and Bos indicus. The positive selective sweep analysis in Weining cattle was analyzed using composite likelihood ratio (CLR) and nucleotide diversity (θπ), resulting in 203 overlapped genes. In addition, we studied the cold adaptation of Weining cattle by comparing with other Chinese cattle (Wannan and Wenshan cattle) by three methods . Of the top 1% gene list, UBE3D and ZNF668 were analyzed, and these genes may be associated with fat metabolism and blood pressure regulation in cold adaptation. Our findings have provided invaluable information for the development and conservation of cattle genetic resources, especially in southwest China.Weining cattle is a Chinese indigenous breed influenced by complex breeding and geographical background. The multi-ethnic breeding culture makes Weining cattle require more attention as livestock resources for its genetic diversity. Here, we used 10 Weining cattle (five newly sequenced and five downloaded) and downloaded another 48 genome data to understand the aspects of Weining cattle: genetic diversity, population structure, and cold-adapted performance. In the current study, a high level of genetic diversity was found in Weining cattle, and its breed comprised two potential ancestries, which were  Bos taurus) and humped cattle (Bos indicus) are two main sub-species of cattle  of Yanbian cattle . This study may potentially reveal the ancestral components, population structure, and genetic diversity of Weining cattle.A scarce number of studies were carried out to explore the knowledge of genomic variation in Weining cattle at the genome level. We analyzed 58 whole-genome data of individuals (including five newly sequenced Weining cattle data) and identified single-nucleotide polymorphisms (SNPs) compared with those of commercial and native populations around the world based on the This study was approved by the Institutional Animal Care and Use Committee of Northwest A&F University following the recommendation of the Regulations for the Administration of Affairs Concerning Experimental Animals of China (Permit number: NWAFAC1019).We sampled five Weining cattle from Bi’jie, Guizhou, China. These samples were collected from villages, and the farmers were interviewed in detail to ensure unrelatedness among the sampled individuals. Genomic DNA was extracted from ear tissues using the standard phenol–chloroform method . Paired-In addition, another five published data of Weining cattle were downloaded, and we also downloaded genome-wide data of 48 cattle for comparison including Wenshan cattle (n = 5), Wannan cattle (n = 5), Guangfeng cattle (n = 4), Hanwoo cattle (n = 10), Brahman (n = 4), Gir (n = 2), Nelore (n = 1), Angus (n = 9), and Simmental (n = 8) . In totaBos taurus reference assembly ARS-UCD1.2 using BWA-MEM (0.7.13- r1126) with default parameters. The average mapping rate of these reads sequenced in this study was 99.28%, and the sequencing coverage was approximately 12 × per individual. Then, potential duplicate reads were filtered by Picard tools (REMOVE_DUPLICATES = true) (http://broadinstitute.github.io/picard). After that, the Genome Analysis Toolkit  was further used for SNP calling   tree, and performed PCA by using genome-wide SNPs within autosomes to determine the population genetic structure. Principal component analysis (PCA) was performed using the smartPCA program in the EIGENSOFT v5.0 software package . Populatmbl.de/) .FROH can accurately calculate the number of inbreeding lines. FROH is calculated by calculating the ratio of the total length of ROH fragments in the genome to the total length (FROH = VCFtools were used to estimate nucleotide diversity (θπ) for each breed or population . The winWithin Weining cattle for genome scans, we have used the nucleotide diversity (θπ) and the composite likelihood ratio (CLR), two statistic methods. θπ was estimated based on a sliding window method with windows of 50 kb and a step of 20 kb using VCFtools . The CLRFST), nucleotide diversity ratio (θπ-ratio), and cross-population extended haplotype homozygosity (XP-EHH) methods were used to select positive natural regions in the Weining cattle genome. These statistics were calculated by using a sliding window approach: a 50-kb window and a step size of 20 kb. We calculated the average FST, θπ-ratio, and XP-EHH values of each SNP window and used the outlier method to obtain the windows with the top 1% values of each method. Finally, three gene clusters of three methods in these outlier windows were carried out.According to the National Cattle Resources of China , Weininghttp://kobas.cbi.pku.edu.cn/)  were taken as significant terms for statistics.In addition, candidate genes overlapped at least in two methods were taken for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses by KOBAS 3.0 (edu.cn/) . FinallyIndividual genomes of five Weining cattle were generated to 10.43 × coverage each and were jointly genotyped with the publicly available genome (ARS-UCD1.2) . To reveROH also showed that Angus and Simmental cattle breeds had significantly higher inbreeding coefficients than other populations , corresponding to taurine and indicine cattle lineages . SimilarDLG2, PRLR, MLH1, CFAP299, GOLGA4, and CCNH were reported as reproductive trait–related candidate genes  related to muscle growth and fat deposition (p < 0.05)  .FST, XP-EHH, and θπ-ratio) , and significant signal regions (top 1%) were obtained by three methods (π-ratio)  and annoπ-ratio) . To detescanning . This reiversity .p < 0.05) were obtained. KEGG pathways are enriched in terms such as “Purine metabolism, bta00230,” “MAPK signaling pathway, bta04010,” “Wnt signaling pathway, bta04310,” and “Endocrine resistance, bta01522”  enriched the genetic diversity of Chinese indicine from the introgression . UBE3D, ubiquitin-protein ligase E3D, is involved in intracellular physiological processes by regulating the ubiquitination process of regulatory proteins. The study has shown that Weining cattle had high unsaturated fatty acids (UFA)  in southwest Chinese native breeds (UBE3D was selected as the understanding of genetic mechanisms of fat composition in sheep (ZNF536 was related to cold tolerance in Chantecler chickens (ZNF668 might be involved in the elevation of blood pressure to hold body temperature when exposed to a cold environment (Comparing two native cattle breeds (Wannan and Wenshan cattle) bred in hot  and humid environments , we found cold-resistant candidate genes in Weining cattle, for instance: e breeds . Also, te breeds . Coincidin sheep . On the in sheep . In addiin sheep . ZNF536 chickens . Herein,ironment . Compareironment .Weining cattle are one of the potential beef cattle breeds in southwest China. The animal breeding culture of different ethnic minorities has contributed to the hybrid of Weining cattle. Meanwhile, the rich genetic diversity causes its remarkable adaptability in cold and humid mountains. Although the impact of the market economy forces the protection and development of Weining cattle under great pressure, the diversity and enrichment of genetic information in the genome of Weining cattle is a valued material for cattle breeding.Analysis of genomic diversity and selection signatures of Weining cattle were carried out at a sequence level, and new insights into the genetic basis of crossbred cattle were provided. By comparing the Weining breed with other cattle populations, we introduce its genetic diversity, population genetic structure, and environmental adaptation characteristics. Moreover, a set of candidate genes were identified and may be related to cold adaptation, low fertility, and fatty acid composition in Weining cattle, although additional physiological and functional experiments are needed for verification. Overall, it is of great significance to understand the genetic diversity and adaptation of cattle breeds in southwest China.


T. gondii infection. The main aims of this study were to assess the seropositivity to toxoplasmosis of an exotic breed of cattle (n = 400) from different farms using the Latex Agglutination Test, validate the Cattle Toxo IgG ELISA kit with the help of the commercially available ELISA kit and compare the efficacy of the LAT and Cattle Toxo IgG ELISA kit for assessing seropositivity of cattle to toxoplasmosis. Four hundred cattle sera were collected from an exotic breed of cattle in Pakistan. These sera were evaluated through an LAT and Cattle Toxo IgG ELISA kit. Of 400 samples, 90 were elected and screened through a commercially available ELISA kit. The sensitivity and specificity of the Cattle Toxo IgG ELISA kit came out to be 100% and 96.15%, and in LAT, it was found as 26.31% and 61.53%, respectively. The Cattle Toxo IgG ELISA kit revealed 29.75% (119/400) seropositivity, 6% less than that found through LAT. The results of this study show that Cattle Toxo IgG ELISA kit is a highly accurate and reliable serodiagnostic tool to diagnose bovine toxoplasmosis.Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis in warm-blooded vertebrates, globally. The main aims of this study were to assess the seropositivity to toxoplasmosis of an exotic breed of cattle (n = 400) from different farms using the Latex Agglutination Test and validate Cattle Toxo IgG ELISA kit. Of a total of 400 cattle sera that were evaluated by LAT, 143 (35.75%) were found positive. Based on these data, 90 samples  were elected for screening through a commercially available ELISA kit. The same 90 samples were screened through a Cattle Toxo IgG ELISA kit for validation purposes. Of 90 samples, 40 were seropositive in the Cattle Toxo IgG ELISA kit (100% sensitivity), and 38 were seropositive in a commercially available ELISA kit. All 50 samples in the Cattle Toxo IgG ELISA kit (96.15% specificity) were also seronegative in the commercially available ELISA kit. Hence, the sensitivity and specificity of the Cattle Toxo IgG ELISA kit came out to be 100% and 96.15%, and in LAT, it was found as 26.31% and 61.53%, respectively. Therefore, the Cattle Toxo IgG ELISA kit is a highly reliable serodiagnostic tool to diagnose bovine toxoplasmosis. Toxoplasma gondii [Felidae, are its definitive hosts and excrete environmentally resistant oocysts in their feces [T. gondii, and it causes subclinical infection in cattle [Toxoplasmosis is an important zoonosis caused by a heteroxenous obligate intracellular protozoan named a gondii . It causa gondii . Cats, iir feces . These oir feces . Humans ir feces ,6,7. Toxir feces . Cattle n cattle . The econ cattle . The sern cattle . In Pakin cattle .Toxoplasma gondii  have also been reported from Pakistan while studying SAG2 sequence on feline fecal samples [T. gondii has genetic diversity due to genomic recombination, distinct population structures, and intercontinental and regional diversity [Atypical strains of  samples  such as  samples . The T. iversity .(DT), Indirect Fluorescent Antibody Test (iFAT), Modified Agglutination Test (MAT), and Enzyme-linked Immunosorbent Assay (ELISA) are used for the detection of antibodies against T. gondii in animals. Subsequently, the OIE Manual declares the DT as the gold standard for detecting antibodies against T. gondii in humans [T. gondii antibodies [Serological surveillance is a good method for identifying and evaluating farm infection ,16,17. Tn humans ,21. It in humans . In LAT,tibodies . It is ctibodies ,25. iFATtibodies . Since ttibodies ,27. Indetibodies .T. gondii in a single form are used to identify anti-toxoplasma antibodies, whereas the combination form of antigens has been reviewed for increasing their sensitivity [T. gondii compared to the Modified Agglutination Test (MAT) in serology [T. gondii [T. gondii [T. gondii is required to coat the ELISA plate. This study was designed to assess the seropositivity of the exotic breed of cattle/bovine toxoplasmosis while simultaneously validating our newly developed ELISA kit named as Cattle Toxo IgG ELISA kit exploiting the commercial ELISA kit as Gold Standard.The different recombinant proteins were previously used to detect specific IgG anti-toxoplasma antibodies in animals. These diagnostic antigens are mainly based on surface antigens such as SAG1, SAG2, SAG3 ,32,33,34sitivity . The ELIserology . In the serology . The SAG. gondii ,46. It i. gondii ,48. Nati. gondii ,49. Subs. gondii . The devn = 400) from four different cattle farms in Pakistan. All the animals were healthy and female belonging to various age groups: 0–1.5 years (n = 400), 2–2.5 years (n = 76), 3–3.5 years (n = 62), 4–4.5 years (n = 50) and ≥5 years (n = 161).The present investigation was conducted on the Holstein Friesians breed of cattle  strain colonies were elected from the LB agar plate (kanamycin 1 mg/mL). The colonies were inoculated in 10 mL of fresh LB broth and incubated  until the OD600 reached 2.0. After attaining the desired concentration, the culture was diluted 100-fold in 50 mL of fresh LB broth, and it was incubated  until the OD600 reached 0.5. After that, 1.0 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture and incubated . Subsequently, 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done. The rSAG1 protein of T. gondii was purified by HisPur nickel-nitrilotriacetic acid (Ni-NTA) affinity columns .The expression and purification of rSAG1 of  earlier . BrieflyThe rSAG1 was quantified by Bicinchoninic Acid (BCA) assay kit . The purified rSAG1 was verified through WB by immunoblotting with Anti-6x His Tag Antibodies .2CO3), and it was incubated at 4 °C overnight. The plate was washed with the washing buffer (0.001 M PBS/0.05% Tween-20) at 300 µL/well. The plate was saturated with 4% BSA in 0.01 M PBS at 200 µL/well, and incubated at 37 °C for 2 h. The plate was rewashed five times with the washing buffer. Four hundred cattle serum samples were screened through Cattle Toxo IgG ELISA kit. The positive and negative control sera were dispensed in duplicate wells. Two wells were put as blanks in the plate. The plate was incubated at 37 °C for 2 h. After rewashing five times, Anti-bovine IgG (Whole molecule)- Alkaline Phosphatase, antibodies produced in rabbits  were dispensed with 1:10,000 dilution at 100 µL/well and the plate was incubated at 37 °C for 2 h. After rewashing five times, the substrate p-nitrophenyl phosphate (pNPP)  was added with 1 mg/mL of Diethanolamine (DEA) substrate buffer  at 100 µL/well, and incubated at 37 °C. The reaction was stopped after 15 min by adding a stop solution (1 M NaOH) at 100 µL/well. The OD was taken at 405 nm by a Microplate ELISA reader .The 400 cattle sera were evaluated through the Cattle Toxo IgG ELISA kit described in the protocol . The rSAAmong the 400 sera, 90 were elected for screening through the commercial ELISA kit. Of these, 10 were positive with Cattle Toxo IgG ELISA kit and LAT. An additional 30 were negative for Cattle Toxo IgG ELISA kit, another 20 were positive for LAT. Another 30 were negative through the Cattle Toxo IgG ELISA kit and LAT. The detail of the election is shown in ID Screen Toxoplasmosis Indirect Multi-species (standard ELISA kit)  was purchased from ID.vet Company. The standard ELISA was used as per the manufacturer’s instructions. The pre-coated plate with rSAG1 was added with diluted sera (1:10). The unbound antibodies were washed away by the washing solution. Conjugate attached with HRP was added in wells for detecting primary antibodies in the test sera. The antibody-antigen complex was revealed by adding Tetramethylbenzidine (TMB) solution, and the reaction was stopped by adding stop solution. The results were compared with controls. Serum samples, and negative and positive controls were added in duplicate with 1:10 dilution. A well was used as a blank. The plate with sera was incubated at 26 °C for 30 min, and washed three times with a washing solution. Then, 100 µL of enzyme conjugate was added to each well. The plate was incubated at 26 °C for 30 min, and washed again three times with a washing solution. The 100 µL TMB substrate was added in each well. The plate was incubated at 26 °C for 15 min. Then, 100 µL of stop solution (1 N HCl) was added to each well. After 15 min of addition of substrate, the reading of OD was taken at 450 nm by a Microplate ELISA reader .The sensitivity of our kit came out to be 100% against 82.48% of the Standard kit. The optimum serum dilution for Cattle Toxo IgG ELISA kit is 1:100, whereas in the Standard ELISA kit, it is 1:10. Thus, our kit can be exploited in case of minimal amount of serum sample. Cattle Toxo IgG ELISA kit exploits an AP-conjugate, whereas the Standard ELISA kit exploits HRP-conjugate. Cattle Toxo IgG ELISA kit can be optimally incubated at 37 °C , whereas for the Standard ELISA kit, it is 26 °C. Stop solution in our kit is NaOH, whereas it is HCl in the Standard kit.The MedCalc statistical software (version 11.4.4.0) was used to compare the performance of the Cattle Toxo IgG ELISA kit, LAT, and the standard ELISA kit by calculating the kappa value. ROC curve was used to determine the cut-off value of the Cattle Toxo IgG ELISA kit . The serFour hundred samples were evaluated through a LAT and a Cattle Toxo IgG ELISA kit. Among these, 143 (35.75%) were positive through LAT, whereas 119 (29.75%) were positive through the Cattle Toxo IgG ELISA kit .p < 0.05 as described in The diagnostic performance of LAT and the Cattle Toxo IgG ELISA kit was assessed through positive and negative sera, identified by performing LAT and Cattle Toxo IgG ELISA kit. LAT’s sensitivity and specificity percentages relative to Cattle Toxo IgG ELISA kit were 24.36 and 59.43, respectively. The kappa value was calculated through SPSS as –0.153. The sensitivity and specificity of LAT were found too low relative to the Cattle Toxo IgG ELISA kit. The Pearson Chi-square (χ2) value was 9.551 and was significant at A total of 90 samples were elected for evaluation through commercial ELISA to check the association of a Cattle Toxo IgG ELISA kit and a standard ELISA kit on the criteria of positive and negative sera by LAT and a Cattle Toxo IgG ELISA kit. The 95% sera were observed positive by the standard ELISA kit for 40 samples. In contrast, for 50 sera, the 100% samples were found negative by the standard ELISA kit as shown in For the validation of the Cattle Toxo IgG ELISA kit, 90 bovine sera were elected. Of these 90 sera, 30 were seropositive and 60 were seronegative as per Latex Agglutination Test. The elected (90) sera were checked/screened through commercial ELISA kit (ID Screen Toxoplasmosis Indirect Multi-species ELISA kit) that found 38 samples as seropositive and 52 as seronegative. These findings of the commercial standard ELISA kit were taken from a gold standard kit. The same 90 sera were then screened through a Cattle Toxo IgG ELISA kit that determined 40 samples as seropositive , whereas 50 were found as seronegative. With this information, we calculated the sensitivity and the specificity of the Cattle Toxo IgG ELISA kit as described in The sensitivity and specificity of Cattle Toxo IgG ELISA kit emerged to be 100% and 96.15%, respectively, as shown in The OD values obtained by the Cattle Toxo IgG ELISA kit and through the standard ELISA kit were fed in MedCalc software to determine the cut-off value of the Cattle Toxo IgG ELISA kit that came out to be 0.431. This cut-off value for the new ELISA kit was used to determine the positive and negative bovine sera tested through the Cattle Toxo IgG ELISA kit.p < 0.001) between the Cattle Toxo IgG ELISA kit and the standard ELISA kit as described in The kappa value, a frequently used statistical test that measures the inter-rater reliability for qualitative items and interprets the chance of agreement, was calculated for the Cattle Toxo IgG ELISA kit through SPSS and came out to be 0.955. It was found to be highly significant (p < 0.001) through ROC. The ROC analysis depicted the importance of the cut-off value of the new ELISA kit. Similarly, the AUC explains the overall performance of diagnostic test as described earlier [The Receiver Operating Characteristic (ROC) curve is a graphical plot that describes the diagnostic ability of the binary classifier system. The AUC of the Cattle Toxo IgG ELISA kit was 0.997. The value of the Area Under Curve (AUC) was shown to be highly significant ( earlier . The higp < 0.001).Using a Cattle Toxo IgG ELISA kit, seroprevalence of bovine toxoplasmosis in the exotic breed of cattle at different farms was detected as 29.75% (119/400) (p < 0.0001)  was seen in the age group of ≥5 years, followed by 7.5% (30/400) in the age group of 2–2.5 years, 5.5% (22/400) in the age group of 3–3.5 years, 3.75% (15/400) in the age group 4–4.5 years and 2.5% (10/400) in the age group 0–1.5 years (001) See .p < 0.001).Using LAT, the seroprevalence of bovine toxoplasmosis in the exotic breed of cattle at different farms was detected as 35.75% (143/400) (p < 0.0001), respectively as described in The highest seroprevalence  was seen in the age group of ≥5 years, followed by 09% (36/400) in the age group of 2–2.5 years, 06% (24/400) in the age group of 3–3.5 years, 05% (20/400) in the age group 4–4.5 years and 3.75% (10/400) in the age group 0–1.5 years (T. gondii infection in animals [T. gondii cannot be detected antemortem. Therefore, detecting antibodies in cattle sera is the exclusive tool for antemortem diagnosis of toxoplasmosis. In our findings, 35.75% of cattle were positive for T. gondii antibodies by using LAT. Other researchers have already used it to detect T. gondii antibodies and found relatively low seropositivity of cattle to toxoplasmosis [T. gondii in cattle could be attributed to the genetic resistance of cattle to T. gondii and a sound management system that lessens the contact of cattle with cat or cat wastes [Serological techniques are used to diagnose  animals  because lasmosis ,58 compat wastes .T. gondii is routinely available. While the animal ages, its cumulative Likelihood of exposure increases. That is why the age of animals is considered one of the most critical factors in prevalence-related studies of Toxoplasmosis in animals [It has been admitted that Toxoplasma infection in animals is acquired through the ingestion of infective oocysts from the environment ,60 as an animals .T. gondii using the same methodology as earlier [In the current study, a Cattle Toxo IgG ELISA kit was developed with rSAG1 of  earlier  but withT. gondii in different animals such as sheep, goats, cattle, buffaloes, dogs, and cats. Its sensitivity and specificity are 82.48% and 97.8%, respectively [The Standard ELISA kit used in this study was used as a yardstick and is a reliable serological method for detecting IgG antibodies of ectively . In a stectively . The staT. gondii infection [T. gondii infection [Toxoplasma gondii. Still another fact that can lie behind this difference in seroprevalence observed between these two tests is that the Cattle Toxo IgG ELISA kit carries only one antigen of T. gondii against the total antigenic extract exploited in LAT able to interact with a multitude of antibodies. The 400 bovine sera were also evaluated through the Cattle Toxo IgG ELISA kit, and 29.75% of cattle were found positive using the Cattle Toxo IgG ELISA kit. The highest seropositivity rate for T. gondii-specific IgG was found in cattle aged ≥5 years.LAT, used as an initial screening and reference test for diagnosis of toxoplasmosis, has 79.1% sensitivity and 86.89% specificity as claimed by the manufacturer. In contrast, in our study, the sensitivity and specificity of the Cattle Toxo IgG ELISA kit surpassed these values of LAT, whereas false positivity of LAT has already been reported , highlignfection . It was nfection . IgM antnfection —most proT. gondii but resistant to the development of clinical signs, hence to the disease. Toxoplasma has been identified as one of the opportunistic pathogens in immunocompromised patients. Cattle have strong immunity and they appear healthy [In this study, no connection was found between health status and seropositivity of toxoplasmosis in sheep. All the seropositive animals displayed satisfactory health status and looked normal. Mostly cattle are susceptible to infection of  healthy .This study concluded that Cattle Toxo IgG ELISA kit is a highly accurate and reliable serodiagnostic tool as the sensitivity and specificity of Cattle Toxo IgG ELISA kit were found as 100% and 96.15%, respectively. This study concluded that the Cattle Toxo IgG ELISA kit is a more accurate and reliable immunodiagnostic test than LAT for diagnosing bovine toxoplasmosis in cattle.


Genomic sequencing is a powerful diagnostic tool in critically ill infants, but performing exome or genome sequencing (ES/GS) in the context of a research study is different from implementing these tests clinically. We investigated the integration of rapid ES into routine clinical care after a pilot research study in a Level IV Neonatal Intensive Care Unit (NICU). We performed a retrospective cohort analysis of infants admitted with suspected genetic disorders to the NICU from December 1, 2018 to March 31, 2021 and compared results to those obtained from a previous research study cohort . Clinical rapid ES was performed in 80/230 infants (35%) with a suspected genetic disorder and identified a genetic diagnosis in 22/80 infants (28%). The majority of diagnoses acutely impacted clinical management (14/22 (64%)). Compared to the previous research study, clinically integrated rapid ES had a significantly lower diagnostic yield and increased time from NICU admission and genetics consult to ES report, but identified four genetic diagnoses that may have been missed by the research study selection criteria. Compared to other genetic tests, rapid ES had similar or higher diagnostic yield and similar or decreased time to result. Overall, rapid ES was utilized in the NICU after the pilot research study, often as the first-tier sequencing test, and could identify the majority of disease-causing variants, shorten the diagnostic odyssey, and impact clinical care. Based on our experience, we have identified strategies to optimize the clinical implementation of rapid ES in the NICU. Traditionally, the genetic diagnostic odyssey has been long, costly, potentially invasive, and difficult for these infants and their families. Recently, there has been increasing use of massively-parallel sequencing tests, namely exome sequencing (ES) and genome sequencing (GS), for infants in intensive care settings. Multiple studies of ES/GS in critically ill infants have demonstrated diagnostic yields of 20–60% (with the highest yields reported with phenotype-driven selection), decreased time to genetic diagnosis , an impact on clinical care, and perception of high utility by clinicians and parents20.Many infants admitted to Neonatal Intensive Care Units (NICUs) have a known or suspected genetic disorder, and these conditions contribute significantly to morbidity and mortality11. Between March 2017 and November 2018 (hereafter referred to as Phase I), 50 infants had rapid ES performed, mainly from the Level IV NICU at Boston Children’s Hospital (BCH). A genetic diagnosis was identified in 29/50 infants (58%), including two infants with variants identified in novel disease genes, and the highest yield was seen in infants with neurological phenotypes, specifically hypotonia and/or seizures. The diagnoses informed management in 24/29 infants (83%).We previously performed a prospective pilot study of rapid ES in critically ill infants using phenotype-based selection criteria, enrolling infants <6 months corrected gestational age (GA) with an ICU indication and new onset (<7 days) of hypotonia, seizures, a complex metabolic phenotype, and/or multiple congenital anomalies, as well as one infant with a disorder of sex development22. We therefore hypothesized that integration of rapid ES into routine clinical care may result in a reduction in rapid ES usage, varied phenotypic criteria applied to patient selection, and an increased time to report compared to rapid ES as part of a research study. We thus investigated our initial clinical implementation of rapid ES and its impact on the genetic diagnostic odyssey for infants in our institution’s NICU in the years following the completion of the pilot rapid ES research study.Subsequently, rapid ES was integrated into routine clinical care in our institution’s NICU. Unlike the research study in which a dedicated research team was readily available to identify and screen infants based on specific inclusion criteria, enroll and consent families, and collect and send samples, routine clinical care involves constantly rotating clinical team members with differing perspectives as well as competing clinical demandsDuring the December 2018 to March 2021 period (hereafter referred to as Phase II), 1230 infants were admitted to the Level IV NICU of our institution and 248 infants had a genetics consult. 18/248 infants (7%) had a known molecular genetic diagnosis (MGD) at the time of initial consult and were excluded from further analysis. Characteristics of the remaining 230 infants are summarized in Table The rapid ES workflow during the pilot research study (Phase I) compared to the rapid ES workflow after integration into routine clinical care in the NICU (Phase II) is summarized in Fig. During Phase II, the neonatology team identified infants suspected to have an underlying genetic disorder and consulted the genetics team. Although a genetics consult is not required to order rapid ES at our institution, in practice the genetics team was always involved and coordinated most of the rapid ES logistics. The geneticists, in discussion with the neonatologists, and if relevant, additional consulting teams like neurology, determined if rapid ES was an appropriate genetic test. We did not employ specific criteria for making this determination but instead depended on the expert opinion of the clinical teams on service. If rapid ES was determined to be an appropriate genetic test, a request form was submitted to a hospital committee made up of representatives from departments including genetics and laboratory medicine. In practice, the genetics team typically submitted this request and the committee decided to approve or deny the request within 24–48 hours.p < 0.001) and to require respiratory support (65/80 (81%) versus 103/150 (69%), p = 0.044) compared to infants who did not have rapid ES performed. In addition, they had a significantly longer length of stay in our NICU (median 21 days (IQR 10–46) versus median 10 days (IQR 3–23.5), p < 0.001) and a significantly higher death rate in the first year of life (21/80 (26%) versus 13/150 (9%), p < 0.001). Age at genetics consult and time from NICU admission to genetics consult were not significantly different between infants who had rapid ES performed in Phase II versus Phase I (Table p < 0.001), congenital anomalies (26/35 (74%) versus 34/80 (43%), p = 0.002), and dysmorphic facial features (23/35 (66%) versus 28/80 (35%), p = 0.004) compared to infants who had rapid ES performed in Phase II.During Phase II, a total of 80/230 infants (35%) with a genetics team consult for a suspected underlying genetic disorder had rapid ES performed. Baseline demographics were similar between infants who did versus did not have rapid ES performed. Age at genetics consult (median 10.5 days (IQR 4–52) versus median 7 days (IQR 3–28)) and time from NICU admission to genetics consult  versus median 2 days (IQR 1–6)) were not significantly different between infants who did versus did not have rapid ES performed, respectively. Infants who had rapid ES performed were significantly more likely to have a neurologic phenotype (31/80 (39%) versus 22/150 (15%), p = 0.119).During Phase I, the research team consented the family, coordinated the patient and parent blood sample collection, and shipped the trio samples directly to the sequencing facility. During Phase II, the genetics team (fellow and/or attending physician) consented the family, the primary neonatology team placed the rapid ES order in the electronic medical record  versus median 3 days (IQR 1–7), 23. Preliminary results were verbally returned within 7 days and a final report was returned within 14 days of receiving all samples (proband and parents where available). During Phase I, preliminary results and the final report were released to the research team, communicated by the research team to the clinical team, and the final report was scanned into the EMR. During Phase II, preliminary results were called to the provider specified in the order , and the final report was released to our institution’s lab control and resulted as a lab in the EMR. Although the neonatology team could return results, in practice, results were returned to the family by the genetics team, often in the setting of a family meeting with the neonatology team also present. If the patient was discharged or had passed away, the genetics team contacted the family and returned results via phone call or clinic visit per family preference. Preliminary positive results were usually returned at the time of the verbal report with the disclaimer that the final report was still pending, and all results were retuned at the time of the final report. All families were offered a genetics clinic visit for further counseling and follow-up. The time from sample collection to final ES report was not significantly different between Phase II and Phase I (median 13 days (IQR 10–16.8) versus median 13 days (IQR 10–14), p = 0.333). The overall time from genetics consult to ES report was significantly longer in Phase II compared to Phase I (median 18 days (IQR 15–35) versus median 16 days (IQR 14–19.5), p = 0.019). The overall time from NICU admission to ES report was also significantly longer in Phase II compared to Phase I (median 20 days (IQR 16–29) versus median 17 days (IQR 15–19), p = 0.016).During both phases, the sequencing facility performed exome sequencing and analysis to detect single nucleotide variants (SNVs), small insertion/deletions (indels), and copy number variants (CNVs)p = 0.028) and BW (median 2945 grams (IQR 2270–3515) versus median 2600 grams (IQR 1550–3050), p = 0.011) compared to infants with non-diagnostic rapid ES (Table p = 0.003).A genetic diagnosis was made in 22/80 infants (28%) who had rapid ES in Phase II  in Phase II compared to Phase I (55% versus 23%), though the significance of this is difficult to interpret due to cohort size. Identification of recessive conditions has particular importance for reproductive counseling for the families. The time from genetics consult to MGD was median 17.5 days (IQR 15–23.5) and the age at MGD was median 30 days (IQR 21–46.8) . Rapid ES was the first genetic test sent based on our institution’s EMR for 16/22 infants diagnosed (73%). The genetic diagnosis was returned before discharge or death for 13/22 infants (59%), and 8/22 infants (36%) passed away in the first year of life. For two infants, rapid ES was sent due to concern for a second genetic etiology after a first genetic etiology had already been identified and did not identify an additional underlying genetic condition.BRCA2 variant identified in a parent. In total, genetic diagnoses impacted acute management in 14/22 infants (64%) . Of note, other genetic tests included non-rapid ES (sent for 12 infants), which was ES sent as an outpatient that took 2–3 months to return. There was not a significant difference in the percentage of infants who received a genetic diagnosis prior to discharge or death between the four types of genetic testing. Time from consult to diagnosis was not significantly different when comparing infants who had to those who did not have rapid ES, but was significant when excluding those infants who had chromosome-level diagnoses  . These remained significant when removing infants who had non-rapid ES performed .Finally, we investigated the genetic tests sent and the genetic diagnoses made in the Phase II cohort overall (Supplementary Table DMPK (OMIM 160900). Two infants had a gene panel or single gene tests sent prior to ES which did not identify a genetic diagnosis that was subsequently identified on rapid ES—an infant diagnosed with pathogenic variants in ALG12  who had negative MPS1 gene testing and an infant with a pathogenic variant in ADNP (OMIM 615873) who had a negative Rubinstein-Taybi syndrome panel. Of the infants with pathogenic CNVs, only one infant had both rapid ES and CMA sent, and both tests identified the pathogenic CNVs.There was only one case in which rapid ES did not identify a pathogenic variant that was identified on a different genetic test—an infant with congenital myotonic dystrophy for whom a myotonic syndrome gene panel identified the pathogenic expansion in Given that we collected data through September 2021 for infants admitted through March 2021 and there were thus some infants with less than 12 months of data, we conducted a sensitivity analysis for diagnostic yield and diagnosis prior to discharge or death restricting to genetic tests in the first six months of life, which did not change the statistical significance (there were only two infants who received a genetic diagnosis between six months and one year).11. These infants were also more likely to require respiratory support, which may reflect a tendency to prioritize rapid ES as a genetic test in more critically ill infants and may also be related to the high utilization among infants with neurological phenotypes . The increased length of stay and death within the first year for these infants likely reflects the known morbidity and mortality of genetic disorders that present in the neonatal period17.In this study, we describe the integration of rapid ES into routine clinical care in our Level IV NICU after the completion of a pilot research study. Overall, 248/1230 infants admitted to our institution’s NICU in Phase II had a genetics team consult, suggesting approximately one fifth of infants had a known or suspected underlying genetic disorder. Approximately one-third of infants with a genetics team consult in Phase II had rapid ES. These infants were more likely to have a neurological phenotype compared to those who did not have rapid ES. Although the infants were selected for rapid ES by the clinical team (neonatologist and consulting geneticist and/or neurologist) based on expert opinion and without specific selection criteria, this may reflect clinician recognition that neurological phenotypes have high yield on rapid ES, as previously demonstrated in Phase I22. We found that the transition of rapid ES from the research study setting in Phase I into routine clinical care in Phase II was associated with a relatively lower diagnostic yield and an increased time from both NICU admission and genetics consult to ES report. We have identified several barriers to the implementation of rapid ES in routine clinical care, highlighted with potential strategies in Fig. 20. The lower diagnostic yield of Phase II compared to Phase I may reflect the shift from eligibility based on specific selection criteria in Phase I to eligibility based on the expert opinion of individual clinicians at the time of an infant’s NICU admission (which includes their knowledge and attitudes on genomic sequencing) and approval by the hospital oversight committee in Phase II. We screened the infants who received a diagnosis by rapid ES in Phase II based on the selection criteria used in Phase I, and found that 4/22 (18%) may not have qualified in Phase I (Supplementary Table ADNP (OMIM 615873) that led to new subspecialty evaluation. Thus, while rapid ES in Phase II had a decreased diagnostic yield compared to Phase I, it also identified genetic diagnoses in infants who may have had a longer or unresolved diagnostic odyssey in Phase I. We are currently implementing a quality improvement project in our NICU to establish optimized multidisciplinary selection criteria24 for rapid ES based on the Phase I and Phase II results and to provide education to neonatology providers on genomic medicine and practical use of these criteria. We acknowledge the need to balance the cost of rapid ES—which is currently bundled into the hospitalization cost at our institution such that the hospital bears the cost burden if insurance reimbursement for this service does not occur—with its ability to rapidly identify a broad range of genetic disorders. It is thus important for selection criteria to also specify when rapid ES is not appropriate—for example, for patients with features highly suggestive of a trisomy or other chromosomal anomaly, CMA is more appropriate as a first-line genetic test.Optimizing the rapid ES workflow is critical to the efficiency and sustainability of genomic sequencing in routine clinical carehat 4/22 8% may noThe longer time from NICU admission and genetics consult to ES report in Phase II compared to Phase I likely reflects the logistics of the rapid ES workflow described above now falling on providers from various teams with competing clinical demands compared to a dedicated, readily available research team. For critically ill infants, each day delay in diagnosis can matter for impact ranging from providing the family diagnostic closure to making decisions regarding goals of care. To understand barriers, we divided the rapid ES process into three major intervals: time from NICU admission to genetics consult, time from genetics consult to sample collection, and time from sample collection to ES report. The time from NICU admission to genetics consult is determined by the time it takes the primary NICU team to identify an admitted infant as having a suspected underlying genetic disorder and consult the genetics team. The time from genetics consult to sample collection is determined by 1) the time to determination of eligibility, 2) the time to consent the family, and 3) the time to place the order and collect trio samples. Determination of eligibility based on a research team performing daily screening using specific selection criteria is more efficient than the determination of eligibility based on the expert opinion of various clinicians and subsequent approval by a hospital committee. We are attempting to increase efficiency by optimizing selection criteria and providing provider education as discussed above, as well as by surveillance by our Neonatal Genomics Program. Consenting the family, which includes getting in contact with the family to arrange a time for consent and the actual consent process, currently falls on the genetics team at our institution. Placing the order and collecting trio samples is often delayed by confusion about specific order details for what is considered a special lab test and coordination of buccal swab sample collection from the parents. Education of primary neonatology providers  who are at the bedside in real-time to consent and streamline sample collection and/or addition of a NICU Genetics Counselor who can focus on coordinating the ordering process are options we are currently exploring to improve efficiency.The time from sample collection to final ES report is determined by 1) the time from sample collection to shipment to the sequencing facility and 2) the sequencing turnaround time at the sequencing facility. In Phase I, the trio samples were personally shipped to the sequencing facility by the research team immediately after collection. In Phase II, the patient sample was sent to lab control and shipped from our institution to the sequencing facility and the parent samples were sent by either the genetics team or the parents (using a prepaid shipping label and box) to the sequencing facility. Practically, it is not feasible for lab control at a large hospital to immediately ship every sample on arrival to the sequencing facility, although rapid genetic tests are expedited for approval and shipping. The turnaround time at the sequencing facility for sequencing and analysis was the same for both phases. Thus, our ongoing QI efforts are focused on the former intervals. Overall, we were encouraged that utilization did not decrease in Phase II and that the median time difference from genetics consult to ES report between the phases was only two days.When comparing 1) rapid ES, 2) gene panel and single gene tests, 3) karyotype, FISH, and CMA, and 4) other genetic tests, several trends emerged. Traditionally, the tests in the second and third groups are the initial tests sent in a stepwise genetic evaluation, and ES may eventually be sent if these are non-diagnostic. On the one hand, rapid ES had a similar yield to gene panel and single gene tests , with a larger difference observed when we performed a sensitivity analysis removing cases where a metabolic diagnosis was known and genetic testing confirmed the diagnosis (26% vs 19%), but a shorter time to result. Of note, our data are likely an underestimate of the time to result for gene panel and single gene tests as the tests are sent only on the proband and thus parental phasing is often required to confirm a genetic diagnosis, and we were not always able to accurately determine the time interval to obtain subsequent phasing. Of the infants who had non-diagnostic gene panel or single gene tests, only two subsequently had rapid ES with diagnoses found and the majority remained undiagnosed at the end of the study period; thus it is unclear how many diagnoses may have been missed by this panel-first approach. Rapid ES had a higher yield than karyotype, FISH, and CMA (28% versus 10%), but a similar time to result.25. Of the 55 infants with non-diagnostic rapid ES in Phase II, 15 (27%) are currently enrolled in the research study.Although we acknowledge that for certain classes of variants, such as short tandem repeat expansions, ES is currently suboptimal for detection, we suggest that rapid ES (or GS) can be an appropriate initial genetic test for the majority of NICU infants suspected to have an underlying genetic condition as a single test that optimizes both diagnostic yield and time to result. However, for infants with features highly suggestive of a trisomy or other chromosomal anomaly, CMA provides similarly rapid results at less cost. An infant with non-diagnostic ES at our institution has access to both reanalysis clinically and enrollment in a research study to perform reanalysis, GS, and/or functional studies, and thus there is the potential to identify a genetic diagnosis from the single ES test in the future as we continue to gain knowledge about disease genes and pathogenic variantsImportant limitations of our study are the retrospective nature and decreased ability to fully capture the genetic diagnostic odyssey for infants who were not transferred to our NICU soon after birth  or who were transferred from our institution to another institution where the genetic diagnosis was made after additional genetic testing. Our study is based on data from a NICU at a quaternary care referral center, which is likely enriched for admissions with congenital anomalies and underlying genetic disorders compared to a NICU at a birth hospital. In addition, we acknowledge the importance of understanding the cost implications of genomic sequencing and are including these measures in future QI efforts. However, our study provides a valuable description of the integration of rapid ES into routine clinical care in a NICU with a high volume of infants with underlying genetic conditions.In conclusion, we find that integration of rapid ES into routine clinical care in the NICU after a pilot research study had a diagnostic yield within the range of previously published studies, rapid return of results and genetic diagnoses, and resulted in timely changes in clinical management in the majority of cases. Although clinically integrated rapid ES had a lower diagnostic yield and longer time to report than research study rapid ES, the diagnostic yield was still relatively high and it identified genetic diagnoses that would have been missed by the research study selection criteria. QI efforts are underway to optimize selection criteria and increase the efficiency of the rapid ES process. For clinicians working in the acute and busy NICU environment, we suggest that rapid ES represents a good choice for the majority of infants with suspected genetic disorders as an initial single genetic test  that captures the vast majority of currently known pathogenic genetic variants and shortens the difficult diagnostic odyssey for patients and their families, especially as the cost of genomic sequencing continues to decrease.11, consent for rapid exome sequencing was performed by the research team and in the clinical integration cohort, consent for rapid exome sequencing was performed by the consulting genetics team.This retrospective study was approved by the Boston Children’s Hospital Institutional Review Board with a waiver of informed consent due to the nature of the study involving retrospective review of EMR. In both the pilot research study cohort and the clinical integration cohort, rapid ES was performed at GeneDx . In the pilot research study cohort26. In addition to basic demographic data, we recorded the date, location, and reason for the initial genetics consult, the genetic testing recommended at the time of the initial consult, and the length of stay in the NICU or hospital; deceased status and date of death were also noted where applicable. Critical illness was defined by the need for respiratory support , vasoactive medications, and/or dialysis. We recorded whether prenatal or other genetic testing was sent before the initial consult and collected details on those genetic tests when available. For each genetic test sent at BCH, we recorded the date collected, the date resulted, and whether the test resulted in a MGD. A MGD was defined when a P or LP variant(s) was identified on any genetic test that explained the infant’s presentation . For each infant, we recorded whether a MGD was made in the first year of life, the testing modality leading to that diagnosis, and the date of diagnosis. For VUS, the clinical notes were reviewed to determine if these variants were considered clinically to represent a MGD. For infants with a MGD, we evaluated the impact on clinical management using the categories from the pilot research study: 1) transition to end-of-life/palliative care, 2) decision to pursue supportive care (tracheostomy and/or gastrostomy tube), 3) medication change or new therapeutic option, and/or 4) new subspecialty evaluation (including tests like echocardiography or magnetic resonance imaging). For the infants enrolled from the BCH NICU in the pilot research study, we reviewed the already collected data and the EMR to collect additional data on demographics and critical illness.We identified all infants admitted to the NICU of our institution between December 1, 2018 and March 31, 2021 who had a genetics team consult for an undiagnosed condition. Data related to the genetic evaluation and genetic diagnosis in the first year of life were abstracted from our EMR for each infant and stored in a Research Electronic Data Capture (REDCap) database hosted at our institutionStatistical analysis was performed using SPSS , using two-tailed Fisher’s exact, Mann-Whitney, or Kruskal-Wallis tests to compare variables as appropriate. Survival analysis was performed in R (Version 1.4.1717) using a Kaplan-Meier estimator and log rank test.Further information on research design is available in the Supplementary Dataset 1Supplementary InformationReporting Summary Checklist